J Biol Chem, 1996 Apr 12, 271(15), 8855 - 62 Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease . A study using oligodeoxynucleotide phosphorothioates; Thorogood H et al.; A set of phosphorothioate-containing oligonucleotides based on pGACGATATCGTC, a self-complementary dodecamer that contains the EcoRV recognition sequence (GATATC), has been prepared . The phosphorothioate group has been individually introduced at the central nine phosphate positions and the two diastereomers produced at each site separated and purified . The Km and Vmax values found for each of these modified DNA molecules with the EcoRV restriction endonuclease have been determined and compared with those seen for the unmodified all-phosphate-containing dodecamer . This has enabled an evaluation of the roles that both of the non-esterified oxygen atoms in the individual phosphates play in DNA binding and hydrolysis by the endonuclease . The results have also been compared with crystal structures of the EcoRV endonuclease, complexed with an oligodeoxynucleotide, to allow further definition of phosphate group function during substrate binding and turnover . For further study, see the related article "Probing the Indirect Readout of the Restriction Enzyme EcoRV: Mutational Analysis of Contacts to the DNA Backbone" (Wenz, A., Jeltsch, A., and Pingoud, A . (1996) J . Biol . Chem . 271, 5565-5573). J Biol Chem, 1996 Apr 12, 271(15), 8692 - 9 Bradyrhizobium japonicum porphobilinogen synthase uses two Mg(II) and monovalent cations; Petrovich RM et al.; Bradyrhizobium japonicum porphobilinogen synthase (B . japonicum PBGS) has been purified and characterized from an overexpression system in an Escherichia coli host (Chauhan, S., and O'Brian, M . R . (1995) J . Biol . Chem . 270, 19823-19827) . B . japonicum PBGS defines a new class of PBGS protein, type IV (classified by metal ion content), which utilizes a catalytic MgA present at a stoichiometry of 4/octamer, an allosteric MgC present at a stoichiometry of 8/octamer, and a monovalent metal ion, K+ . However, the divalent MgB or ZnB present in some other PBGS is not present in B . japonicum PBGS . Under optimal conditions, the Kd for MgA is <0.2 microM, and the Kd for MgC is about 40 microM . The response of B . japonicum PBGS activity to monovalent and divalent cations is mutually dependent and varies dramatically with pH . B . japonicum PBGS is also found to undergo a dynamic equilibrium between active multimeric species and inactive monomers under assay conditions, a kinetic characteristic not reported for other PBGSs . B . japonicum PBGS is the first PBGS that has been rigorously demonstrated to lack a catalytic ZnA . However, consistent with prior predictions, B . japonicum PBGS can bind Zn(II) (presumably as ZnA) at a stoichiometry of 4/octamer with a Kd of 200 microM; but this high concentration is outside a physiologically significant range. J Biol Chem, 1996 Apr 12, 271(15), 8605 - 11 Structure-function relations of smooth muscle calponin . The critical role of serine 175; Tang DC et al.; Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin . Both properties are lost following phosphorylation (primarily at serine 175) by protein kinase C or calmodulin-dependent protein kinase II . To evaluate further the functional importance of serine 175, wild-type calponin and three site-specific mutants (S175A, S175D, and S175T) were expressed in Escherichia coli and compared with calponin purified from chicken gizzard smooth muscle in terms of actin binding, actomyosin MgATPase inhibition, and phosphorylation by protein kinase C and calmodulin-dependent protein kinase II . The affinities of skeletal muscle F-actin for wild-type and S175T calponins were similar to that for the tissue-purified protein (Kd = 0.8, 1.3, and 1.0 microM, respectively), whereas the affinities for S175A and S175D calponins were much lower (Kd = 26.8 and 44.2 microM, respectively) . Tissue-purified, wild-type, and S175T calponins displayed comparable inhibition of the smooth muscle actin-activated myosin MgATPase, whereas S175A and S175D calponins were much less effective . Phosphorylation confirmed serine 175 as the principal site of phosphorylation by both kinases . These results indicate that the hydroxyl side chain at position 175 of calponin plays a critical role in the binding of calponin to actin and inhibition of the cross-bridge cycling rate. Nature, 1996 Apr 11, 380(6574), 548 - 50 Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2; Colas P et al.; A network of interacting proteins controls the activity of cyclin-dependent kinase 2 (Cdk2) (refs 1,2) and governs the entry of higher eukaryotic cells into S phase . Analysis of this and other genetic regulatory networks would be facilitated by intracellular reagents that recognize specific targets and inhibit specific network connections . We report here the expression of a combinatorial library of constrained 20-residue peptides displayed by the active-site loop of Escherichia coli thioredoxin, and the use of a two-hybrid system to select those that bind human Cdk2 . These peptide aptamers were designed to mimic the recognition function of the complementarity-determining regions of immunoglobulins . The aptamers recognized different epitopes on the Cdk2 surface with equilibrium dissociation constant in the nanomolar range; those tested inhibited Cdk2 activity . Our results show that peptide aptamers bear some analogies with monoclonal antibodies, with the advantages that they are isolated together with their coding genes, that their small size should allow their structures to be solved, and that they are designated to function inside cells. Mol Gen Genet, 1996 Apr 10, 250(6), 705 - 14 Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli; Olsson CL et al.; To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 in Escherichia coli, the infC, rpmI and rpIT genes encoding IF3, L35 and L20, respectively, were placed under the control of lac promotor/operator sequences . Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium . Lysogenic strains were constructed with recombinant lambda phages that express either rpmI and rplT or infC and prmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations . At low cellular concentration of IF3, but not L20, decreases and the growth rate slows . Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo . During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis . As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation of infC . The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein . In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41-43S is seen . Previous studies have shown that the L20 protein negatively controls its own gene expression . Reduction of the cellular concentration of L20 derepresses the expression of an rplT-lacZ gene fusion, thus confirming autogenous regulation by L20. Mol Gen Genet, 1996 Apr 10, 250(6), 674 - 80 The isfA mutation inhibits mutator activity and processing of UmuD protein in Escherichia coli recA730 strains; Bebenek A et al.; Further studies on the isfA mutation responsible for anti-SOS and antimutagenic activities in Escherichia coli are described . We have previously shown that the isfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity . The isfA mutation has now been demonstrated also to suppress mutator activity in E . coli recA730 and recA730 lexA51(Def) strains that constitutively express RecA coprotease activity . We further show that the antimutator activity of the isfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD . Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in the recA730 isfA strain and partially restores its mutator activity . On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability. Biochemistry, 1996 Apr 9, 35(14), 4609 - 18 Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding; Thrall SH et al.; A host cell-derived tRNA3Lys molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome . We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA3Lys, and to address RT's putative role in the required and specific packaging of tRNA3Lys into the budding virus . Titration of RT with tRNA3Lys resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (Kd) of 57.6 +/- 7.5 nM was derived . Titration of RT with Escherichia coli tRNA2Glu, E . coli tRNA2Tyr, E . coli tRNALys, yeast tRNAPhe, or in vitro-synthesized human tRNA3Lys (no base modifications) resulted in similar fluorescence changes and Kd values as obtained for the natural tRNA3Lys . The specific interaction between RT and tRNA3Lys during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT . Other factors during viral assembly must therefore cooperate in the packaging of tRNA3Lys . The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding . The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNAPhe was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA . The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites . Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands . The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both tRNA and p/t binding . Reverse transcriptase was found to bind to the mutant tRNA 10-fold more tightly than to the unaltered tRNA (Kd = 4.5 +/- 1.0 and 44.6 +/- 6.6 nM, respectively) . Further analyses revealed that the tighter affinity is probably due to a preferred p/t binding mode and not to one expected if separate tRNA and p/t binding regions are accessed simultaneously by the same molecule. Biochemistry, 1996 Apr 9, 35(14), 4591 - 601 Active site of bee venom phospholipase A2: the role of histidine-34, aspartate-64 and tyrosine-87; Annand RR et al.; In bee venom phospholipase A2, histidine-34 probably functions as a Bronsted base to deprotonate the attacking water . Aspartate-64 and tyrosine-87 form a hydrogen bonding network with histidine-34 . We have prepared mutants at these positions and studied their kinetic properties . The mutant in which histidine-34 is changed to glutamine is catalytically inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50-100-fold) or in which tyrosine-87 is changed to phenylalanine (no change in turnover number) retain good activity . The interfacial Michaelis constants are changed by less than 10-fold for all mutants . Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 should yield enzymes that retain a native-like structure and support catalysis . The pKa of the histidine-34 imidazole was deduced from the pH-rate profile and from the pH dependence of the rate of histidine-34 alkylation by 2-bromo-4'-nitroacetophenone . The pKa is increased about one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the aspartate-64 to alanine mutant the pKa is unchanged . These pKas are generally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not unusually strong . The hydrogen bonding network linking tyrosine-87 to aspartate-64 and aspartate-64 to histidine-34 is not critical for catalysis. Biochemistry, 1996 Apr 9, 35(14), 4468 - 79 Identification of active site residues of chorismate mutase-prephenate dehydrogenase from Escherichia coli; Christendat D et al.; Chemical modification studies of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase and mass spectral analysis of peptide fragments containing modified residues are presented . The reaction with diethyl pyrocarbonate (DEPC) results in the modification of several enzymic groups, including a single histidine group essential for dehydrogenase activity and a single lysine residue essential for mutase activity . This conclusion is based on the following evidence . (1) Hydroxylamine rapidly restores dehydrogenase activity to the DEPC-inactivated enzyme without restoring mutase activity . (2) Mutase activity is also lost upon treatment of the enzyme with trinitrobenzene sulfonate . (3) The reactivity of the dehydrogenase to DEPC increases with pH, suggesting the participation of a group with a pKa of 7.0 in the dehydrogenase reaction . (4) Two peptides identified by differential peptide mapping had mass values matching those calculated for peptides comprising residues 127-135 (containing His131) and residues 36-48 (containing Lys37) . In support of the idea that the residues being modified are within the active sites, we show that the substrates prephenate and nicotinamide adenine dinucleotide (NAD+) offer protection against inactivation of dehydrogenase activity while inactivation of mutase activity can be prevented by prephenate and a transition state analogue (3-endo-8-exo)-8-hydroxy-2-oxabicyclo{3.3.1}-non-6-ene-3,5-dicarboxylic acid (endo-oxabicyclic diacid) . Lys37 is conserved among several chorismate mutases and may participate in catalysis by interacting with an ether oxygen between C-5 and C-8 of chorismate in the transition state . His131 may be assisting in a hydride transfer from prephenate to NAD+ in the dehydrogenase reaction. Biochemistry, 1996 Apr 9, 35(14), 4427 - 33 The role of the insertion loop around tryptophan 148 in tthe activity of thrombin; DiBella EE et al.; Thrombin has trypsin-like specificity for Arg-Xaa and Lys-Xaa peptide bonds; however, it is much more specific than trypsin, cleaving far fewer peptide bonds in macromolecular substrates . To probe the nature of the specificity of thrombin, a mutant has been constructed in which the Trp148 loop of thrombin has been replaced with the same loop of bovine trypsin . This mutant was expressed in Escherichia coli as prethrombin-2(148) using a T7 expression system previously described for wild-type prethrombin-2 {DiBella et al . (1995) J . Biol . Chem . 270, 163-169} . After refolding and purification, prethrombin-2(148) was activated to thrombin(148) with Echis carinatus snake venom . The k(cat)/K(m) for the release of fibrinopeptide A from fibrinogen was 4.5 +/- 0.5 microM(-1)s(-1) for thrombin(148), which was approximately 20% of that of recombinant thrombin (25 +/- 2.0 microM(-1)s(-1)) . Thrombin(148) was inhibited less well by hirudin with a K(i) of 500 pM compared to a value of 12 pM determined for recombinant thrombin . The mutant thrombin was also compared to trypsin and wild-type recombinant thrombin for the ability to cleave small peptide substrates . The Michaelis constants (K(m)) were found to be between 5- and 10-fold higher for thrombin(148) relative to wild-type recombinant thrombin, although the catalytic constants (k(cat)) for thrombin(148) and recombinant thrombin remained relatively unchanged for all three substrates . Thrombin(148) had a specificity constant (k(cat)/K(m)) 2-fold higher for the hydrolysis of H-D-phenyalanyl-L- pipecolyl-L-arginine-p-nitroaniline (a thrombin substrate) than that of trypsin . For N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine- p-nitroaniline (a trypsin substrate) and N-carbobenzoxyglycylprolyl-L-arginine-p-nitroaniline (a substrate for both enzymes), the specificity constants for trypsin were 1000- and 16-fold higher, respectively . Although replacement of the Trp(148) loop does not yield an enzyme with more trypsin-like specificity, the Trp(148) loop is important in the substrate binding and specificity of thrombin (on the basis of K(m) and K(i)). Biochemistry, 1996 Apr 9, 35(14), 4359 - 64 His257 is a uniquely important histidine residue for tetracycline/H+ antiport function but not mandatory for full activity of the transposon Tn10-encoded metal-tetracycline/H+ antiporter; Yamaguchi A et al.; His257 is the only histidine residue located in the putative transmembrane region of the Tn10-encoded metal-tetracycline/H+ antiporter (TetA) and contributes to the substrate/H+ coupling {Yamaguchi, A. . Adachi, K., Akasaka . T., Ono . N., & Sawai, T . (1991) J . Biol . Chem . 266, 6045-6051} . Tn10-TetA contains five histidine residues, including His257 . When these histidine residues were replaced by alanine one by one, only the His257Ala mutant showed almost complete loss of the tetracycline transport activity, whereas the other four His --> Ala mutants, H42A, H158A, H329A, and H359A, retained transport activity comparable to that of the wild type . The mutant which contains only one histidine, His257, retained about 80% of the wild-type activity, whereas the histidine-less mutant, in which all five histidine residues were replaced by Ala, exhibited little activity . These results clearly indicated that His257 is a unique histidine residue in TetA responsible for the transport activity . The His257Tyr mutant, irrespective of the presence or absence of the other four histidine residues, retained about 30% of the wild-type tetracycline transport activity and showed corresponding tetracycline-coupled H+ translocation, indicating that an imidazole group is not necessary at position 257 for the substrate/H+ coupling . A histidine-specific reagent, diethyl pyrocarbonate (DEPC), equally inactivated the wild-type and one-histidine mutant TetA, whereas the H257Y mutant was hardly inactivated by DEPC irrespective of the presence or absence of the other four histidine residues, indicating that the inactivation by DEPC is due to the modification of His257. Biochemistry, 1996 Apr 9, 35(14), 4326 - 33 Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras; Pan CQ et al.; The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites . Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid . The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome . All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses . A newly discovered site resides in the lac operon of Escherichia coli . The binding of Fis to this site may antagonize repression by the LacI repressor . These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference. Biochemistry, 1996 Apr 9, 35(14), 4279 - 86 Structure of the LexA repressor-DNA complex probed by affinity cleavage and affinity photo-cross-linking; Dumoulin P et al.; The structure of the complex of full-length Escherichia coli LexA repressor with a consensus operator DNA fragment has been probed by affinity photo-cross-linking and affinity cleavage . These methods allow the determination of approximate intermolecular distances between a given protein residue and a base or sugar moiety within the operator . In a first step unique cysteine residues were introduced in positions 7, 28, 38, or 52 of the protein . In all four cases, the original amino acid was an arginine . The four amino acids in these positions were expected to be situated on the surface of LexA interacting with DNA, as inferred from the structure of the LexA DNA binding domain {Fogh et al . (1994) EMBO J . 13, 3936-3944} . In a second step, these unique cysteine side chains of the purified proteins were chemically modified either with 4-azidophenacyl bromide or with S-(2-pyridylthio)cysteaminyl-EDTA . The first set of derivatives gives rise to UV-induced cross-linking which may be revealed by alkali/heat treatment; the second leads to direct DNA cleavage in the proximity of the derivatized amino acid . To reduce hydroxyl radical diffusion, the EDTA-iron cleavage reactions were done in the presence of high amounts of glycerol . The results indicate that amino acids 7 and 52 are near nucleotide pairs 8-12 of the operator and that amino acids 28 and 36 of LexA are near nucleotide pairs 5-8 of the operator . The results unambiguously define the orientation of the LexA DNA binding domain relative to the operator and provide support for the model of the LexA-operator complex proposed by Knegtel et al . {(1995) Proteins 21, 226-236} . Ethylation interference experiments further suggest that Arg-7 contacts the phosphate group between nucleotides 8 and 9 as predicted by the model. J Comp Neurol, 1996 Apr 8, 367(3), 329 - 41 Interphotoreceptor retinoid-binding protein (IRBP): expression in the adult and developing Xenopus retina; Hessler RB et al.; Apposition of the neural retina and pigment epithelium is critical to photoreceptor development and function . Interphotoreceptor retinoid-binding protein (IRBP) is a major component of the extracellular matrix separating these epithelia in the African clawed frog Xenopus laevis (Gonzalez-Fernandez et al., {1993}, J . Cell Sci . 105:7-21) . In the adult retina, IRBP appears to mediate the transport of hydrophobic molecules, particularly retinoids and fatty acids, within the hydrophilic extracellular domain . In this paper, we compare the distribution of IRBP and its mRNA in adult and embryonic Xenopus retina . Xenopus IRBP antisense RNA, labeled with tritium or digoxigenin, was used for in situ hybridizaton studies . For immunohistochemistry, we used an antiserum against Xenopus IRBP expressed in Escherichia coli . In the adult, we found that IRBP is synthesized at similar levels by both rods and cones . The protein is restricted to the interphotoreceptor matrix, with lesser amounts in the pigment epithelial cytoplasm . In the embryo, expression of the mRNA for IRBP is restricted to the central retina, where photoreceptor differentiation has taken place . By contrast, the protein is distributed throughout the embryonic subretinal space . Therefore, the presence of IRBP precedes photoreceptor differentiation . In summary, IRBP is synthesized by both rods and cones and may be internalized by the pigment epithelium . In the embryo, IRBP is synthesized by the central retina and diffuses through the matrix, reaching the undifferentiated peripheral retina . In view of its ligand-binding properties, diffusion of IRBP may provide the peripheral neural retina with a vehicle to transport retinoids and docosahexaenoic acid (molecules critical to normal retinal development) from the pigment epithelium. J Chromatogr A, 1996 Apr 5, 729(1-2), 113 - 24 Analysis of recombinant human interleukin-11 fusion protein derived from Escherichia coli lysate by combined size-exclusion and reversed-phase liquid chromatography; Amari JV et al.; A two-dimensional size-exclusion-reversed-phase high-performance liquid chromatographic assay has been developed for the quantitation of recombinant human interleukin-11 fusion protein (rhIL-11 FP) expressed in E . coli cells . The sample preparation procedure included the optimization of lysis buffer components to achieve maximum rhIL-11 FP recovery through the disruption of associations between rhIL-11 FP and E . coli components . The E . coli cells were dialyzed into lysis buffer and lysed by a French Press prior to two-dimensional chromatographic analysis . A size-exclusion column was used first to remove high- and low-molecular-mass E . coli components . Then reversed-phase chromatography was used to separate and quantify the rhIL-11 FP . The assay was linear over the range of 0.0294 to 0.235 mg/ml . The limit of quantitation, 0.0294 mg/ml, was based on % normalized residuals and precision criteria not exceeding 10% . The reproducibility of the assay for lysate samples was good on a daily (% R.S.D . = 1.0; n = 5) and a day-to-day reproducibility was good (% R.S.D . = 2.2; n = 9) . Selectivity and chromatographic peak identification were based upon gel electrophoresis and N-terminal sequencing of the rhIL-11 FP peak collected from the reversed-phase column. J Mol Biol, 1996 Apr 5, 257(3), 700 - 15 Kinetic and structural consequences of replacing the aspartate bridge by asparagine in the catalytic metal triad of Escherichia coli alkaline phosphatase; Tibbitts TT et al.; In each subunit of the homodimeric enzyme Escherichia coli alkaline phosphatase, two of the three metal cofactors Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51 . Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively . Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn2+ or Mg2+ . Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme . At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme . Upon the addition of excess Mg2+ at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme . The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg2+ increased . Under optimal conditions, the k(cat)/K(m) ratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme . To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 angstroms resolution at pH 7.5, and then to 2.1 angstroms resolution at pH 9.2 with 20 mM MgCl2 . The two structures were then refined . The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51 . At this pH the phosphate anion was bound via one oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5 . In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone . Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site. J Mol Biol, 1996 Apr 5, 257(3), 574 - 85 Uneven distribution of GATC motifs in the Escherichia coli chromosome, its plasmids and its phages; Henaut A et al.; This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coli genome, compared to its distribution in phages and plasmids . At first sight the distribution of GATC words looks random . But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromasome), is used as a theoretical reference, strong biasesare observed . GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp . The last class is the only one present in E . coli parasites . It can be ascribed to the triggering sequences of the long-patch mismatch repair system . The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection . The other classes, which could be targets for a nucleic acid-binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs . Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions . In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions . In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift. J Mol Biol, 1996 Apr 5, 257(3), 550 - 60 Replication of plasmid R6K gamma origin in vivo and in vitro: dependence on IHF binding to the ihf1 site; Dellis S et al.; The gamma origin of plasmid R6K requires the specific initiator protein pi for initiation of replication . However, increased pi concentrations inhibit replication . The host-encoded integration host factor (IHF) protein permits gamma origin replication at otherwise inhibitory pi levels . IHF is thought to mediate this positive effect by directly binding to the gamma origin . In this study we demonstrate that IHF binding to one IHF site in the gama origin, ihf1, but not to the other side, ihf2, is necessary for the gamma origin to replicate at high pi protein levels . We also show that in vitro replication of the gamma origin plasmid requires IHF binding to the ihf1 site . Finally, we demonstrate both in vivo and in vitro that, when mutant pi proteins (hyperactive) are provided instead of wild-type pi, gamma origin plasmids can replicate in the absence of IHF . This supports a previously proposed hypothesis that the pi mutants can bypass the IHF requirement for gamma origin replication. J Mol Biol, 1996 Apr 5, 257(3), 473 - 8 The side-chain of the amino acid residue in position 110 of the Lac repressor influences its allosteric equilibrium; Muller-Hartmann H et al.; Binding of the Lac repressor to its operator DNA controls the expression of the genes of the lac operon of Escherichia coli . Lac repressor's affinity for the lac operator is diminished by an inducer that affects the structure of the repressor tetramer . Here we report the cloning and sequencing of the mutant Lac repressor i-t gene, whose product, the LacR-t repressor, shows a higher affinity for the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and a lower affinity for the lac operator than the wild-type repressor . We show that the altered phenotype is due to a single amino acid residue replacement; the alanine residue at position 110 in the wild-type is replaced by threonine in i-t . Other amino acid residues in position 110 have been shown to result in an i-s phenotype . For the i-s-substitution of alanine 110 with lysine we demonstrate an increase in the affinity for operator DNA and a decrease in the affinity for IPTG . Thus, A110--> K shows the opposite effect to A110-->T on the repressor protein . We explain the phenotype of the LacR mutants by displacements of the conformational equilibrium for the dimeric repressor unit between RR (high operator affinity, low inducer affinity) and R*R* (low operator affinity, high inducer affinity) towards R*R* in the i-t and towards RR in the i-s mutant in position 110 with respect to the wild-type . The putative structures of the wild-type and mutant Lac repressors confirm this conclusion. J Biol Chem, 1996 Apr 5, 271(14), 8502 - 8 The peripheral complex of the tobacco hornworm V-ATPase contains a novel 13-kDa subunit G; Lepier A et al.; A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit . {14C}N,N'-dicyclohexylcarbodiimide labeling and its position on SDS-electrophoresis gels revealed that this protein was different from the 17-kDa proteolipid . A cDNA clone encoding a highly hydrophilic protein with a calculated molecular mass of 13,692 Da was obtained by immunoscreening . Monospecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa protein of the purified V-ATPase, confirming that a cDNA encoding this protein had been cloned . In vitro translation of the cRNA showed that the cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electrophoresis gels . The cloned protein showed 37% amino acid sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases . In contrast to the Vma10p protein, which behaved like a V0 subunit, the M . sexta 13-kDa protein behaved like a V1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold inactivation . When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M . sexta V-ATPase could be purified by gel chromatography . This V1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peripheral V1 complex of the V-ATPase . We designate this new V1 component subunit G. J Biol Chem, 1996 Apr 5, 271(14), 8152 - 6 Characterization of active recombinant his-tagged oxygenase component of Comamonas testosteroni B-356 biphenyl dioxygenase; Hurtubise Y et al.; Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 . The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FERBPH, and a ferredoxin reductase REDBPH . REDBPH and FERBPH transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate . In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system . His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active . Its major features were compared to the untagged enzyme . In both cases, the native form is an alpha3beta3 heteromer, with each alphabeta unit containing a {2Fe-2S} Rieske center (epsilon455 = 8,300 M-1 cm-1) and a mononuclear Fe2+ . Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak . However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E . coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin. J Biol Chem, 1996 Apr 5, 271(14), 8126 - 32 Dual regulation of a chimeric plant serine/threonine kinase by calcium and calcium/calmodulin; Takezawa D et al.; A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca2+-binding domain was recently cloned from plants (Patil, S., Takezawa, D., and Poovaiah, B . W . (1995) Proc . Natl . Acad . Sci . U . S . A . 92, 4797-4801) . The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca2+/calmodulin-dependent manner . The calmodulin-binding region of CCaMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII) . CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of 32P/mol) that is stimulated 3.4-fold by Ca2+ (0.339 mol of 32P/mol), while calmodulin inhibits Ca2+-stimulated autophosphorylation to the basal level . A deletion mutant lacking the visinin-like domain did not show Ca2+-stimulated autophosphorylation activity but retained Ca2+/calmodulin-dependent protein kinase activity at a reduced level . Ca2+-dependent mobility shift assays using E . coli-expressed protein from residues 358 520 revealed that Ca2+ binds to the visinin-like domain . Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca2+-induced conformational changes in the visinin-like domain . Autophosphorylation of CCaMK increases Ca2+/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its Ca2+-independent activity . This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca2+ and Ca2+/calmodulin . The presence of a visinin-like Ca2+-binding domain in CCaMK adds an additional Ca2+-sensing mechanism not previously known to exist in the Ca2+/calmodulin-mediated signaling cascade in plants. J Biol Chem, 1996 Apr 5, 271(14), 8121 - 5 Elements within the first 17 amino acids of human osteonectin are responsible for binding to type V collagen; Xie RL et al.; The region in human osteonectin (ON) responsible for binding to type V collagen has been identified as the first 17 NH2-terminal residues . This conclusion is based upon binding studies with deletion mutants of ON produced in Escherichia coli, in which parts of the first 17 amino acids have been removed . Wild-type ON from E . coli and mammalian cell-derived nonglycosylated ON bind identically to type V collagen and at least twice as effectively as mammalian cell-derived N-glycosylated ON . In previous studies, it was shown that N-glycosylation at residue 99 significantly reduces the capacity of ON to bind to type V collagen . Results reported in this communication demonstrate that the actual binding site on ON for type V collagen is distal from the site of N-glycosylation in terms of amino acid sequence but may be proximal in the folded, fully glycosylated, three-dimensional structure . Consistent with this conclusion is the ability of a synthetic peptide consisting of amino acids 1-17 to specifically inhibit the binding of ON to type V collagen. J Biol Chem, 1996 Apr 5, 271(14), 8095 - 100 Characterization of crystalline formate dehydrogenase H from Escherichia coli . Stabilization, EPR spectroscopy, and preliminary crystallographic analysis; Gladyshev VN et al.; The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex . The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme . FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable . The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined . Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400 . The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A . There is one monomer of FDH per crystallographic asymmetric unit . Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen . By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH . This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation . A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes . These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme. J Biol Chem, 1996 Apr 5, 271(14), 8046 - 52 Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies; Betton J et al.; The maltose-binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides . We have examined the characteristics of a Mal- mutant of malE corresponding to the double substitution Gly32 --> Asp/Ile33 --> Pro, MalE31, previously obtained by random mutagenesis . In vivo, the MalE31 precursor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm . Furthermore, the accumulation of insoluble MalE31 is independent of its cellular localization; MalE31 lacking its signal sequence forms inclusion bodies in the cytoplasm . The native MalE31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea . The renatured protein exhibits full maltose binding affinity (Kd= 9 x 10(-7) M), suggesting that its folded structure is similar to that of the wild-type protein . Unfolding/refolding experiments show that MalE31 is less stable (-5 . 5 kcal/mol) than the wild-type protein (-9.5 kcal/mol) and that folding intermediates have a high tendency to form aggregates . In conclusion, the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein. J Biol Chem, 1996 Apr 5, 271(14), 8022 - 7 Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods . Cloning, heterologous expression, and localization; Frins S et al.; The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction . It is highly sensitive to the free calcium concentration ({Ca2+}) . The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein) . We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene . The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis . Recombinant GCAP was functionally expressed in Escherichia coli . Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free {Ca2+} with a Hill coefficient of 2.5 . Activation by recombinant nonacylated GCAP showed a lower degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free {Ca2+} . Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina. J Biol Chem, 1996 Apr 5, 271(14), 8015 - 21 Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase; Zhang Y et al.; Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli . We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols . The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1 . The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme . Size exclusion chromatography and spectroscopic measurements at intermediate {GdnHCl} revealed two intermediate folding states . The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein . CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state . The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states. J Biol Chem, 1996 Apr 5, 271(14), 7978 - 85 Identification of novel Pax-2 binding sites by chromatin precipitation; Phelps DE et al.; The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues . The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation . In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned . Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells . In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain . The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2 . The genomic clones can now be used as sequence tags to identify potential target loci. J Biol Chem, 1996 Apr 5, 271(14), 7923 - 6 Novel properties of L-type polypeptide subunits in mouse ferritin molecules; Beaumont C et al.; Properties of the L- and H-type polypeptide subunits forming ferritin 24-mer molecules in mice were investigated, using the products of in vitro transcription and translation from the two cloned genes, and recombinant ferritin molecules (H24L0 or H0L24) produced by transformation in Escherichia coli . Several different conditions for analytical electrophoresis reproducibly show that the relative migration position of the two mouse ferritin subunits is reversed from that reported for ferritin H- and L-subunits in all other mammals; since mouse and human H-polypeptides almost co-migrate, this unusual relative mobility is due largely to novel properties of the murine L-subunit . This unusual electrophoretic property of the mouse L-subunit has led to conflicting reports about the subunit composition of natural mouse ferritin . Here, we show that the single major electrophoretic band given by liver ferritin purified from mice having a short-term iron overload matches that produced by the genetically defined L-polypeptide and that some bona fide H-subunits are also detected . In conclusion, it is reasonable to assume that, when mouse ferritin samples will be analyzed under the same conditions as those described here, the slower species will correspond to the L-type subunit . However, when dealing with ferritin from species other than human or mouse, it should be kept in mind that upon electrophoretic analysis of ferritin polypeptide, the designation of an electrophoretic band as being H- or L-type subunits will be very uncertain without corroboration from genetic, immunological, or amino acid sequencing data. Cell, 1996 Apr 5, 85(1), 71 - 81 Inversion of the membrane topology of SecG coupled with SecA-dependent preprotein translocation; Nishiyama K et al.; E . coli preprotein translocase comprises SecA and SecY/E/G complex . SecA delivers the preprotein to the putative protein-conducting channel formed by SecY/E by undergoing ATP-driven cycles of membrane insertion and deinsertion . SecG renders the translocase highly efficient . An antibody raised against the C-terminal region of SecG inhibits preprotein translocation into everted membrane vesicles despite the exposure of this region to the inside of membrane vesicles in the absence of preprotein translocation . When preprotein translocation was started with ATP and then blocked by the inhibition of ATP hydrolysis, the C-terminal region was exposed to the outside of membrane vesicles . Another region of SecG showed a change in membrane sidedness upon preprotein translocation, indicating that SecG undergoes topology inversion . This topology inversion was tightly coupled to the SecG function and linked with the insertion-deinsertion cycle of SecA. Anal Biochem, 1996 Apr 5, 236(1), 101 - 6 Single-step synthesis and characterization of biotinylated nitrilotriacetic acid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose; McMahan SA et al.; Using a one-step reaction, a bifunctional compound was synthesized for detecting histidine-tagged proteins immobilized on nitrocellulose . This compound has a biotin as one functional group and a nitrilotriacetic acid as the other . The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites . The remaining two sites are available for binding to a histidine tag . The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence . Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit . This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose . The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot. Eur J Pharmacol, 1996 Apr 4, 300(1-2), 99 - 104 Comparison of the effects of aminoguanidine and N omega-nitro-L-arginine methyl ester on the multiple organ dysfunction caused by endotoxaemia in the rat; Wu CC et al.; This study compares the effects of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide (NO) synthase, and N omega-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of endothelial NO synthase, on hypotension and multiple organ dysfunction caused by endotoxaemia in the anaesthetised rat . In the sham-operated rats, L-NAME, but not aminoguanidine, caused a dose-dependent increase in blood pressure . Endotoxin caused hypotension, increased in plasma nitrite (an indicator of inducible NO synthase activity), and dysfunction of kidney, liver and pancreas . Treatment of endotoxic rats with aminoguanidine or L-NAME caused significant and sustained rises in blood pressure . The increase in plasma nitrite caused by endotoxin was inhibited by aminoguanidine, but not by L-NAME . Aminoguanidine, but not L-NAME, attenuated the renal, liver and pancreatic dysfunction caused by endotoxaemia . Thus, selective inhibition of inducible (aminoguanidine), but not endothelial NO synthase (L-NAME) attenuates the circulatory failure and the multiple organ failure caused by endotoxaemia. Nature, 1996 Apr 4, 380(6573), 451 - 3 Direct observation of single kinesin molecules moving along microtubules; Vale RD et al.; Kinesin is a two-headed motor protein that powers organelle transport along microtubules . Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule . Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead) . The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle . Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl . Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism. Biochim Biophys Acta, 1996 Apr 3, 1280(1), 41 - 50 Rapid transmembrane movement of C6-NBD-labeled phospholipids across the inner membrane of Escherichia coli; Huijbregts RP et al.; In this study we have investigated the transmembrane movement of short chain fluorescently labeled phospholipids across the inner membrane of Escherichia coli . Exogenously added C6-NBD-labeled phospholipids rapidly flip across the inner membrane of E . coli, as was shown by a dithionite reduction assay applied to inverted inner membrane vesicles (IIMV) isolated from wild type E . coli cells . The rate of transmembrane movement of the phospholipid probes incorporated into IIMV is temperature dependent, and shows no phospholipid head group specificity . C6-NBD-labeled phospholipids translocate across the membrane of IIMV incubated at 37 degrees C with a t1/2 of 7 min . After the incorporation into IIMV C6-NBD-PG is partially converted to CL by CL-synthase . If IIMV are pretreated with proteinase K the conversion of this fluorescent probe to C6-NBD-CL is not observed anymore, suggesting that the catalytic domain of CL-synthase is at the cytoplasmic site of the plasma membrane of E . coli . Newly synthesized C6-NBD-CL also flips across the inner membrane although at a slower rate than the other phospholipid probes . The transmembrane movement occurs in both directions and is not influenced by treatment of the IIMV with a sulfhydryl reagent or a proteinase, nor by the presence of ATP, or a deltapH across the membrane of the IIMV . However, the transmembrane movement of the C6-NBD-labeled phospholipid probes is not observed in LUVETs (large unilamellar vesicles made by extrusion technique) prepared of wild type E . coli lipids, indicating that the rapid transmembrane movement of phospholipids across the inner membrane of E . coli is a protein-mediated process. Biochemistry, 1996 Apr 2, 35(13), 4231 - 40 Roles of surface hydrophobic residues in the interfacial catalysis of bovine pancreatic phospholipase A2; Lee BI et al.; The interfacial