Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7357 - 62 The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase; de Nadal E et al.; Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes . Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity . These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells . Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway . Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts . In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed . In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p . Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1) . Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit. Biochemistry, 1998 Jun 23, 37(25), 9193 - 202 Strong selective pressure to use G:U to mark an RNA acceptor stem for alanine; Chihade JW et al.; The identity of alanine tRNAs is dependent on a G:U base pair at the 3:70 position of the acceptor helix . This system of molecular recognition is widely distributed from bacteria to human-cell cytoplasm . In contrast, some mitochondrial alanine acceptor helices are markedly different and contain nucleotides known to block aminoacylation by a nonmitochondrial enzyme . Thus, acceptor helix recognition may differ in these systems and may not depend on G:U . Here we report an example of a Caenorhabditis elegans mitochondrial system where the G:U pair is preserved but where proximal nucleotides known to block charging by a nonmitochondrial enzyme are also present . We show that, as expected, the mitochondrial substrate is not charged by the bacterial enzyme . In contrast, the cloned mitochondrial enzyme charged both mitochondrial and bacterial microhelices . Strikingly, charging of each required the G:U pair . Thus, G:U recognition persists even with an acceptor helix context that inactivates nonmitochondrial systems . The results suggest strong selective pressure to use G:U in a variety of contexts to mark an acceptor stem for alanine . Separate experiments also demonstrate that, at least for the mitochondrial enzyme, helix instability or irregularity is not important for recognition of G:U. Biochemistry, 1998 Jun 23, 37(25), 9052 - 7 Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis; Lee AY et al.; Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase . Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein . To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry . The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding . The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses . Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes . Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process. Biochemistry, 1998 Jun 23, 37(25), 9033 - 7 Glutamate-459 is important for Escherichia coli branching enzyme activity; Binderup K et al.; The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan . The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site . By primary structure analysis, a conserved position unique to branching enzymes has been identified . This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering) . Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action.We used site-directed mutagenesis of the Glu-459 residue in the E . coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes . A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme . Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin. Biochemistry, 1998 Jun 23, 37(25), 8832 - 8 Autocatalytic formation of a hydroxy group at C beta of trp171 in lignin peroxidase; Blodig W et al.; In the high-resolution crystal structures of two lignin peroxidase isozymes from the white rot fungus Phanerochaete chrysosporium a significant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group . To further clarify the nature of this feature, we carried out tryptic digestion of the enzyme and isolated the Trp171 containing peptide . Under ambient conditions, this peptide shows an absorbance spectrum typical of tryptophan . At elevated temperature, however, the formation of an unusual absorbance spectrum with lambda max = 333 nm can be followed that is identical to that of N-acetyl-alpha, beta-didehydrotryptophanamide, resulting upon water elimination from beta-hydroxy tryptophan . The Trp171 containing tryptic peptide isolated from the recombinant and refolded lignin peroxidase produced from Escherichia coli does not contain the characteristic 333 nm absorbance band at any temperature . However, treatment with 3 equiv of H2O2 leads to complete hydroxylation of Trp171 . Reducing substrates compete with this process, e.g., in the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for complete modification . We conclude that the hydroxylation at the C beta of Trp171 is an autocatalytic reaction which occurs readily under conditions of natural turnover, e.g., in the ligninolytic cultures of P . chrysosporium, which are known to contain an oxidase-based H2O2-generating system . No dependence on dioxygen was found for this oxidative process . Chemical modification of fungal lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads to a drastically reduced activity with respect to the substrate veratryl alcohol . This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady-state conditions. Biochemistry, 1998 Jun 23, 37(25), 8825 - 31 Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis; Thoden JB et al.; Carbamoyl phosphate synthetase from Escherichia coli catalyzes the production of carbamoyl phosphate from two molecules of Mg2+ATP, one molecule of bicarbonate, and one molecule of glutamine . The enzyme consists of two polypeptide chains referred to as the large and small subunits . While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia . From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes . Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis . Here we describe the X-ray crystallographic structure of carbamoyl phosphate synthetase from E . coli in which His 353 has been replaced with an asparagine residue . Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution . The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family. Biochemistry, 1998 Jun 23, 37(25), 8817 - 24 Solution structure of the transmembrane H+-transporting subunit c of the F1F0 ATP synthase; Girvin ME et al.; Subunit c is the H+-translocating component of the F1F0 ATP synthase complex . H+ transport is coupled to conformational changes that ultimately lead to ATP synthesis by the enzyme . The properties of the monomeric subunit in a single-phase solution of chloroform-methanol-water (4:4:1) have been shown to mimic those of the protein in the native complex . Triple resonance NMR experiments were used to determine the complete structure of monomeric subunit c in this solvent mixture . The structure of the protein was defined by >2000 interproton distances, 64 (3)JN alpha, and 43 hydrogen-bonding NMR-derived restraints . The root mean squared deviation for the backbone atoms of the two transmembrane helices was 0.63 A . The protein folds as a hairpin of two antiparallel helical segments, connected by a short structured loop . The conserved Arg41-Gln42-Pro43 form the top of this loop . The essential H+-transporting Asp61 residue is located at a slight break in the middle of the C-terminal helix, just prior to Pro64 . The C-terminal helix changes direction by 30 +/- 5 degrees at the conserved Pro64 . In its protonated form, the Asp61 lies in a cavity created by the absence of side chains at Gly23 and Gly27 in the N-terminal helix . The shape and charge distribution of the molecular surface of the monomeric protein suggest a packing arrangement for the oligomeric protein in the F0 complex, with the front face of one monomer packing favorably against the back face of a second monomer . The packing suggests that the proton (cation) binding site lies between packed pairs of adjacent subunit c. Biochemistry, 1998 Jun 16, 37(24), 8803 - 7 Mutagenic specificity of model estrogen-DNA adducts in mammalian cells; Terashima I et al.; Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic properties of the model estrogen-DNA adducts N2-{3-methoxyestra-1,3,5(10)-trien-6(alpha, beta)-yl}-2'-deoxyguanosine (dG-N2-3MeE) and N6-{3-methoxyestra-1,3, 5(10)-trien-6(alpha,beta)-yl}-2'-deoxyadenosine (dA-N6-3MeE) in simian kidney (COS-7) cells . Oligodeoxynucleotides (5'TCCTCCTCXCCTCTC; X = dG, dA, dG-N2-3MeE, or dA-N6-3MeE) containing an unmodified or model estrogen lesion were inserted into single-stranded (ss) phagemid vectors . These ss vectors were transfected into COS-7 cells . The progeny plasmid obtained were used to transform Escherichia coli DH10B . The transformants were analyzed by oligodeoxynucleotide hybridization and sequencing to determine the mutation frequency and spectrum . Preferential incorporation of dCMP, the correct base, was observed opposite the dG-N2-3MeE lesion . Targeted mutations showing G --> T transversions were detected, along with a small number of G --> C transversions . When a dA-N6-3MeE-modified oligodeoxynucleotide was used, preferential incorporation of dTMP, the correct base, was also observed . Targeted mutations representing A --> T transversions were detected, accompanied by a small amount of A --> G transitions . The frequency of mutation observed opposite dA-N6-3MeE (17.5%) was 2.3 times higher than that observed opposite dG-N2-3MeE (7.5%) . These results indicate that estrogen DNA adducts have mutagenic potential in mammalian cells. Biochemistry, 1998 Jun 16, 37(24), 8776 - 82 Evaluation of the kinetic mechanism of Escherichia coli glycinamide ribonucleotide transformylase; Shim JH et al.; A kinetic scheme is presented for Escherichia coli glycinamide ribonucleotide transformylase (GAR transformylase, EC 2.1.2.2) based on a steady-state and pre-steady-state kinetic analysis of the reaction in both directions employing stopped-flow absorbance and fluorescence spectroscopy . Steady-state parameters showed that kcat for the reverse direction is about 10 times lower than that for the forward direction although the Km values for formyl dideazafolate and dideazafolate or for glycinamide ribonucleotide and formyl glycinamide ribonucleotide are similar . No pre-steady-state transient was observed in either direction, and the single-turnover rate constant under saturating levels of substrates in each direction was found to be very close to the respective steady-state kcat value . This indicates that steps involving ternary complexes are rate-determining for steady-state turnover in each direction . By conducting the single-turnover reactions under various preincubation and mixing conditions, a random sequential kinetic mechanism was implicated in which the enzyme binds glycinamide ribonucleotide or formyl dideazafolate productively in no obligatory order . The collective data provided a quantitative kinetic scheme to serve as a basis for the analysis of mutations. Biochemistry, 1998 Jun 16, 37(24), 8735 - 42 Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency; Klabe RM et al.; Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR) . Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors . Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor . The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity . The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups . The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90 . Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold) . However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme . Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance . Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene. Biochemistry, 1998 Jun 16, 37(24), 8653 - 8 The influence of the regulatory chain amino acids Glu-62 and IIe-12 on the heterotropic properties of Escherichia coli aspartate transcarbamoylase; Dutta M et al.; In the structure of Escherichia coli aspartate transcarbamoylase with CTP bound {Kosman, R . P., Gouaux, J . E., and Lipscomb, W . N . (1993) Proteins, Struct . Funct . Genet . 15, 147-177} Lys-6 and Glu-62 form a salt-link between two regulatory chains . However, recent X-ray structural studies suggest that side chain and backbone interactions existed between Glu-62 and Ile-12 . Thus the interaction between Glu-62 and Ile-12 may help to establish the correct conformation of the nucleotide binding site . The present study of two single-site mutant enzymes, Glu-62r-->Ala and Ile-12r-->Ala, was undertaken to investigate whether the role of Glu-62 is to maintain the stability of the interface between the regulatory chains in the dimer, or interacts with the side chain of Ile-12r to define the nucleotide binding site . For both the mutant enzymes, the maximal velocity, the aspartate saturation at half the maximal velocity, and Hill coefficient were close to wild-type values . The Glu-62r-->Ala enzyme showed enhanced regulatory effects with ATP, CTP, and UTP . As a result of this mutation the enzyme losses its ability to discriminate between CTP and UTP . For the Ile-12r-->Ala enzyme, the heterotropic properties were reduced or eliminated . The enhanced regulatory effects observed with the Glu-62r-->Ala enzyme do not seem to be consistent with the presence of a salt-link between Glu-62r and Lys-6r . However, based upon kinetic data of the unique but completely opposite heterotropic properties of the two mutant enzymes, it is suggested that the side chain interaction between Glu-62r and Ile-12r helps to define the conformation of the effector binding pocket . In this study, we report the properties of both the Glu-62r-->Ala and Ile-12r-->Ala enzymes and their importance for the heterotropic activation and inhibition of aspartate transcarbamoylase. Biochim Biophys Acta, 1998 May 20, 1392(1), 119 - 26 Molecular cloning, expression in Escherichia coli, and characterization of a novel L-3-hydroxyacyl coenzyme A dehydrogenase from pig liver; He XY et al.; A novel L-3-hydroxyacyl-CoA dehydrogenase from pig liver has been cloned, expressed, purified, and characterized . This enzyme is a homodimer with a molecular mass of 65.6 kDa, and is distinguished from the dehydrogenase of pig heart by its structural features and catalytic properties . Its subunit, consisting of 302 amino acid residues, has two additional residues in a key region of the active center while it lacks a sequence of seven residues in the NAD+-binding domain, when compared with the subunit of pig heart enzyme . In addition, there are substitutions of four single residues . The catalytic efficiency of pig liver dehydrogenase was significantly greater than that of the heart enzyme for short-chain substrate, but its catalytic rates declined with an increase in substrate chain-lengths . The distinction between pig liver and heart dehydrogenases cannot be attributed to a species difference, and thus it is concluded that there exist different isoforms of monofunctional L-3-hydroxyacyl-CoA dehydrogenases in pig . High level expression of mitochondrial L-3-hydroxyacyl-CoA dehydrogenase in Escherichia coli has provided a very convenient way to purify this important beta-oxidation enzyme . There is substantial homology between pig liver dehydrogenase and various multifunctional beta-oxidation enzymes in the active center of these enzymes; a consensus sequence, HX3PX1-3MXLXE, is proposed as the signature sequence of l-3-hydroxyacyl-CoA dehydrogenases . Brain Res Mol Brain Res, 1998 Apr, 55(2), 181 - 97 Identification of a novel serine protease-like molecule in human brain; Meckelein B et al.; Proteolysis of the amyloid beta protein precursor (APP) is a key event in the development of Alzheimer's disease . In our search for proteases that can cleave APP and liberate the amino terminus of the amyloidogenic beta protein, we characterized a calcium-dependent serine protease (CASP) which is present in reactive astrocytes and cross-reacts with anti-cathepsin G antibodies . We wanted to take advantage of this cross-reactivity to clone the cDNA of CASP and eventually evaluate its tissue distribution . Screening of two human fetal brain cDNA libraries with anti-cathepsin G antibodies led to the identification of a cDNA coding for a novel protein whose only homology to known proteins is to the active site of trypsin-type serine proteases . We called this protein the novel serine protease (NSP) . NSP exists in at least three differentially spliced forms, one of which is expressed predominantly in brain and testis . Immunohistochemistry and immunoprecipitation with antibodies generated against NSP show that it is expressed and secreted by a variety of cells and that, in brain, it is found primarily in cerebrovascular smooth muscle cells and reactive astrocytes . Biophys J, 1998 Jun, 74(6), 2889 - 902 Electromechanical coupling model of gating the large mechanosensitive ion channel (MscL) of Escherichia coli by mechanical force; Gu L et al.; We have developed a theoretical electromechanical coupling (EMC) model of gating of the large-conductance mechanosensitive ion channel (MscL) . The model presents the first attempt to explain the pressure-dependent transitions between the closed and open channel conformations on a molecular level by assuming 1) a homohexameric structural model of the channel, 2) electrostatic interactions between various domains of the homohexamer, 3) structural flexibility of the N-terminal portion of the monomer, and 4) mechanically and electrostatically induced displacement of the N-terminal domain relative to other structural domains of the protein . In the EMC model, 12 membrane-spanning alpha-helices (six each of the M1 and M2 transmembrane domains of the MscL monomer), are envisaged to line the channel pore with a diameter of 40 A, whereas the N- and C-termini are oriented toward each other inside the pore when the channel is closed . The model proposes that stretching the membrane bilayer by mechanical force causes the monomers to be pulled away from and slightly tilted toward each other . This relative movement of alpha-helices could serve as a trigger to initiate a "swing-like" motion of the N-terminus around the glycine residue G14 that may act as a pivot . The analysis of the attractive and repulsive coulomb forces between all domains of the channel homohexamer suggested that an inclination angle of approximately 3.0 degrees - 4.1 degrees between the oppositely oriented channel monomers should suffice for the N-terminus to turn away from other domains causing the channel to open . According to the EMC model the minimal free energy change, deltaG, that could initiate the opening of the channel was 2 kT . Also, the model predicted that the negative pressure required for channel open probability, Po = 0.5, should be between 50 and 80 mmHg . These values were in a good agreement with the experimentally estimated pressures of 60-70 mmHg obtained with the MscL reconstituted in liposomes . Furthermore, consistent with a notion that the N-terminus may present a mechanosensitive structural element providing a mechanism to open the MscL by mechanical force, the model provides a simple explanation for the variations in pressure sensitivity observed with several MscL mutants having either deletions or substitutions in N- or C-terminus, or site-directed mutations in the S2-S3 loop. Biophys J, 1998 Jun, 74(6), 2786 - 801 A molecular dynamics study of the pores formed by Escherichia coli OmpF porin in a fully hydrated palmitoyloleoylphosphatidylcholine bilayer; Tieleman DP et al.; In this paper we study the properties of pores formed by OmpF porin from Escherichia coli, based on a molecular dynamics simulation of the OmpF trimer, 318 palmitoyl-oleoyl-phosphatidylethanolamine lipids, 27 Na+ ions, and 12,992 water molecules . After equilibration and a nanosecond production run, the OmpF trimer exhibits a C-alpha root mean square deviation from the crystal structure of 0.23 nm and a stable secondary structure . No evidence is found for large-scale motions of the L3 loop . We investigate the pore dimensions, conductance, and the properties of water inside the pore . This water forms a complicated pattern, even when averaged over 1 ns of simulation time . Around the pore constriction zone the water dipoles are highly structured in the plane of the membrane, oriented by the strong transversal electric field . In addition, there is a net orientation along the pore axis pointing from the extracellular to the intracellular side of the bilayer . The diffusion coefficients of water inside the pore are greatly reduced compared to bulk . We compare our results to results from model pores (Breed et al., 1996 . Biophys . J . 70:1 643-1 661; Sansom et al . 1997 . Biophys . J . 73:2404-241 5) and discuss implications for further theoretical work. Trends Genet, 1998 Jun, 14(6), 236 - 43 Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis; Patton EE et al.; The ubiquitin-dependent proteolytic pathway targets many key regulatory proteins for rapid intracellular degradation . Specificity in protein ubiquitination derives from E3 ubiquitin protein ligases, which recognize substrate proteins . Recently, analysis of the E3s that regulate cell division has revealed common themes in structure and function . One particularly versatile class of E3s, referred to as Skp1p-Cdc53p-F-box protein (SCF) complexes, utilizes substrate-specific adaptor subunits called F-box proteins to recruit various substrates to a core ubiquitination complex . A vast array of F-box proteins have been revealed by genome sequencing projects, and the early returns from genetic analysis in several organisms promise that F-box proteins will participate in the regulation of many processes, including cell division, transcription, signal transduction and development. Biochem Mol Biol Int, 1998 Jun, 45(1), 155 - 61 A novel method for selection of chymotrypsin inhibitors from a phage peptide library; Liu Y et al.; A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library . In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates . After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing . These peptides share a consensus sequence motif as (S/T)RVPR(R/H) . Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin . The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure. Biotechnology (N Y), 1995 Aug, 13(8), 801 - 4 Improved mimicry of a foot-and-mouth disease virus antigenic site by a viral peptide displayed on beta-galactosidase surface; Benito A et al.; A major antigenic site (site A) of foot-and-mouth disease virus includes multiple overlapping epitopes located within the flexible G-H loop of capsid protein VP1 . We have studied the antigenicity of several recombinant E . coli beta-galactosidases displaying the site A from a serotype C virus in different surface regions of the bacterial enzyme . In each one of the explored insertion sites, the recombinant peptide shows different specificity with a set of anti-virus monoclonal antibodies directed to site A . In some of them, the inserted stretch mimics better than free or haemocyanin-coupled peptide the antigenicity of site A in the intact virus . In particular, an insertion within an exposed loop involved in the activating interface of beta-galactosidase (amino acids 272 to 287) led to a significant improvement of the overall reactivity . Since insertions at this site renders proteins enzymatically active, the activating interface could be an adequate place for the presentation of foreign antigens in correctly assembled beta-galactosidase tetramers . These results also suggest that anti-virus antibodies directed against the major antigenic site of FMDV recognize different conformations of the G-H loop, which are better reproduced in some of the recombinant proteins because of the dissimilar restrictions imposed by each particular insertion site. Biotechnology (N Y), 1995 May, 13(5), 504 - 6 Improved N-terminal processing of recombinant proteins synthesized in Escherichia coli; Sandman K et al.; Preparations of rHMfA (recombinant histone A from Methanothermus fervidus) synthesized in E . coli by the heterologous expression of the hmfA gene were found to contain a mixture of rHMfA molecules, approximately 40% that retained the N-terminal formyl-methionyl residue (f-met-rHMfA), approximately 50% that lacked the formyl moiety but retained the methionyl residue (met-rHMfA), and only approximately 10% that had lost both components of the protein synthesis initiating amino acid residue and therefore had the same N-terminal sequence as native HMfA molecules synthesized in Mt . fervidus . Expression of the hmfA gene in E . coli cells grown in the presence of trimethoprim and thymidine, coupled with the concurrent over-expression of a methionine aminopeptidase-encoding map gene, has been shown to overcome this N-terminal heterogeneity problem and to result in rHMfA preparations in which > 85% of the molecules have the fully processed, native N-terminal sequence . This procedure should be generally useful for ensuring N-terminal processing of recombinant proteins synthesized in E . coli. Biotechnology (N Y), 1995 May, 13(5), 475 - 9 Antibody VH domains as small recognition units; Davies J et al.; To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH . Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface . These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners . The third hypervariable loop of the modified VH was then randomised to yield a repertoire of 2 x 10(8) independent clones, which was displayed on phage and selected through antigen binding . VH clones specific for hapten and protein antigens were isolated . Soluble VH was expressed with an isoleucine residue at position 47 to improve expression and stability compared to VH containing a glycine residue at this position, which however was preferable for phage selection . Affinities of soluble VH for hapten were between 100 nM and 400 nM . The VH domains were highly specific, stable and well expressed in Escherichia coli . These positive biophysical properties and their small size make them attractive for biotechnological applications. Biotechnology (N Y), 1995 Apr, 13(4), 373 - 7 Calmodulin as a versatile tag for antibody fragments; Neri D et al.; Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner . We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments . Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose) . The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis . Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin. Biotechnology (N Y), 1995 Apr, 13(4), 366 - 72 Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions; Lu Z et al.; We have developed a system for probing protein/protein interactions which makes use of the bacterial flagellum to display random peptide libraries on the surface of E . coli . In developing the system the entire coding sequence of E . coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E . coli flagellum . The resulting fusion protein (FLITRX) was efficiently exported and assembled into partially functional flagella on the bacterial cell surface . A diverse library of random dodecapeptides were displayed in FLITRX on the exterior of E . coli as conformationally constrained insertions into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin . To demonstrate that members of this library could be bound and selected via specific protein/protein interactions to a target protein, a method was devised to enable efficient isolation of those bacteria displaying peptides with affinity to immobilized antibodies . We have unambiguously mapped three different antibody epitopes using this method . Peptides selected as FLITRX active-site fusions retain their binding specificity when made as native thioredoxin active-site loop fusions . This will facilitate future structural characterizations and broaden the general utility of the system for exploring other classes of protein-protein interactions. Biotechnology (N Y), 1995 Mar, 13(3), 276 - 8 A computer program to determine a protein sequence from an amino acid analysis; Cordier-Ochsenbein F et al.; We have developed a computational method for analyzing the proteolytic products of a protein, knowing its sequence and the amino acid percentages of its products . For all fragments, amino acid percentages are calculated and compared to the experimental results (calculating the error within the experiment) . The program keeps the best fitted fragment using a least squared method . This program was written to determine the sequence of the proteolytic products that appeared during the purification of annexin I domain 2 . The reliability of the method was verified in this case . However the latter depends on the length and on the amino acid composition of the entire protein and of its fragments . This program may be suitable for analyzing the sequence of the products in any protease digestion, whether designed or accidental. Biotechnology (N Y), 1995 Feb, 13(2), 175 - 9 Tight transcriptional control mechanism ensures stable high-level expression from T7 promoter-based expression plasmids; Mertens N et al.; One of the more efficient systems for high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulatable transcription unit supplying the specific T7 RNA polymerase . Using various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase, we document that leaky expression of the latter is responsible for the frequently observed loss of the culture's ability to express genes of interest . We further show that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system . We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is attenuated by a series of tandemly arranged transcription terminators . The plasmids also incorporate the phage lambda-derived nutL/N protein antitermination function, allowing conditional reversion of attenuation upon induction . The applicability of the system is illustrated by the strictly regulatable, high-level production of several cytokines of human and murine origin. Biotechnology (N Y), 1995 Feb, 13(2), 151 - 4 Red-shifted excitation mutants of the green fluorescent protein; Delagrave S et al.; Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP . Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins . Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus . These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism . In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays. Biotechnology (N Y), 1995 Jan, 13(1), 53 - 7 Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus; Turpen TH et al.; Using malaria as a model disease, we engineered the surface of tobacco mosaic tobamovirus (TMV) for presentation of selected epitopes to the mammalian immune system . The TMV coat protein is a well-characterized and abundant self-assembling polymer previously shown to be a highly immunogenic carrier . Selected B-cell epitopes were either inserted into the surface loop region of the TMV coat protein or fused to the C terminus using the leaky stop signal derived from the replicase protein reading frame . Tobacco plants systemically infected with each of these constructs contained high titers of genetically stable recombinant virus, enabling purification of the chimeric particles in high yield . Symptoms induced in tobacco ranged from a normal mosaic pattern similar to that induced by the parental U1 strain to a unique bright yellow mosaic . As measured by quantitative ELISA against synthetic peptide standards, wild type TMV coat protein and fusion protein synthesized by the leaky stop mechanism coassembled into virus particles at the predicted ratio of approximately 20:1 . Recombinant plant viruses have the potential to meet the need for scalable and cost effective production of subunit vaccines that can be easily stored and administered. Q Rev Biophys, 1997 Nov, 30(4), 333 - 64 From membrane to molecule to the third amino acid from the left with a membrane transport protein; Kaback HR et al.; The lac permease of E . coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient . This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer . The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane . Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover . Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E . coli is proposed . Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII) . In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles . Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa . Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa . In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex . In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325 . The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane . Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism . On the other hand, widespread, cooperative conformational changes appear to be involved in turnover . As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover . This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e . helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism . (ABSTRACT TRUNCATED) Plant Cell, 1998 Jun, 10(6), 981 - 93 Pollen profilin function depends on interaction with proline-rich motifs; Gibbon BC et al.; The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells . Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms . Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L-proline and monomeric actin from maize pollen . In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen . The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen . In contrast, the affinity of ZmPRO4 for poly-L-proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms . When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1 . A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L-proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4 . In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L-proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells. Plant Cell, 1998 Jun, 10(6), 905 - 15 Srchi13, a novel early nodulin from Sesbania rostrata, is related to acidic class III chitinases; Goormachtig S et al.; On the tropical legume Sesbania rostrata, stem-borne nodules develop after inoculation of adventitious root primordia with the microsymbiont Azorhizobium caulinodans . A cDNA clone, Srchi13, with homology to acidic class III chitinase genes, corresponds to an early nodulin gene with transiently induced expression during nodule ontogeny . Srchi13 transcripts accumulated strongly 2 days after inoculation, decreased from day 7 onward, and disappeared in mature nodules . Induction was dependent on Nod factor-producing bacteria . Srchi13 was expressed around infection pockets, in infection centra, around the developing nodule and its vascular bundles, and in uninfected cells of the central tissue . The specific and transient transcript accumulation together with the lipochitooligosaccharide degradation activity of the recombinant protein hint at a role of Srchi13 in normal nodule ontogeny by limiting the action of Nod factors. J Biol Chem, 1998 Jun 19, 273(25), 15860 - 5 Biochemical properties of two protein kinases involved in disease resistance signaling in tomato; Sessa G et al.; In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein . In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule . Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathione S-transferase fusion proteins, respectively . The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping . In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay . Moreover, an active glutathione S-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto . Pti1 phosphorylation by Pto was also characterized and found to occur with a Km of 4.1 microM at sites similar to those autophosphorylated by Pti1 . Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping . This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites. J Biol Chem, 1998 Jun 19, 273(25), 15675 - 81 A revised model for the oligomeric state of the N-ethylmaleimide-sensitive fusion protein, NSF; Fleming KG et al.; The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking . Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions . However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers . We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques . Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer . Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains . The sedimentation coefficient, s20,w0, of 13.4 (+/-0 . 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape . The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions. J Biol Chem, 1998 Jun 19, 273(25), 15546 - 52 The ATPase activity of Hsp104, effects of environmental conditions and mutations; Schirmer EC et al.; Hsp104 is crucial for stress tolerance in Saccharomyces cerevisiae, and both of its nucleotide-binding domains (NBD1 and NBD2) are required . Here, we characterize the ATPase activity and oligomerization properties of wild-type (WT) Hsp104 and of NBD mutants . In physiological ionic strength buffers (pH 7.5, 37 degreesC) WT Hsp104 exhibits Michaelis-Menten kinetics between 0.5 and 25 mM ATP (Km approximately 5 mM, Vmax approximately 2 nmol min-1 microg-1) . ATPase activity is strongly influenced by factors that vary with cell stress (e.g . temperature, pH, and ADP) . Mutations in the P-loop of NBD1 (G217V or K218T) severely reduce ATP hydrolysis but have little effect on oligomerization . Analogous mutations in NBD2 (G619V or K620T) have smaller effects on ATPase activity but impair oligomerization . The opposite relationship was reported for another member of the HSP100 protein family, the Escherichia coli ClpA protein, in studies employing lower ionic strength buffers . In such buffers, the Km of WT Hsp104 for ATP hydrolysis decreased 10-fold and its stability under stress conditions increased, but the effects of the NBD mutations on ATPase activity and oligomerization remained opposite to those of ClpA . Either the functions of the two NBDs in ClpA and Hsp104 have been reversed or both contribute to ATP hydrolysis and oligomerization in a complex manner that can be idiosyncratically affected by such mutations. J Biol Chem, 1998 Jun 19, 273(25), 15487 - 93 Defining the interleukin-8-binding domain of heparan sulfate; Spillmann D et al.; Interleukin-8, a member of the CXC chemokine family, has been shown to bind to glycosaminoglycans . It has been suggested that heparan sulfate on cell surfaces could provide specific ligand sites on endothelial cells to retain the highly diffusible inflammatory chemokine for presentation to leukocytes . By using selectively modified heparin and heparan sulfate fragments in a nitrocellulose filter trapping system, we have analyzed sequence requirements for interleukin-8 binding to heparin/heparan sulfate . We demonstrate that the affinity of a monomeric interleukin-8 molecule for heparin/heparan sulfate is too weak to allow binding at physiological ionic strength, whereas the dimeric form of the protein mediates binding to two sulfated domains of heparan sulfate . These domains, each an N-sulfated block of approximately 6 monosaccharide units, are contained within an approximately 22-24-mer sequence and are separated by a region of </=14 monosaccharide residues that may be fully N-acetylated . Binding to interleukin-8 correlates with the occurrence of the di-O-sulfated disaccharide unit -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- . We suggest that the heparan sulfate sequence binds in horseshoe fashion over two antiparallel-oriented helical regions on the dimeric protein. J Biol Chem, 1998 Jun 19, 273(25), 15479 - 86 RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region; Yamashita T et al.; The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase . We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010) . The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells . The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties . Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity . The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells . These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs. Biochem Pharmacol, 1998 May 15, 55(10), 1673 - 81 Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells; Parker WB et al.; Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer . Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently . The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively . F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells . Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses . CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively . The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr . Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells . These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E . coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E . coli cytosine deaminase . These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment. Arch Biochem Biophys, 1998 Jun 1, 354(1), 158 - 64 Cross-linking and disulfide bond formation of introduced cysteine residues suggest a modified model for the tertiary structure of URF13 in the pore-forming oligomers; Rhoads DM et al.; URF13 is a mitochondrially encoded protein in the inner mitochondrial membrane of maize (Zea mays L.) carrying the cms-T cytoplasm . This protein is responsible for Texas-type cytoplasmic sterility and is a ligand-gated, pore-forming receptor for the pathotoxins of fungal pathogens Bipolaris maydis race T and Phyllosticta maydis . URF13 contains three transmembrane alpha-helices, with amphipathic helices II and III likely involved in pore formation, and is present as oligomers in cms-T maize mitochondria and when expressed in Escherichia coli cells . To study tertiary and quaternary structures of URF13 oligomers, we employed combinations of site-directed mutagenesis and chemical cross-linking . We introduced Cys residues individually into consecutive positions 78-82, believed to be in helix III . We expressed these proteins in E . coli cells and tested for cross-linking through disulfide bond formation or by using Cys-Cys cross-linkers . URF13-R79C, URF13-R81C, and URF13-T82C were cross-linked using Cys-Cys-specific cross-linkers, as were double mutants URF13-C27R/R79C, URF13-C27R/R81C, and URF13-C27R/T82C, indicating that the cross-linking was between introduced Cys residues on adjacent URF13 molecules . Disulfide bond formation, induced by diamide, was seen only in URF13-T82C and URF13-C27R/T82C, indicating that Cys residues introduced into position 82 are closely juxtaposed in the oligomers . Based on these observations, we modified the models for the secondary structure of URF13 and the tertiary structure of the URF13 oligomers . Sequential cross-linking of URF13-R81C oligomers with bismaleimidohexane (Cys-Cys cross-linker) and N,N'-dicyclohexylcarbodiimide (Lys-Asp/Glu cross-linker) suggests that URF13 oligomers consist of an even number of monomers. J Biol Chem, 1998 Jun 26, 273(26), 16339 - 45 The mechanism of action of steroidogenic acute regulatory protein (StAR) . StAR acts on the outside of mitochondria to stimulate steroidogenesis; Arakane F et al.; Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450scc on the inner mitochondrial membrane . StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH2-terminal mitochondrial targeting sequence . To explore the mechanism of StAR action, we produced 6-histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the NH2-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria . His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system . His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria . There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes . In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment . In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin . Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed . To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli . Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations . A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein . This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria . Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol . The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants. Immunogenetics, 1998 Jul, 48(2), 108 - 15 High RAD51 mRNA levels in young rabbit appendix . A role in B-cell gene conversion? Schiaffella E, Fuschiotti P, Bensinger SJ, Mage RG. The rabbit has a limited number of VH genes that rearrange . As in the chicken, the 3'-most VH1 gene is rearranged in most B lymphocytes . This laboratory reported that by 6 weeks after birth, diversification of rearranged VH genes occurs, at least in part, by gene conversion-like events in the appendix, suggesting that this organ is a homologue of the avian bursa of Fabricius . Rad51 contributes to the repair of double-strand breaks in DNA during somatic and meiotic recombination . The gene was first identified in lower eukaryotes, and later in vertebrates including chicken, as encoding an Escherichia coli RecA-like protein . We report the cloning and sequencing of RAD51 from the rabbit . Because the chicken bursa was shown to express high levels of RAD51 message, we investigated the expression of RAD51 in the rabbit appendix and other tissues . Using a quantitative polymerase chain reaction mimic assay and conventional northern analyses, we found high RAD51 expression in young rabbit appendix comparable to levels in testis where there is an abundance of meiotic recombination . RAD51 levels were three times higher in appendix B lymphocytes compared with T lymphocytes and were lower in adult appendix, as well as in spleen and Peyer's patches of young rabbits . We measured the levels of message in several appendix cell sub-populations obtained by fluorescence-activated cell sorting and found that sub-populations of B lymphocytes corresponding to different stages of B-cell development as well as B cells undergoing isotype switch did not have significantly different mRNA levels. Sex Transm Infect, 1998 Feb, 74(1), 11 - 9 Influence of ovarian hormones on urogenital infection; Sonnex C; Numerous studies have examined the influence of hormones on infectious diseases and there is now a wealth of data relating to the more specific effect of the sex hormones, oestrogen and progesterone, on urogenital infections . The interaction between these hormones and the immune system is complex and the variation of hormonal effect between species further complicates the true picture as related to humans . Although it is difficult therefore to draw general conclusions regarding predominant effects of specific hormones, there is the suggestion that oestrogen enhances the pathogenicity of many urogenital micro-organisms . Our understanding of the influential role played by sex hormones in disease pathogenesis is at an early stage and illustrates well the importance of drawing together and interpreting as a whole both epidemiological and molecular studies. J Gen Virol, 1998 Jun, 79 ( Pt 6), 1531 - 7 Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR; Barthe GA et al.; Citrus psorosis virus (CPV) is a multicomponent ssRNA virus with a coat protein of approximately 48 kDa . The viral genome is encapsidated in short and long particles that are readily separated by sucrose density-gradient centrifugation . CPV particles are spiral filaments that are referred to as spiroviruses (SV) . A cDNA library of purified short particles from isolate CPV-4 was prepared in a Lambda vector and screened for expression of the coat protein gene (CPG) with a monoclonal antibody to the coat protein . Sequencing of immunopositive clones indicated a single ORF encoding a 49 kDa protein . This ORF, when expressed in E . coli, gave a protein identical in size and immunoreactivity to the CPV coat protein . A full-length clone of the CPG was transcribed and used in Northern hybridization assays to establish that short particle RNA of CPV is negative sense and contains the CPG . Moreover, the CPG was not found on RNA extracted from long particles or on the sedimentable dsRNA from CPV infected tissue . RT-PCR assays were developed for the amplification of a 600 bp fragment of CPG and for the complete CPG (1317 bp) . The 600 bp fragment from a biologically and serologically different isolate, CPV-6, was cloned, sequenced and found to share 86% (nucleotide) and 96% (amino acid) identity with CPV-4 . BLAST analysis of sequences from CPV-4 and CPV-6 detected no significant nucleic acid or protein similarity with any known viral sequences. J Gen Virol, 1998 Jun, 79 ( Pt 6), 1509 - 17 Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants; Jacquet C et al.; Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene . Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus . In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions . These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy . Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission . However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs . Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT . Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation. J Gen Virol, 1998 Jun, 79 ( Pt 6), 1487 - 94 Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7, S8, S9 and S10; Isogai M et al.; The proteins encoded by rice black streaked dwarf fijivirus (RBSDV) genomic segments 7-10 (S7-S10) were characterized . Open reading frames (ORFs) from these segments were expressed as fusion proteins in Escherichia coli . Antibodies raised against the expressed products were used as probes to determine whether the viral ORFs encode structural proteins . In Western blots, antibodies to the expressed S8 and S10 products reacted with a core capsid (65 kDa) and a major outer capsid (56 kDa) protein, respectively, while none of the antibodies to S7 and S9 products reacted with structural proteins . Antisera to RBSDV S7 ORF1 and S9 ORF1 each detected a single protein of the predicted size in total protein extracts from infected rice plants and viruliferous Laodelphax striatellus . Immunoelectron microscopy revealed that antibodies to RBSDV S7 ORF1 and RBSDV S9 ORF1 reacted with tubular structures and viroplasm, respectively, in sections of both infected maize plants and viruliferous L . striatellus . Antisera to ORF2 of S7 and S9 failed to detect any proteins in the infected tissue using either Western blotting or immuno-electron microscopic techniques. Circ Res, 1998 Jun 15, 82(11), 1173 - 88 Increased protein kinase C activity in myotrophin-induced myocyte growth; Sil P et al.; Myotrophin, a novel protein that has been shown to stimulate myocyte growth, has been isolated, purified, and sequenced from the hearts of spontaneously hypertensive rats and dilated cardiomyopathic human tissue . Recently, the cDNA clones encoding myotrophin have been isolated and expressed in Escherichia coli, and the recombinant myotrophin was found to be as biologically and immunologically active as natural myotrophin . The mechanism by which myotrophin stimulates protein synthesis and initiates myocardial hypertrophy is not known . To evaluate the involvement of protein kinase C (PKC) in myotrophin-induced hypertrophy, PKC activity and its distribution in the subcellular fraction were determined in cultured neonatal and adult myocytes . PKC activity was determined by measuring the incorporation of 32P into histone type III-S and PKCepsilon substrate peptide (epsilon(pep)) from {gamma-32P}ATP in neonatal myocytes . Myotrophin significantly stimulated PKC activity in neonatal myocytes and was associated with a significant increase in protein synthesis . The effect of myotrophin on the stimulation of PKC activity and {3H}leucine incorporation was abolished by pretreatment with either staurosporine or H-7, two selective, pharmacological PKC inhibitors . Pretreatment of myocytes with staurosporine also reduced the myotrophin-induced mRNA levels of c-fos and beta-myosin heavy chain . To evaluate the subcellular events whose occurrence was due to myotrophin and translocation of PKC, we studied the effect of genistein, a tyrosine kinase inhibitor, on myotrophin-induced neonatal myocyte growth . Genistein attenuated the {3H}leucine incorporation induced by myotrophin . To define the specificity of the PKC isoform(s) involved in myotrophin-stimulated myocyte growth, both neonatal and adult myocytes were treated with myotrophin, and Western blot analyses were performed by using the antibodies of different PKC isoforms . Results showed that both PKCalpha and PKCepsilon isoforms participated in the myotrophin-induced neonatal myocyte growth, whereas only the PKCepsilon isoform was involved in myotrophin-induced adult myocyte hypertrophy . PKCdelta and PKCzeta do not seem to participate in either neonatal or adult myocyte growth induced by myotrophin . Treatment with antisense oligonucleotides specific for PKCalpha and PKCepsilon isoforms further supported this result . PKCalpha is the major PKC isoform in neonatal myocytes and needs Ca2+ and phospholipids for its activation, and PKCepsilon (the Ca2+-independent PKC isoform) is present in both neonatal and adult myocytes; the 15-mer antisense oligodeoxynucleotides of each were used for this study . Treatment of neonatal myocytes with the PKCalpha and PKCepsilon antisense oligodeoxynucleotides for 5 days significantly reduced Ca2+-dependent and Ca2+-independent PKC activity, respectively, as well as the {3H}leucine incorporation induced by myotrophin . Furthermore, myotrophin-induced PKC activity was primarily located in the particulate fraction and did not result in a concomitant decrease in the cytosolic fraction . Myotrophin does not change PKC isoform expression (both Ca2+ dependent and independent PKC isoforms used in this study) in rat neonatal cardiac fibroblasts . Our data suggest that myotrophin exerts its action on protein synthesis, possibly through a tyrosine kinase-coupled pathway and translocation of PKC from the cytosol to the cell membrane. J Appl Microbiol, 1998 Apr, 84(4), 585 - 92 Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water; Tsen HY et al.; Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E . coli (STEC) strains, including enterohaemorrhagic E . coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII . Both ETEC and STEC are pathogenic to humans, pigs and cattle . As contamination of environmental water by any of these pathogenic E . coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E . coli was developed . The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories . The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E . coli cells as well as non-E . coli bacteria . Its detection limit was 10(2)-10(3) cfu each of the target cells per assay . When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10(0) cells 100 ml-1 of the water sample could be detected . Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples. Radiats Biol Radioecol, 1998 Mar-Apr, 38(2), 232 - 7 {Features of modifications of cytotoxic consequences of microwave and thermal heating}; Morozov II et al.; The incubation of bacteria Escherichia coli B/r and Escherichia coli Bs-1, preliminarily heated by microwave (frequency 7 GHz) or equal usual heat in water isotonic (0.9%) or hypertonic (10%) solution of sodium chloride resulted in decrease or increase of the cell lethality independently from heating manner . Hypertonic solutions of this compound, as was established, protected the cells against heat damages during the microwave heating less effectively than during thermal heating. Arch Biochem Biophys, 1998 Jun 1, 354(1), 24 - 30 The fibronectin-like domain is required for the type V and XI collagenolytic activity of gelatinase B; O'Farrell TJ et al.; Gelatinase B (matrix metalloproteinase-9) is able to degrade several extracellular matrix proteins, including gelatin, elastin, and collagen types IV, V, XI, and XIV . This enzyme contains a "fibronectin-like" domain which is composed of three tandem copies of a fibronectin type 2 homology unit inserted into its catalytic domain . We have studied the involvement of this domain in the substrate specificity of gelatinase B by expressing a mutant of the enzyme, in Escherichia coli, in which this domain has been deleted . This mutant enzyme retained its ability to cleave the peptide substrate Mca-PLGL(Dpa)AR-NH2, possessing K(m) and kcat values similar to those of the wild-type enzyme . In addition, the NH2-terminal, 14-kDa, inhibitory domain of recombinant tissue inhibitor of metalloproteinase-2 was able to inhibit the mutant and the wild-type enzymes with the same potency . The mutant's gelatinolytic activity was also retained but reduced in comparison to that of the wild-type enzyme . However, contrary to the wild-type enzyme, the mutant was not able to digest or bind fibrillar collagen types V and XI . These data indicate that the fibronectin-like domain of gelatinase B is an important determinant of the enzyme's fibrillar collagen substrate specificity . It allows the enzyme to bind to and cleave collagen types V and XI, events which are thought to be involved in several normal physiological and pathological processes such as metastasis and arthritis. Exp Cell Res, 1998 May 25, 241(1), 23 - 35 Myristoyl-coA:protein N-myristoyltransferase from bovine cardiac muscle: molecular cloning, kinetic analysis, and in vitro proteolytic cleavage by m-calpain; Raju RV et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides . Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities . Northern blot analysis of bovine heart poly(A)+ RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA . Western blot analysis of cardiac muscle extracts with human NMT antibody indicated a prominent immunoreactive band with a molecular mass of 50 kDa . The expression of mRNA and protein levels in cardiac muscle is not correlated with NMT activities, suggesting the presence of regulators of the enzyme activity . We have isolated the cDNA encoding bovine cardiac muscle NMT (cNMT) by reverse transcription polymerase chain reaction . The single long open reading frame of 1248 bp of bovine cNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da . The cDNA clone expressed in Escherichia coli resulted in the production of functionally active 50-kDa NMT . Ultrastructural and immunolocalization of NMT utilizing the immunogold labeling technique demonstrated cytoplasmic distribution with occasional mitochondrial and myofilaments localization of the NMT antibody . Cardiac muscle NMT has a higher affinity for myristoyl-CoA than toward palmitoyl-CoA . Substrate specificity indicated that cNMT has a higher affinity toward pp60src and M2 gene segment of reovirus type 3-derived peptide substrates than toward cAMP-dependent protein kinase-derived peptide . Primary translational product of cNMT sequence contained several regions rich in proline, glutamic acid, serine, and threonine, which are known as "PEST" regions . PEST-FIND analysis of the amino acid sequences indicated eight PEST regions were present in the cNMT . These PEST regions are suggested to be recognized by specific proteases, particularly Ca(2+)-dependent neutral proteases, calpains, which are responsible for the degradation of PEST-containing proteins . We have demonstrated the abolishment of NMT activity and NMT protein degradation in vitro by m-calpain . The proteolysis of cNMT by m-calpain and the abolishment of NMT activity was prevented by the calpain inhibitor, calpastatin . These observations indicate that calpains may regulate NMT activity. Lett Appl Microbiol, 1998 Apr, 26(4), 275 - 8 The production of D-acetoin by a transgenic Escherichia coli; Ui S et al.; A 6 kbp SphI fragment encoding genes for the enzymes alpha-acetolactate synthase (ALS), alpha-acetolactate decarboxylase (ALDC) and meso-2,3-butanediol dehydrogenase (meso-BDH), involved in the formation of meso-2,3-butanediol (meso-BD) from pyruvic acid, was cloned into plasmid pUC118 . When derivatives of this plasmid were introduced into Escherichia coli JM109, the transformants were able to produce D-acetoin (D-AC) without contamination with L-acetoin (L-AC). Biochemistry (Mosc), 1998 May, 63(5), 579 - 83 Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant Escherichia coli cells expressing firefly luciferase; Romanova NA et al.; Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant E . coli cells expressing firefly luciferase was investigated . It was shown that bioluminescence intensity and time-course of the bioluminescent signal changed upon immobilization and depended on intracellular ATP concentration and permeability of the cell membrane. J Biol Chem, 1998 Jun 26, 273(26), 16615 - 20 TorD, a cytoplasmic chaperone that interacts with the unfolded trimethylamine N-oxide reductase enzyme (TorA) in Escherichia coli; Pommier J et al.; Reduction of trimethylamine N-oxide (TMAO) in Escherichia coli involves the terminal molybdoreductase TorA, located in the periplasm, and the membrane anchored c type cytochrome TorC . In this study, the role of the TorD protein, encoded by the third gene of torCAD operon, is investigated . Construction of a mutant, in which the torD gene is interrupted, showed that the absence of TorD protein leads to a two times decrease of the final amount of TorA enzyme . However, specific activity and biochemical properties of TorA enzyme were similar to those of the enzyme produced in the wild type . Excess of TorD protein restores the normal level of TorA enzyme, and also, leads to the appearance of a new cytoplasmic form of TorA on SDS-polyacrylamide gel electrophoresis using gentle conditions . This probably indicates a new folding state of the cytoplasmic TorA protein when TorD is overexpressed . BIAcore techniques demonstrated direct specific interaction between the TorA and TorD proteins . This interaction was enhanced when TorA was previously unfolded by heating . Finally, as TorA is a molybdoenzyme, we demonstrated that TorD can interact with TorA before the molybdenum cofactor has been inserted . As TorD homologue encoding genes are found in various TMAO reductase loci, we propose that TorD is a chaperone protein specific for the TorA enzyme . It belongs to a family of TorD-like chaperones present in several bacteria, and, probably, involved in TMAO reductase folding. J Biol Chem, 1998 Jun 26, 273(26), 16555 - 60 Thiamin biosynthesis in Escherichia coli . Identification of this thiocarboxylate as the immediate sulfur donor in the thiazole formation; Taylor SV et al.; ThiFSGH and ThiI are required for the biosynthesis of the thiazole moiety of thiamin in Escherichia coli . The overproduction, purification, and characterization of ThiFS and the identification of two of the early steps in the biosynthesis of the thiazole moiety of thiamin are described here . ThiS isolated from E . coli thiI+ is post-translationally modified by converting the carboxylic acid group of the carboxyl-terminal glycine into a thiocarboxylate . The thiI gene plays an essential role in the formation of the thiocarboxylate because ThiS isolated from a thiI- strain does not contain this modification . ThiF catalyzes the adenylation by ATP of the carboxyl-terminal glycine of ThiS . This reaction is likely to be involved in the activation of ThiS for sulfur transfer from cysteine or from a cysteine-derived sulfur donor. J Biol Chem, 1998 Jun 26, 273(26), 16544 - 50 Critical role for the 310 helix region of p57(Kip2) in cyclin-dependent kinase 2 inhibition and growth suppression; Hashimoto Y et al.; Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A . A., Jeffrey, P . D., Pattern, A . K., Massague, J., and Pavletich, N . P . (1996) Nature 382, 325-331) has suggested that the 310 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis . In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3(10) helix region of this protein is indispensable for the inhibition of these complexes . In contrast, the 3(10) helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined . Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3(10) helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3(10) helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP . Mutations within the 3(10) helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G1 in vivo . These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2+ activities . Thus the 3(10) helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities. J Biol Chem, 1998 Jun 26, 273(26), 16476 - 86 The reduced levels of chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo; Arnold DA et al.; Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme and is stimulated by an 8-nucleotide element known as Chi (chi) . We present a detailed biochemical characterization of a mutant RecBCD enzyme, designated RecBC1004D, that displays a reduced level of chi site recognition . Initially characterized genetically as unable to respond to the chi sequence, we provide evidence to indicate that the ability of this mutant enzyme to respond to chi is reduced rather than lost; the mutant displays about 20-fold lower chi recognition than wild-type RecBCD enzyme . Although this enzyme exhibits wild-type levels of double-stranded DNA exonuclease, helicase, and ATPase activity, its ability to degrade single-stranded DNA is enhanced 2-3-fold . The data presented here suggest that the reduced recombination proficiency of the recBC1004D strain observed in vivo results from a basal level of modification of the RecBC1004D enzyme at both chi-specific, as well as nonspecific, DNA sequences. J Biol Chem, 1998 Jun 26, 273(26), 16400 - 8 Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells; Halfon S et al.; We describe a new cystatin in both mice and humans, which we termed leukocystatin . This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions . As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs . In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns . The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAGTM fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins . The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system. J Biol Chem, 1998 Jun 26, 273(26), 16358 - 65 Synthesis of polyribonucleotide chains from the 3'-hydroxyl terminus of oligodeoxynucleotides by Escherichia coli primase; Sun W et al.; Escherichia coli primase synthesizes RNA primers on DNA templates for the initiation of DNA replication . The sole known activity of primase is to catalyze synthesis of short RNA chains de novo . We now report a novel activity of primase, namely that it can synthesize RNA from the 3'-hydroxyl terminus of a pre-existing oligodeoxynucleotide . The oligonucleotide-primed synthesis of RNA by primase occurs in both of the G4oric-specific priming system and the dnaB protein associated general priming system . This priming reaction of primase is verified by a number of biochemical methods, including inhibition by modified 3'-phosphate of oligonucleotides and deoxyribonuclease I and ribonuclease H cleavages . We also show that the primed RNA is an effective primer for the synthesis of DNA chain by E . coli DNA polymerase III holoenzyme . The significance of this finding to primases generating multimeric length RNA is discussed. J Biol Chem, 1998 Jun 26, 273(26), 16273 - 80 Role of electrostatic interactions on the affinity of thioredoxin for target proteins . Recognition of chloroplast fructose-1, 6-bisphosphatase by mutant Escherichia coli thioredoxins; Mora-Garcia S et al.; Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme . Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin . To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E . coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines . After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants . However, the conversion of cysteine residues back to cystine depended on the target protein . Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts . Moreover, the affinity of E30K, L94K, and E30K/L94K E . coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM) . We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f . While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues . These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange. J Biol Chem, 1998 Jun 26, 273(26), 16241 - 7 Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase; Valiyaveetil FI et al.; Subunit a is the least understood of the three subunits that compose the F0 sector in the Escherichia coli F0F1 ATP synthase . In this study, we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical information on the folding of subunit a . The extent of labeling of the substituted Cys residues by fluorescein-5'-maleimide was determined . The localization of reactive Cys residues was inferred from differences in the extent of labeling in inside out and right side out membrane vesicles . The NH2-terminal segment of subunit a was localized to the outside (periplasmic) surface and the COOH terminus to the cytoplasmic surface by these procedures . Loop residues in two periplasmic extramembranous loops and in two cytoplasmic extramembranous loops were also localized . The localization of two cytoplasmic Cys residues was confirmed by using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to block fluorescein-5'-maleimide labeling . From the localization of the Cys residues, a model for the topography is proposed that consists of five transmembrane segments with the NH2 terminus periplasmic and the COOH terminus cytoplasmic . The positions of second site suppressors, including several isolated here to the nonfunctional E219C and H245C substitutions, provide support for the topographical model proposed. J Biol Chem, 1998 Jun 26, 273(26), 16235 - 40 Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues; Long JC et al.; The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin . Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag) . None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium . Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell . Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid . After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography . The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase . Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131 . On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed. J Biol Chem, 1998 Jun 26, 273(26), 16229 - 34 Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel; Vik SB et al.; Subunit a of the E . coli F1F0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu219 and His245 . A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 238, 243, and 245 . The mutants were tested for growth yield, binding of F1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase activity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F1 . Significant loss of function was seen only with insertions after positions 238 and 243 . In contrast, both insertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type . The other insertions showed an intermediate loss of function . Missense mutations of His245 to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function . Replacement of Leu162 by histidine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate . An interaction between Trp241 and His245 may be involved in gating a "half-channel" from the periplasmic surface of F0 to Asp61 of subunit a. J Biol Chem, 1998 Jun 26, 273(26), 16098 - 103 TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads; Kinoshita T et al.; Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface . However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP . In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2 . The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner . In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations . TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA . A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available . Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F. J Biol Chem, 1998 Jun 26, 273(26), 16067 - 74 Novel recombinant analogues of bovine placental lactogen . G133K and G133R provide a tool to understand the difference between the action of prolactin and growth hormone receptors; Helman D et al.; Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form . Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity . The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration . Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested . Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR- and GHR-ECDs . The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished . Both analogues exhibited antagonistic activity in the latter cells . To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization. J Biol Chem, 1998 Jun 26, 273(26), 16000 - 4 Ambiguities in mapping the active site of a conformationally dynamic enzyme by directed mutation . Role of dynamics in structure-function correlations in Escherichia coli adenylosuccinate synthetase; Wang W et al.; On the basis of ligated crystal structures, Asn21, Asn38, Thr42, and Arg419 are not involved in the chemical mechanism of adenylosuccinate synthetase from Escherichia coli, yet these residues are well conserved across species . Purified mutants (Asp21 --> Ala, Asn38 --> Ala, Asn38 --> Asp, Asn38 --> Glu, Thr42 --> Ala, and Arg419 --> Leu) were studied by kinetics, circular dichroism spectroscopy, and equilibrium ultracentrifugation . Asp21 and Arg419 are not part of the active site, yet mutations at positions 21 and 419 lower kcat 20- and 10-fold, respectively . Thr42 interacts only through its backbone amide with the guanine nucleotide, yet its mutation to alanine significantly increases Km for all substrates . Asn38 hydrogen-bonds directly to the 5'-phosphoryl group of IMP, yet its mutation to alanine and glutamate has no effect on Km values, but reduces kcat by 100-fold . The mutation Asn38 --> Asp causes 10-57-fold increases in Km for all substrates along with a 30-fold decrease in kcat . At pH 5.6, however, the Asn38 --> Asp mutant is more active, yet binds IMP 100-fold more weakly, than the wild-type enzyme . Proposed mechanisms of ligand-induced conformational change and subunit aggregation can account for the properties of mutant enzymes reported here . The results underscore the difficulty of using directed mutations alone as a means of mapping the active site of an enzyme. J Biol Chem, 1998 Jun 26, 273(26), 15980 - 4 Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle; Rivers A et al.; Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete . In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska, A., Gietzen, K . F., Rivers, A., and Virshup, D . M . (1998) J . Biol . Chem . 273, 1357-1364) . However, we now report that while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases . Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation . Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo. J Biol Chem, 1998 Jun 26, 273(26), 15940 - 5 Unisite catalysis without rotation of the gamma-epsilon domain in Escherichia coli F1-ATPase; Garcia JJ et al.; Unisite {gamma-32P}ATP hydrolysis was studied in ECF1 from the mutant betaE381C after generating a single disulfide bond between beta and gamma subunits to prevent the rotation of the gamma/epsilon domain . The single beta-gamma cross-link was obtained by removal of the delta subunit from F1 and then treating with CuCl2 as described previously (Aggeler, R., Haughton, M . A., and Capaldi, R . A . (1996) J . Biol . Chem . 270, 9185-9191) . The mutant enzyme, betaE381C, had an increased overall rate of unisite hydrolysis of {gamma-32P}ATP compared with the wild type ECF1 due to increases in the rate of ATP binding (k+1), Pi release (k+3), and ADP release (k+4) . Release of bound substrate ({gamma-32P}ATP) was also increased in the betaE381C mutant . Cross-linking between Cys-381 and the intrinsic Cys-87 of gamma caused a further increase in the rate of unisite catalysis, mainly by additional effects on nucleotide binding in the high affinity catalytic site (k+1 and k+4) . In delta-subunit-free ECF1 from wild type or betaE381C F1, addition of an excess of ATP accelerated unisite catalysis . After cross-linking, unisite catalysis of betaE381C was not enhanced by the cold chase . The covalent linkage of gamma to beta increased the rate of unisite catalysis to that obtained by cold chase of ATP of the noncross-linked enzyme . It is concluded that the conversion of Glu-381 of beta to Cys induces an activated conformation of the high affinity catalytic site with low affinity for substrate and products . This state is stabilized by cross-linking the Cys at beta381 to Cys-87 of gamma . We infer from the data that rotation of the gamma/epsilon rotor in ECF1 is not linked to unisite hydrolysis of ATP at the high affinity catalytic site but to ATP binding to a second or third catalytic site on the enzyme. J Magn Reson, 1998 Jun, 132(2), 191 - 6 13C-NOESY-HSQC with Split Carbon Evolution for Increased Resolution with Uniformly Labeled Proteins; Baur M et al.; Two new pulse sequences are presented for the recording of 2D 13C-HSQC and 3D 13C-NOESY-HSQC experiments, containing two consecutive carbon evolution periods . The two periods are separated by a z-filter which creates a clean CxHz-quantum state for evolution in the second period . Each period is incremented (in a non-constant-time fashion) only to the extent that the defocusing of carbon inphase magnetization through J-coupling with neighboring carbons remains insignificant . Therefore, 13C homonuclear J-couplings are rendered ineffective, reducing the loss of signal and peak splitting commonly associated with long 13C evolution times . The two periods are incremented according to a special acquisition protocol employing a 13C-13C gradient echo to yield a data set analogous to one obtained by evolution over the added duration of both periods . The spectra recorded with the new technique on uniformly 13C-labeled proteins at twice the evolution time of the standard 13C-HSQC experiment display a nearly twofold enhancement of resolution in the carbon domain, while maintaining a good sensitivity even in the case of large proteins . Applied to the IIAMan protein of E . coli (31 kDa), the 13C-HSQC experiment recorded with a carbon evolution time of 2 x 8 ms showed a 36% decrease in linewidths compared to the standard 13C-HSQC experiment, and the S/N ratio of representative cross-peaks was reduced to 40% . This reduction reflects mostly the typical loss of intensity observed when recording with an increased resolution . The 13C-NOESY-HSQC experiment derived from the 13C-HSQC experiment yielded additional NOE restraints between resonances which previously had been unresolved . Microb Pathog, 1998 Jun, 24(6), 361 - 72 Cloning and molecular analysis of genes encoding two immunodominant antigens of Ehrlichia risticii; Vemulapalli R et al.; Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism . To understand the role of 55 and 51 kilodalton immunodominant antigens of E . risticii in strain variation, their genes from the 25-D and 90-12 strains were cloned, sequenced, and expressed in E . coli . Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a ;10 kDa protein . Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues of E . coli GroEL and GroES proteins, respectively . There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains of E . risticii . The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively . The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences . The difference in some of the amino acids may be responsible for variation in their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a ;2 kDa lower molecular weight region . In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified . The 55 and 51 kDa antigens were overexpressed in E . coli using a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S . A.) . The purified recombinant proteins cross-reacted with antisera to E . canis and E . sennetsu . Biochemistry, 1998 May 26, 37(21), 7885 - 91 Delineation of the Cdc42/Rac-binding domain of p21-activated kinase; Thompson G et al.; p21-activated kinases (PAKs) serve as effector proteins for the GTP-binding proteins Cdc42 and Rac . They are serine/threonine kinases containing the Cdc42/Rac interactive binding (CRIB) motif . The main aim of this study was to define the minimal domain of alphaPAK required for Cdc42/Rac binding . Eight stable PAK fragments of varying lengths, each containing the CRIB motif (residues 75-88), were expressed in Escherichia coli, and their ability to interact with Cdc42 and Rac was assessed using scintillation proximity assays, isothermal titration calorimetry, and fluorescence techniques . The shortest fragments examined (residues 70-94 and 75-94) bound only weakly to either Cdc42 or Rac . A longer fragment starting at residue 75 and ending at residue 105 showed binding to Q61L Rac.GTP with Kd = 1.9 microM . Highest affinity binding (Kd approximately 0.05 microM) was seen with longer fragments ending at residue 118 or 132 . A small increase in affinity was seen with those fragments starting at residue 70 rather than residue 75 . PAK fragments bound with approximately 3-10-fold higher affinity to Cdc42 than to Rac and bound Q61L variants with 5-10-fold higher affinity than wild type . The dissociation rates of Q61L Rac.mant-GTP and of Q61L Cdc42 . mant-GTP from PAK fragment residues 70-132 were measured to be 0.66 and 0.25 min-1, respectively, which are 100-fold lower than dissociation rates for Ras:Ras-effector domains, although their affinities are similar . Calorimetric measurements revealed that binding was associated with a relatively slow heat change . It is suggested that these PAK fragments (in the absence of Cdc42 or Rac) might exist predominantly in an inactive conformation that slowly interconverts with an active conformation and/or a slow conformational change may occur upon binding to Cdc42/Rac . In conclusion, the PAK CRIB motif itself is insufficient for high-affinity binding to Cdc42/Rac, but a 30 amino acid region of PAK (residues 75-105), containing this motif, is sufficient. Biochemistry, 1998 May 26, 37(21), 7850 - 8 Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate; Shao X et al.; A monomeric version of the dimeric tryptophan repressor from Escherichia coli, L39E TR, has previously been shown to resemble a transient intermediate that appears in the first few milliseconds of folding {Shao, X., Hensley, P., and Matthews, C . R . (1997) Biochemistry 36, 9941-9949} . In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the monomeric form with those of the native, dimeric form . The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Trp99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circular dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C . Coincident normalized transitions show that the urea denaturation process for each single-tryptophan mutant follows a two-state model involving monomeric native and unfolded forms . The free energies at standard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer . Fluorescence and near-UV circular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well-structured environments that are distinctively different from those found in their dimeric counterparts . Acrylamide quenching experiments show that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure . Measurements of the steady-state anisotropies of Trp19/L39E and Trp99/L39E TR demonstrate that the motions of both tryptophan side chains are restricted in the folded conformation . On the basis of these data, it can be concluded that this monomeric form of the tryptophan repressor adopts a well-folded, stable conformation with nonnative tertiary structure . When combined with previous results, the current findings demonstrate that the development of higher order structure during the folding of this intertwined dimer does not follow a simple hierarchical model. Biochemistry, 1998 May 26, 37(21), 7844 - 9 Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate deaminase; Montero-Moran GM et al.; The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was first proposed on the basis of a theoretical analysis of the sequence and demonstrated by spectrophotometric experiments . Two tyrosine residues, Tyr121 and Tyr254, were indicated as involved in the mechanism of cooperativity and in the allosteric regulation of the enzyme {Altamirano et al . (1994) Eur . J . Biochem . 220, 409-413} . Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T --> R transition {Altamirano et al . (1995) Biochemistry 34, 6074-6082} . From crystallographic data of the R allosteric conformer, Tyr254 has been shown to be part of the allosteric pocket {Oliva et al . (1995) Structure 3, 1323-1332} . Although it is not directly involved in binding the allosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydrogen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain . Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotropic effects . Homotropic cooperativity diminished and the allosteric activation pattern changed from one of the K-type in the wild-type deaminase to a mixed K-V pattern . On the other hand, Tyr254-Trp deaminase is kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein . These results show that the interactions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E . coli glucosamine 6-P deaminase. Biochemistry, 1998 May 26, 37(21), 7778 - 86 Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association of ClpA and clpP; Maurizi MR et al.; The ClpAP protease from Escherichia coli consists of the ATP-binding regulatory component, ClpA (subunit Mr 84 165), and the proteolytic component, ClpP (subunit Mr 21 563) . Our hydrodynamic studies demonstrate that the predominant forms of these proteins in solution correspond to those observed by electron microscopy . ClpP and proClpP(SA), which in electron micrographs appear to have subunits arranged in rings of seven subunits, were found by ultracentrifugation to have s20,w values of 12.2 and 13.2 S and molecular weights of 300 000 and 324 000 +/- 3000, respectively, indicating that the native form of each consists of two such rings . The two intact rings of ClpP were separated in the presence of >/= 0.1 M sulfate at low temperatures, suggesting that ring-ring contacts are polar in nature and more easily disrupted than subunit contacts within individual rings . Sedimentation equilibrium analysis indicated that ClpA purified without nucleotide exists as an equilibrium mixture of monomers and dimers with Ka = (1.0 +/- 0.2) x 10(5) M-1 and