binding is a unique and important step in the phospholipase A2 (PLA2) catalyzed hydrolysis of phospholipids which is distinct from the binding of a substrate to the active site . To assess the roles of surface hydrophobic residues of PLA2 in these processes, we selectively mutated Leu-19 and Leu-20 of bovine pancreatic PLA2 to charged (L19K and L20K), uncharged polar (L19S and L20S), and amphiphilic (L19W and L20W) groups and measured their kinetic and binding properties using various phospholipid aggregates, including micelles, monolayers, and polymerized mixed liposomes . The mutations of Leu-19 and Leu-20 did not significantly change either the tertiary structure or the thermodynamic stability of bovine pancreatic PLA2 . Toward monomeric 1,2-dihexanoyl-sn-glycero-3-phosphocholine, all Leu-20 mutants (L20S, L20W, and L20K) showed activities comparable to that of wild type whereas the substitution of Leu-19 with less hydrophobic side chains (L19S and L19K) reduced the activity to 70% and 50% . Toward zwitterionic 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC8PC) micelles, L20S and L20K mutants showed only 30% and 35% of the wild-type activity, respectively, whereas L20W was about twice as active as wild type . Also, L19S and L19K showed 75% and 15% of the wild-type activity, respectively . Toward anionic Trition X-100/sodium deoxycholate/diC8PC (4:2:1) mixed micelles, L20W and L20K were 2.6 times and twice more active than wild type . To determine the sn-2 acyl group selectivity of wild type and mutants, polymerized mixed liposomes were used which contained 1,2-bis{12-(lipoyloxy)-dodecanoyl}-sn-glycero-3-phosphoglycerol and 1 mol % of either 1-2{12-(1-pyrenebutanoyloxy)dodecanoyl}-2-hexanoyl-sn-glycero-3-++ +phosphocholine or 1-{12-(1-pyrenebutanoyloxy)dodecanoyl}-2-dodecanoyl-sn-glycero-3-+ ++phosphocholine . These measurements showed that Leu-19 was involved in the substrate binding and the sn-2 acyl group selectivity of bovine pancreatic PLA2 and that Leu-20 made a direct contact with the surface of phospholipid aggregates . The binding affinities of mutants to micelles, polymerized liposomes, and monolayers were well consistent with their kinetic behaviors, supporting the notion that the altered activities of Leu-19 mutants and Leu-20 mutants were due to the change in their substrate binding and interfacial binding, respectively . Finally, the L20W mutant represents the first example of protein engineering of PLA2 which results in a significant increase in interfacial binding to densely packed neutral monolayers and bilayers. Biochemistry, 1996 Apr 2, 35(13), 4199 - 210 An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies; Marzabadi MR et al.; A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P) . A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme . The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site . A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding . This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding . Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM . As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate . Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date . However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P . The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite . Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site . However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P . Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite . As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site . Consequently, 4 is best described as an S3P-based substrate-analog inhibitor . These combined results corroborate the previous kinetic model {Gruys, K . J., Marzabadi, M . R., Pansegrau, P . D., & Sikorski, J . A . (1993) Arch . Biochem . Biophys . 304, 345-351}, which suggested that 4 interacts well with the S3P subsite but has little, if any, interaction at the expected glyphosate phosphonate or phosphoenolpyruvate-Pi subsites. Biochemistry, 1996 Apr 2, 35(13), 4161 - 8 Membrane topology of the melibiose permease of Escherichia coli studied by melB-phoA fusion analysis; Pourcher T et al.; In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity . In general agreement with a previously suggested secondary structure model of melibiose permease {Botfield, M . C., Naguchi, K., Tsuchiya, T., & Wilson, T.H . (1992) J . Biol . Chem . 267, 1818}, clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence . Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops . Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane . Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it {Calamia, T., & Manoil, C . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 4937}, the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments . However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter. Biochemistry, 1996 Apr 2, 35(13), 4146 - 54 Mutation spectra of M13 vectors containing site-specific Cis-Syn, Trans-Syn-I, (6-4), and Dewar pyrimidone photoproducts of thymidylyl-(3'-->5')-thymidine in Escherichia coli under SOS conditions; Smith CA et al.; The mutations spectra of cis-syn, trans-syn-I, (6-4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (-) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions . Oligonucleotides containing site-specific photoproducts were annealed to a complementary uracil-containing (+) strand that contained one or more unique pairs of nucleotide mismatches and used to prime (-) strand synthesis with a DNA polymerase and dNTPs . Following DNA synthesis, the reaction mixtures were incubated with T4 DNA ligase and ATP and then used to transfect SOS-induced competent CSRO6F' cells (uvrA6 and phr-1) . The transfectants were plated, gridded, and probed by oligonucleotides specific for progeny of the (-) and (+) strands . Individual progeny of the photoproduct-containing (-) strands were plaque purified and sequenced by the dideoxy method . The cis-syn and trans-syn-I dimers were found not to be very mutagenic (<9%), the Dewar product more so (<33%), and the (6-4) product the most mutagenic (<73%) . The mutation spectra were similar to those previously reported for the same photoproducts of the TT site of AGTTGG in the (+) strand of an M13 vector {Lawrence, C . W., et al . (1990) Mol . Gen Genet . 222, 166-168; LeClerc, J . E., et al . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 9685-9689} except that -1 deletion mutations were not observed for the trans-syn-I photoproducts, and a lower frequency of 3'-T-->C mutations was observed for the (6-4) photoproduct . Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side of the photoproduct . Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side was obtained from transfection experiments in which a second double mismatch was introduced opposite or flanking the photoproduct . Analysis of the minor tandem mutations induced by the (6-4) and Dewar products suggests that the SOS polymerase complex is able to elongate what amounts to double mismatches opposite these photoproducts and is consistent with the action of a highly processive polymerase that lacks proofreading ability. Biochemistry, 1996 Apr 2, 35(13), 4139 - 45 C-terminal zinc-containing peptide required for RNA recognition by a class I tRNA synthetase; Glasfeld E et al.; Escherichia coli isoleucyl-tRNA synthetase is one of five closely related class I tRNA synthetases . The active site of the 939 amino acid polypeptide is in an N-terminal domain which contains an insertion believed essential for interactions with the tRNA acceptor helix . The enzyme was shown previously to contain an essential (for function in vivo) zinc bound to a Cys4 cluster at the C-terminal end of the polypeptide . The specific function of this zinc has been unknown . We show here that aminoacylation activity can be reconstituted in vitro by combining a 53 amino acid zinc-containing C-terminal peptide with a protein consisting of the remaining 886 amino acids . Reconstitution of aminoacylation is zinc-dependent . In contrast, the zinc-containing peptide is dispensable for synthesis of isoleucyl adenylate . Affinity coelectrophoresis showed that the 53 amino acid C-terminal peptide is required specifically for tRNA binding . We propose that the zinc-containing peptide curls back to the active site to make contact with the acceptor helix of bound tRNA, but not with isoleucine or ATP . It is the first example of a zinc-containing peptide in a class I tRNA synthetase that is essential for tRNA binding interactions . The design of this enzyme may be part of a more general scheme for class I tRNA synthetases to acquire acceptor helix binding elements during the development of the genetic code. Biochemistry, 1996 Apr 2, 35(13), 4128 - 38 Targeted A --> T and G --> T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol epoxide of dibenz{a,j}anthracene in M13mp7L2; Min Z et al.; The studies described in this report directly examined the mutagenicity in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguanosine (dGuo) adduct derived from (+)-anti-dibenz{a,j}-anthracene-3,4-diol 1,2-epoxide {(+)anti-DB{a,j}A-DE} that were site-specifically placed in a single-stranded M13mp7L2 replication vector . An 11-base oligonucleotide (5'-CTC ACG CTT CT-3') containing either a single (+)anti-DB{a,j}A-DE--trans-N2-dGuo or (+)anti-DB{a,j}A-DE--trans-N6dAdo adduct was successfully incorporated into single-stranded M13mp7L2 plasmid via ligation . In vitro studies using E . coli DNA polymerase I (Klenow fragment)indicated that both adducts were effective blocks for polymerase action . E . coli strains JM103 and JM103 uvrA6 were subsequently transformed with control (unadducted) and adduct-containing M13mp7L2 constructs followed by analysis of progeny DNA . In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors . Activation of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacterial strain transformed with adduct-containing vectors . Targeted mutations were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors . In JM103 cells, (+)anti-DB{a,j}A-DE--N6-dAdo induced exclusively A --> t transversions and (+)anti-DB{a,j}A-DE--N2-dGuo induced exclusively G --> T transversions . In JM103 uvrA6 cells, similar targeted transversion mutations were also obtained except that a few C deletions (i.e., aprroximately 10% of the mutations) were detected immediately 3' to the dAdo adduct . While mutagenesis was SOS dependent in JM103 cells {<0.15% (-SOS) vs approximately 1.3% (+SOS)}, it appeared to be SOS independent in JM103 uvrA6 cells (approximately 1-2% in the presence or absence of SOS induction) . It is argued that adduct-induced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms . In contrast, A --> T mutations are unlikely to arise via a misinformational pathway, which provides the strongest support to date that bulky DNA adducts can induce mutations via a noninformational pathway. Biochemistry, 1996 Apr 2, 35(13), 4079 - 83 Reversible oligomerization and denaturation of the chaperonin GroES; Seale JW et al.; The chaperonin GroEL can assist protein folding and normally acts with the co-chaperonin GroES . These Escherichia coli proteins are encoded on the same operon, with GroES positioned first . In this report, we have investigated the reversible folding of GroES . Using fluorescence anisotropy of dansyl-labeled GroES, intrinsic fluorescence, bis-ANS binding, sedimentation velocity, and limited proteolysis, we show that GroES unfolds in a single, two-state transition . Importantly, intrinsic fluorescence and sedimentation velocity analyses show that GroES is capable of refolding and reassembling from a urea denatured state . The refolded GroES is fully active as shown by its ability to assist GroEL in the refolding of rhodanese . These results indicate that chaperonins may not require other chaperonins for successful folding/assembly . We also show that GroES is capable of assisting in the refolding/reassembly of fully denatured GroEL . The reversible folding of GroES coupled with the ability of GroES to assist the refolding/reassembly of GroEL suggest that the groE operon may be organized in a manner that provides a structural role in GroES/GroEL assembly as well as a functional role. Biochemistry, 1996 Apr 2, 35(13), 4034 - 45 Equilibrium DNA binding of Sac7d protein from the hyperthermophile Sulfolobus acidocaldarius: fluorescence and circular dichroism studies; McAfee JG et al.; The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to double-stranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA . Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound . Reverse titration data have been fit directly to the McGhee-von Hippel model {McGhee, J., & von Hippel, P . (1974) J . Mol . Biol . 86, 469-489} using nonlinear regression . Sac7d binds noncooperatively to poly(dGdC) x poly(dGdC) with an intrinsic affinity of 6.5 x 10(6) M(-1) and a site size of 4 base pairs in 1 mM KH2PO4 and 50 mM KC1 (pH 6.8) . Some binding sequence preference is noted, with the binding to poly(dIdC) x poly(dIdC) over 10-fold stronger than to poly(DAdT) x poly(dAdT) . The binding is largely driven by the polyelectrolyte effect and is consistent with a release of 4.4 monovalent cations from DNA upon complex formation or the formation of 5 ion pairs at the protein-DNA interface . Extrapolation of salt back-titration data to 1 M KC1 indicates a -2.2 kcal/mol nonelectrostatic contribution to the binding free energy . A van't Hoff analysis of poly(dGdC) x poly(dGdC) binding shows that the binding enthalpy is approximately zero and the process is entropically driven . The affinity decreases slightly between pH 5.4 and 8.0 . There is no significant difference between the binding parameters of recombinant Sac7d and native Sac7 proteins, indicating that methylation of the native protein has no effect on the DNA binding function . The binding of Sac7d to various DNAs leads to a significant increase in the DNA long-wavelength circular dichroism (CD) band, the intensity of which shows a sigmoidal dependence on Sac7d concentration . The sigmoidal CD binding isotherm can be quantitatively modeled by a conformational transition in the DNA that is cooperatively induced when protein monomers are bound within a given number of base pairs, ranging from zero for poly(dIdC) x poly(dIdC) to 8 or less for poly(dAdG) x poly(dCdT). Biochemistry, 1996 Apr 2, 35(13), 3950 - 6 Probing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modification; Frillingos S et al.; By using site-directed chemical labeling of lactose permease, conformational changes induced by ligand binding are observed in the native membrane of Escherichia coli . Membranes containing permease mutants with a single-Cys residue and a biotin-acceptor domain were labeled with radioactive N-ethylmaleimide (NEM) in the presence or absence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or a proton electrochemical gradient, followed by solubilization in n-dodecyl beta-D-maltopyranoside and adsorption to avidin . TDG-induced enhancement of the reactivity of membrane-embedded Val315-->Cys (helix X) permease is observed, while the reactivity of Val331-->Cys (helix X) permease is inhibited by ligand binding or imposition of a proton electrochemical gradient . In contrast, the reactivity of permease with a single native Cys residue at position 148 (helix V) is blocked by TDG, but unaffected by the proton electrochemical gradient . Furthermore, as shown with right-side-out and inside-out membrane vesicles, the accessibility of Cys148 to either NEM or impermeant methanethiosulfonate derivatives is comparable from both sides of the membrane . On the other hand, TDG protects Cys148 from alkylation more effectively in right-side-out vesicles (apparent KD = 20-50 microM) than inside-out vesicles (apparent KD ca . 1.0 microM) . The findings provide strong support for the conclusion that the permease retains close to native conformation in n-dodecyl-beta-D-maltopyranoside . In addition, the results are consistent with the idea that lactose permease has two binding sites: one with higher affinity on the periplasmic surface of the membrane and another with lower affinity on the cytoplasmic surface. Biochemistry, 1996 Apr 2, 35(13), 3933 - 43 Partitioning of HIV-1 Gag and Gag-related proteins to membranes; Ehrlich LS et al.; The binding of HIV-1 Gag and Gag-related proteins to model membranes was examined using three experimental systems: (i) large unilamellar phospholipid vesicles (LUVs) and recombinant Gag purified from Escherichia coli; (ii) LUVs added to a mammalian cell extract in which Gag proteins were expressed by a coupled transcription/translation system; and (iii) inside-out plasma membrane vesicles purified from human red blood cells (RBC) and recombinant, purified Gag from E . coli . Several novel aspects of HIV-1 Gag membrane interactions were observed: (i) Gag proteins bound with high affinity to both model membranes with a negatively charged surface and to RBC membranes . (ii) Binding of the Gag precursor and mature Gag proteins exhibited different sensitivities to ionic strength indicating that the precursor directed membrane binding through interactions that were qualitatively and quantitatively distinct from those of any of its individual domains . Studies using energy transfer between tryptophan residues in the proteins and anthroyloxy-containing probes inserted in the LUVs indicated that the orientation of the precursor and of the mature proteins on the membrane surface were distinct; (iii) Gag oligomers appear to have facilitated high-affinity binding under high salt conditions, suggesting that protein-protein interactions led to formation of stronger electrostatic or new hydrophobic membrane binding determinants . Since binding studies with model membranes permit quantitative analysis, these experimental approaches may permit identification of interactions that drive Gag assembly on the membrane. Biochemistry, 1996 Apr 2, 35(13), 3925 - 32 Modulation of protein function by exogenous ligands in protein cavities: CO binding to a myoglobin cavity mutant containing unnatural proximal ligands; Decatur SM et al.; A variety of heterocyclic ligands can be exchanged into the proximal cavity of sperm whale myoglobin mutant H93G, providing a simple method for introduction of the equivalent of unnatural amino acid side chains into a functionally critical location in this protein . These modified proteins bind CO on the distal side . 1H NMR data on H93G(Im)CO, where Im is imidazole, demonstrate that the structure of the distal heme pocket in H93G(Im)CO is very similar to that of wild type; thus, the effects of the proximal ligand's properties on CO binding can be studied with minimal perturbation of distal pocket structure . The exogenous proximal ligands used in this study include imidazole (Im), 4-methylimidazole (4-MeIm), 4-bromoimidazole (4-BrIm), N-methylimidazole (N-MeIm), pyridine (Pyr), and 3-fluoropyridine (3-FPyr) . Substitution of the proximal ligand is found to produce substantial changes in the CO on and off rates, the equilibrium binding constant, and the vibrational stretch frequency of CO . Many of the changes are as large as those reported for distal pocket mutants prepared by site-directed mutagenesis . The ability to systematically vary the nature of the proximal ligand is exploited to test the effects of particular properties of the proximal ligand on CO binding . For example, 4-MeIm and 4-BrIm are similar in size and shape but differ significantly in pKa . The same relationship is true for Pyr and 3-FPyr . By comparison of the IR spectra and CO recombination kinetics of these complexes, the effects of proximal ligand pKa on the CO binding are assessed . Likewise, N-MeIm and 4-MeIm are similar in size and pKa but differ in their ability to hydrogen bond to amino acid residues in the proximal cavity . Comparisons of IR spectra and CO binding kinetics in these complexes reveal that proximal ligand conformation and hydrogen bonding affect the kinetics of CO binding . The mechanism of proximal ligand exchange between solution and the proximal cavity in CO complexes was investigated by obtaining the 19F NMR spectrum of H93G(3-FPyr)CO, whose 19F signal can be observed without interference from resonances of the protein . The proximal ligand is found to exchange within a few seconds by saturation transfer . This exchange rate is about 2 orders of magniture faster than what is observed for the isoelectronic metcyano complex {Decatur, S . M., & Boxer, S . G . (1995) Biochemistry 34, 2122-2129}; in both the ferrous CO and ferric cyano complexes, the proximal ligand exchange rate is independent of ligand concentration . These results suggest that the rate-limiting step in proximal ligand exchange is breakage of the iron-ligand bond, followed by rapid diffusion of the ligand through the protein to bulk solution. Biochemistry, 1996 Apr 2, 35(13), 3880 - 5 Effects of protein RNase inhibitor and substrate on the quaternary structures of bovine seminal RNase; Murthy BS et al.; The effect of the protein RNase inhibitor (PRI) on the activity of bovine seminal RNase (BS-RNase) was investigated using the isolated quaternary forms, MxM and M=M, of the enzyme reported earlier {Piccoli, R., et al., (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 1870-1874} . We found that the inhibitor does not interact with the intact isolated forms but has dramatic, differential effects on the two forms when the assays are performed under reducing conditions . These conditions, which are essential for full activity of the inhibitor, and are typical of its cytosolic localization, also promote monomerization of the M=M form, while under identical conditions the MxM form becomes a noncovalent dimer (NCD) . The sensitivity of BS-RNase or that of the isolated quaternary forms under reducing conditions thus appears to be related to differential monomerization of the two forms of the enzyme; monomer being sensitive to PRI . The present study also shows that the interconversion between the two forms in equilibrium occurs at much higher rates in a reducing environment and that PRI further affects the interconversion and alters the equilibrium favoring monomerization of the protein . An opposite effect on the equilibrium between the forms is played by the substrate, which is found to stabilize the NCD form of the protein with a shift in the equilibrium between the two forms towards the dimer . These results are analyzed in the light of the antitumor action of the enzyme which is exerted in the cytosol, i.e., in the compartment housing the PRI and the ribosomal RNA, the molecular target of the enzyme. Biochemistry, 1996 Apr 2, 35(13), 3875 - 9 Differentiation of catalytic sites on Escherichia coli F1ATPase by laser photoactivated labeling with {3H}-2-Azido-ATP using the mutant beta Glu381Cys:epsilonSer108Cys to identify different beta subunits by their interactions with gamma and epsilon subunits; Gruber G et al.; The ATP binding affinities of the catalytic sites in the three beta subunits of the Escherichia coli F1 ATPase (ECF1) have been explored in relation to the interaction of these subunits with the small subunits gamma and epsilon . ECF1 from the mutant beta E381C:epsilonS108C was reacted with different concentrations of {3H}-2-azido-ATP and covalent insertion of the nucleotide analogue induced by photoactivation of the azide group to a nitrene with single-pulse UV laser excitation . The enzyme showed cooperative binding of {3H}-2-azido-ATP in the presence of Mg2+ . The highest affinity site was located at betafree, the one of the three beta subunits in the mutant that does not form disulfide bonds with either the gamma or the epsilon subunit . This beta subunit is, therefore, the site of unisite catalysis in the enzyme . The second mole of {3H}-2-azido-ATP to bind was located in the beta subunit that links to epsilon (betaepsilon), while the lowest affinity binding of the substrate analogue was with the beta subunit that links to gamma (betagamma) . In the absence of Mg2+, all three beta subunits bound {3H}-2-azido-ATP with a similar, low affinity . The results show that binding of MgATP is determined by, and/or must determine, the interactions of the different alpha-beta subunit pairs with the single-copy subunits gamma, delta, and epsilon of the enzyme. Mutat Res, 1996 Apr 2, 362(3), 261 - 8 The Escherichia coli DNA repair protein UvrA can re-associate with the UvrB: aflatoxin B1-DNA complex in vitro; Allan JM et al.; The UvrA and UvrB proteins form part of the UvrABc endonuclease, which is responsible for nucleotide excision repair in Escherichia coli . Using a mobility shift gel assay we have studied the binding of UvrA dimer, UvrB monomer and UvA(2)B trimer complexes with 40, 50 and 136 bp (32)P-end-labelled DNA fragments adducted with aflatoxin B(1) . UvrA was shown to re-associate with adduct specific UvrB: DNA complexes, a phenomenon which could be reversed by the addition of 500 mM potassium chloride or anti-UvrA anti-sera . Re-association was shown to be UvrA concentration dependent . Re-association of UvrA(2)B to the UvrB:DNA complex was not seen . We have also shown that the UvrB:DNA complex, in the case of aflatoxin B(1), is extremely stable with a half-life excess of 400 min and that fragment termini are not a specific substrate for UvrA binding. Mutat Res, 1996 Apr 2, 362(3), 249 - 59 A comparison of the genotoxic effects of carboplatin and cisplatin in Escherichia coli; Overbeck TL et al.; cis-Diammine(1,1,-cyclobutanedicarboxylato)platinum(II) (carboplatin) is a second generation platinum anticancer agent with antineoplastic properties like that of its parent compound, cis-diamminedichloroplatinum(II) (cisplatin) but with substantially less deleterious side effects in treated patients with cisplatin . We compared their genotoxic effects in Escherichia coli and found carboplatin to be less cytotoxic (measured as loss of colony forming ability) that cisplatin in that equitoxic doses required greater than 60 time more carboplatin . However, solutions of carboplatin containing chloride ion became more cytotoxic to E . coli after a 24 h incubation period than similar freshly made solutions . Two platinum conversion products which were neither present in freshly made solutions nor in solutions lacking chloride were resolved by thin-layer chromatography (TLC) . One of the conversion products migrated like cisplatin and its occurrence in carboplatin solutions was associated with cisplatin-like properties, enhanced cytotoxicity and ability to induce the SOS responses in E . coli . The SOS-inducing abilities were determined by induction of a sulA::lacZ fusion . Likewise, adducts formed in end-labeled oligonucleotides treated with carboplatin appeared identical to those caused by cisplatin when carboplatin was preincubated in chloride-containing solutions but not by carboplatin in freshly made solutions . It is likely that responses evoked by carboplatin in biological systems are partly due to activation of carboplatin by its conversion of cisplatin. Mutat Res, 1996 Apr 2, 362(3), 219 - 26 The ultraviolet-sensitizing function of plasmid R391 interferes with a late step of postreplication repair in Escherichia coli; Wang TC et al.; The conjugative plasmid R391 increases the UV radiation sensitivity of wild-type, uvrA, and lexA cells of Escherichia coli, but not recA strains . To investigate the UV-sensitizing function of R391, we examined the effect of R391 on the repair of DNA daughter-strand gaps and on the UV radiation sensitivities of various repair and/or recombination-deficient mutants . The presence of R391 did not significantly inhibit the repair of DNA daughter-strand gaps in uvrB cells . The presence of R391 increased the UV radiation sensitivity of uvrA, uvrA recF, uvrB, uvrB recF, uvrB recB, and uvrB ssb-113 cells to UV irradiation, but did not significantly increase the UV radiation sensitivity of uvrA ruvA and uvrA ruvC strains . Based on these results, we propose that the UV-sensitizing activity of R391 acts by inhibiting or interfering with the ruvABC-mediated postsynapsis step of recombinational repair. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 3007 - 10 The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets; Kato Y et al.; Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis . The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method . The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution . The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside . There is a 5-angstrom-diameter "hole" through the middle of the barrel . The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor . Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs . These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2969 - 74 Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide; Patel R et al.; Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site . During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template . The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide . The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored . The sequence requirements for template switching are compared to those for transposon excision. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2920 - 5 In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities; Sera T et al.; A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities . A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10 . These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3' . Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A) . Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions . The mutant proteins showed high specificity in a functional transcription interference assay . A model for the interaction of these mutants with the target DNA sequences is discussed. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2856 - 61 Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe; Fijalkowska IJ et al.; The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity . We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity . When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product) . When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable . However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E . coli mutL+ gene . These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe) . Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair . The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2801 - 6 Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse; Morgan BA et al.; Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA . The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use . For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids . In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity . In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice . Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively . With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription . Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation . Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2755 - 8 Protein synthesis editing by a DNA aptamer; Hale SP et al.; Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions . One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions . Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions . Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector . We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase . The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity . The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure . These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing . Thus, specific bases in a nucleic acid effector trigger the editing response. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2680 - 5 DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos; Walter J et al.; In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos . We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins . In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo . This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2670 - 4 The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle; Scherf U et al.; Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1 . CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis . Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells . Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS . CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell . The intracellular distribution of CAS resembles that of tubulin . In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle . CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells . CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles . Thus, CAS appears to be associated with but not to be an integral part of microtubules . Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon . CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum . These cells contain many microtubules . The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2652 - 7 Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites; Herzberg O et al.; The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites approximately 45 angstroms apart . The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase . Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain . While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers . This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate . The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins. Cell Stress Chaperones, 1996 Apr, 1(1), 78 - 89 Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer; Leung SM et al.; Differential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70) . Hsc70 had endothermic transitions with midpoints (Tm) at 59 degrees C and 63 degrees C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan . Combined with increased exposure at 60 degrees C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70 . An exothermic transition (Tm = 66 degrees C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes . An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c . A novel finding was an exothermic transition of Hsc70 beginning at about 30 degrees C (Tm = 41 degrees C) . No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range . Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation . However, Hsc70 was protease-resistant at 20 degrees C, sensitive at 40 degrees C and resistant when returned to 20 degrees C, indicating that this exotherm is associated with a reversible conformational change . As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c, a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK . For all of these substrates, the amount of complex formed increased with increasing temperature over the same range as the 41 degrees C exotherm . It is proposed that a conformational change exposes polar and charged residues in Hsc70 which subsequently become hydrated, resulting in an active chaperone . Hsc70 may be a thermal sensor that matches the supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells . Thermal activation of Hsc70 may also have a role in acquired thermotolerance. Wei Sheng Wu Xue Bao, 1996 Apr, 36(2), 158 - 9 {Study on the death of Escherichia coli induced by hydrostatic pressures}; Qin L et al.; The effect of hydrostatic pressure on the death of E . coli was studied in this paper . The results indicated that E . coli could be killed by hydrostatic pressure above 800 bar . At 2300 bar E . coli was totally killed in 30 minutes . The time course of E . coli death induced by pressure indicated that the most E . coli was killed in the first 10 minutes after the pressure was applied . It was also found that the lower temperature favored killing E . coli under pressure. Microb Drug Resist, 1996 Spring, 2(1), 155 - 7 Affinity chromatography as a means to study multienzyme complexes involved in murein synthesis; von Rechenberg M et al.; The interaction of murein hydrolases and synthases was studied by affinity chromatography . The lytic transglycosylases Slt70 and MltB of E . coli were purified and covalently linked to CNBr-activated Sepharose . Membrane extracts were analyzed for proteins that interact with the immobilized murein hydrolases . Slt70-Sepharose was found to retain the PBPs 1b, 1c, 2, and 3 . Likewise MltB-Sepharose enriched PBP 1b, 1c, and 3 . Thus both lytic transglycosylases have an affinity for a transpeptidase, PBP2 and/or 3, as well as for the bifunctional transpeptidase/transglycosylase 1b . Interestingly, in addition, the poorly characterized PBP 1c interacts strongly with both Slt70 and MltB . It is speculated that the lytic transglycosylases assemble a multienzyme complex consisting of hydrolases and synthases, which is involved in growth of the stress-bearing murein sacculus. Microb Drug Resist, 1996 Spring, 2(1), 131 - 4 Inhibition of peptidoglycan hydrolase activity in vivo and in vitro by energy uncouplers in Escherichia coli; Rodionov DG et al.; The effects of energy uncouplers on in vivo and in vitro peptidoglycan hydrolase activities in Escherichia coli were determined . Sodium azide, potassium cyanide, and carbonyl cyanide m-chlorophenylhydrazone all inhibited ampicillin-induced lysis of exponential phase cultures, even when they were added to lysis-committed cultures . These energy uncouplers also inhibited the solubilization of radiolabeled peptidoglycan by bacterial suspensions that had been treated with 5% trichloroacetic acid by the method of Hartmann et al.3 to activate the peptidoglycan hydrolases . Therefore, the in vivo and in vitro activities of peptidoglycan hydrolases in E . coli are dependent on membrane energization. Microb Drug Resist, 1996 Spring, 2(1), 99 - 103 Molecular interplay of murein synthases and murein hydrolases in Escherichia coli; Holtje JV; Affinity chromatography using different lytic transglycosylases as a specific ligand revealed an interaction of both murein hydrolases and murein synthases . This interaction is taken as evidence for the assemblage into a multienzyme complex that could function as a murein replicase precisely copying the given three-dimensional structure of the murein sacculus . The sacculus of the mother cell would function as a template, which is identically replicated by copying the lengths of the existing glycan strands and the pattern of crosslinkages . A hypothetical enzyme complex specifically involved in cell division and a complex specifically involved in cell elongation are presented . It is postulated that PBPs 1a and/or 1b are present in both complexes, whereas the presence of PBP2 or PBP3 defines the specificity of the murein-synthesizing machinery as being involved in either cell elongation or septation . Moreover, the proposed "holoenzyme" suprastructure could explain why the specific inhibition of PBPs 1a/1b results in bacteriolysis and why inhibition of PBP2 and PBP3 causes the well-known morphological alterations, spherical growth, and filamentation, respectively. Microb Drug Resist, 1996 Spring, 2(1), 55 - 61 How does FtsZ find its location? Voskuil JL, Nanninga N. The conformational flexibility of FtsZ and the properties of its epitopes have been studied . Cellular fractions of Escherichia coli have been treated with Triton X-114 . FtsZ distributed in the polar as well as in the non-polar phase . This has been interpreted to mean that FtsZ can change its conformation . For the nonpolar conformation it has been assumed that the putative hydrophobic pocket of FtsZ (cf . Voskuil et al., J . Bacteriol . 176:1886-1893) is being turned inside out upon interaction with the cytoplasmic membrane . In a tentative model we suggest that FtsA mediates this interaction . Immunoprecipitations of FtsZ with various monoclonal antibodies in the presence or absence of 1 M NaCl gave a clue concerning the hydrophobicity and hydrophilicity of FtsZ's epitopes . Immunogold-labeling also showed differences with respect to the accessibility of FtsZ. Microb Drug Resist, 1996 Spring, 2(1), 51 - 4 Study of the reaction mechanism of the D-glutamic acid-adding enzyme from Escherichia coli; Vaganay S et al.; The D-glutamic acid-adding enzyme of Escherichia coli, or MurD, was purified from an overproducing strain and a few aspects of its reaction mechanism were studied . The existence of a reactive cysteinyl residue was shown by the following experiments: (1) two thiol-modifying reagents, (5,5'-dithiobis)2-nitrobenzoic acid and 2-nitro-5-thiocyanobenzoic acid, inactivated the enzyme; (2) incubation with tetranitromethane led to inactivation and to the appearance of cysteic acid (not to 3-nitrotyrosine); (3) in each case, ATP or UDP-MurNAc-L-Ala (but not D-glutamic acid) protected the enzyme from inactivation . The existence of a reactive lysyl residue was shown by the action of 2,4,6-trinitrobenzenesulfonic acid, a reagent specific for lysyl residues present in phosphate-binding sites . The formation of an acyl phosphate intermediate was consistent with three types of results: (1) the molecular isotope exchange reaction, which took place only in the presence of phosphate, but which was not strictly dependent on the presence of ADP; (2) a release of phosphate, measured by the molybdate assay, observed when the enzyme was incubated with ATP and UDP-MurNAc-L-Ala (without D-glutamic acid); (3) the appearance of a new radioactive compound (besides ATP and Pi) after incubation for a few minutes with UDP-MurNAc-L-Ala and {gamma-32P}ATP . Finally, the fact that phosphinate 1 was a good inhibitor of the enzyme (IC50 = 0.7 microM) strongly suggested that a tetrahedral transition state follows the acyl phosphate in the reaction pathway. Microb Drug Resist, 1996 Spring, 2(1), 25 - 7 Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli; Liger D et al.; The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan . It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine . The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc . The murC gene product appears as a 50-kDa protein accounting for ca . 50% of total cell proteins . A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture . The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene . The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol . The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively . The specificity of the enzyme for its substrates was investigated with various analogues . Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate. J Mol Endocrinol, 1996 Apr, 16(2), 159 - 70 Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells; Nicholson LB et al.; We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease . Recombinant TSH-R.E used as immunogen, was produced in E . coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein . To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized . The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor . The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals) . Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells . The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule . None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG . The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function . The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity. Mol Chem Neuropathol, 1996 Apr, 27(3), 249 - 58 The in vitro formation of recombinant tau polymers: effect of phosphorylation and glycation; Ledesma MD et al.; Tau Isolated from paired helical filaments, aberrant structures that appear in Alzheimer disease (AD) patients' brains, show at least two posttranslational modifications: phosphorylation (Grundke-Iqbal et al., 1986; Ihara et al., 1986) and glycation (Ledesma et al., 1994; Yan et al., 1994) . To test whether these modifications could affect the capacity of tau to self-aggregate, recombinant tau was phosphorylated and glycated, and its capacity to form polymers analyzed . Our results indicate that on phosphorylation and glycation, the capacity of tau to form aggregates increases, and that glycation of tau could stabilize the assembled polymers and could facilitate formation of bundles from these polymers. Biochem Mol Biol Int, 1996 Apr, 38(5), 957 - 64 Molcecular cloning of human antizyme cDNA; Yang D et al.; We have cloned the cDNA encoding the human ornithine decarboxylase antizyme from a 5'-stretch cDNA library of human B-cell lymphoma Daudi . The cloned human antizyme cDNA fragment consists of 1063 bp, has 80% homology to the rat antizyme cDNA, but shows almost no homology to the E . coli antizyme gene . Northern hybridization analysis shows that this gene is expressed in a number of human cell lines with an estimated mRNA transcript size of about 1.1 kb . The size of the mRNA suggests that the cloned cDNA fragment probably represents the full length of human antizyme mRNA transcript . Comparison of the human and rat antizymes demonstrates that they are highly conserved at both nucleotide and peptide levels. Int J Biochem Cell Biol, 1996 Apr, 28(4), 451 - 6 Peptide degradation: effect of substrate phosphorylation on aminopeptidasic hydrolysis