J Biol Chem, 1994 Sep 23, 269(38), 23471 - 6 Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo; Faulkner KM et al.; Several manganic porphyrins, with substituents on the methine bridge carbons, were prepared and examined for stability, redox behavior, catalysis of the dismutation of superoxide radical (O2-), and the ability to protect a superoxide dismutase (SOD)-null strain of E . coli against dissolved oxygen and a SOD-competent strain against paraquat . All of the compounds tested exhibited reversible redox behavior and were stable to EDTA in both the oxidized and reduced states, and several were able to catalyze the dismutation of O2- with the rate constants of approximately 10(7) M-1 s-1 . The marked protective effects of some of these compounds exceeded that which could be anticipated on the basis of such rate constants . The tetrakis (1-methyl-4-pyridyl) compound was reduced enzymatically at the expense of NADPH and nonenzymatically by GSH and was kept in the reduced state within E . coli . Since the rate constant for reoxidation of the reduced form by O2- is 4 x 10(9) M-1 s-1, it appears that this compound acts in vivo as an NADPH/GSH:O2- oxidoreductase rather than as an SOD mimic . Its ability to facilitate aerobic growth of the SOD-null strain can be explained on this basis. J Biol Chem, 1994 Sep 23, 269(38), 23437 - 43 The glucose transporter of Escherichia coli . Overexpression, purification, and characterization of functional domains; Buhr A et al.; The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar . It consists of a transmembrane subunit (IICBGlc) and a hydrophilic subunit (IIAGlc) . The IICBGlc subunit consists of two domains . The NH2-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site . The COOH-terminal hydrophilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane . It contains the phosphorylation site (Cys421) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlc subunit to the transported solute . Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography . Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be purified from 1 liter of culture . Cells expressing IIBGlc-6H and IICGlc-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBGlc . The rate of IIBGlc-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBGlc . The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of the control . IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain . Based on this work, the secondary structure of the IIBGlc domain has been determined by isotope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B . (1994) Eur . J . Biochem . 219, 945-952). J Biol Chem, 1994 Sep 23, 269(38), 23824 - 9 Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli; Petit MA et al.; Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response . Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria . The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M . F., Echols, H . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 10777-10781) . In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro . We treated purified and inactive UmuC with Hsp70 and Hsp60 . After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not . However, lesion bypass activity was recovered upon further treatment with Hsp60 . The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis. J Biol Chem, 1994 Sep 23, 269(38), 23808 - 16 Cloning and expression of the mammalian multifunctional protein CAD in Escherichia coli . Characterization of the recombinant protein and a deletion mutant lacking the major interdomain linker; Guy HI et al.; The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells . Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers . One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species . A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli . The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography . The purified protein had kinetic parameters that were close to those obtained for native CAD . Moreover, the CPSase activity was allosterically regulated . Gel filtration showed that the recombinant protein had the same molecular mass as native CAD . Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact . A deletion mutant that lacked the DA linker was then constructed . The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains . However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex . Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed . Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD. J Biol Chem, 1994 Sep 23, 269(38), 23575 - 82 Rapid degradation of an abnormal protein in Escherichia coli involves the chaperones GroEL and GroES; Kandror O et al.; In Escherichia coli, the molecular chaperones (DnaK, DnaJ, and GrpE) are essential for the rapid degradation of certain proteins . To see if chaperones are involved more generally in proteolysis, we studied the degradation of a short-lived fusion protein, CRAG, which associates with DnaK and GroEL in vivo . Its rapid degradation requires ATP and ClpP, the proteolytic subunit of protease Ti (Clp) . However, this process is not reduced in strains lacking the complementary ATPase subunit, ClpA, or its homologs, ClpB and ClpX . At 37 degrees C, but not at 42 degrees C, protease La also contributes partially to CRAG degradation . Nevertheless, CRAG is not degraded in cell-free extracts or upon incubation with ClpP or protease La . We tested whether the chaperones associated with CRAG might be involved in its degradation . CRAG breakdown was accelerated 2-3-fold in strains with high levels of heat-shock proteins (hsps), i.e . in those that overproduce the hsp transcription factor (sigma 32) or carry a dnaK deletion . A similar stimulation of proteolysis was observed in cells overproducing GroEL or both GroEL and GroES; in these cells, more CRAG was associated with GroEL than in the wild type . In a temperature-sensitive groEL44 mutant at the nonpermissive temperature, CRAG breakdown was accelerated, and more CRAG was found complexed with GroEL . However, in a temperature-sensitive groES mutant, CRAG was completely stable at the nonpermissive temperature and accumulated bound to GroEL . These findings indicate that the association of CRAG with GroEL is a rate-limiting step in CRAG degradation, which also requires a subsequent action of GroES . We propose that if the hsp60/hsp10 chaperonins fail to catalyze the proper folding of a protein, they can facilitate its rapid degradation. J Biol Chem, 1994 Sep 23, 269(38), 23655 - 60 Epitope mapping and functional characterization of monoclonal antibodies specific for the alpha subunit of Escherichia coli RNA polymerase; Sharif KA et al.; The epitopes have been localized for a set of monoclonal antibodies specific for the alpha subunit of the Escherichia coli RNA polymerase . The antibodies are classified into three groups based on their epitopic assignments . Group 1, mAb 123C2, maps in the N terminus of alpha between amino acids 1 and 23; Group 2 antibodies (mAb 129C4, mAb 124D1 and mAb 121C5) map in the central region between amino acids 190 and 210; Group 3 antibodies (mAb 130B1 and mAb 125C6) map in the C terminus between amino acids 310 and 320 . mAb 130C2 is anomalous since it maps to the N terminus between amino acids 1 and 23 as well as to the C terminus between amino acids 320 and 329 . The antibodies were used to investigate the role of alpha in transcription activation with cAMP receptor protein-dependent promoters . Three antibodies (130C2, 121C5, and 125C6) inhibited cAMP receptor protein-dependent initiation with lac P+ but not with lac UV5 or gal P+ . Inhibition was observed with free RNA polymerase and the closed promoter complex; the preformed open promoter complex was insensitive . Only lac P+ was sensitive to these anti-alpha antibodies supporting the concept that the mode of interaction of RNA polymerase with cAMP receptor protein differs between lac P+ and gal P+. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 65 - 74 Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana; Horn MA et al.; The RLK5 gene of Arabidopsis thaliana encodes a novel receptor-like protein kinase . DNA sequence analysis suggests that the RLK5 protein contains an extracellular domain that has 21 tandemly repeated leucine-rich motifs linked, via a transmembrane hydrophobic region, to a protein kinase catalytic domain that is related to the serine/threonine family of protein kinases . To study the intrinsic biochemical properties of this protein kinase we have expressed the catalytic domain as two different recombinant fusion proteins in Escherichia coli . Both hybrid proteins have similar kinetic properties, autophosphorylate on serine and threonine residues and have significantly greater activity in the presence of Mn2+ than Mg2+ . A lysine to glutamic acid substitution in the catalytic domain of RLK5 results in the catalytically inactive protein RLK5(Cat)K711E . The active RLK5 protein can phosphorylate the inactive K711E protein and the K711E protein can partially inhibit the autophosphorylation of RLK5 . Tryptic cleavage of the autophosphorylated proteins followed by two-dimensional thin layer electrophoresis indicates that several sites in the catalytic domain are phosphorylated. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 55 - 64 Bimodal action of spermine on ribosomal peptidyltransferase at low concentration of magnesium ions; Drainas D et al.; At 6 mM Mg2+, submillimolar concentrations of spermine affect the end-point as well as the kinetic phase of puromycin reaction in a cell-free system from Escherichia coli . When the ternary complex AcPhe-tRNA-poly(U)-ribosome (complex C) is formed in the absence of ribosomal wash (FWR fraction), the final degree of AcPhe-puromycin synthesis is raised from 12% to 60%, as the concentration of spermine increases from zero to 200 microM . However, spermine displays partial noncompetitive inhibition at the kinetic phase of the reaction . The inhibitory effect of spermine is related with its binding to AcPhe-tRNA . When complex C is formed in the presence of FWR fraction, spermine slightly affects the final degree of puromycin synthesis is markedly stimulated by the addition of relatively low concentrations of spermine . Kinetic analysis of the activation phase revealed that spermine attached on a specific site of complex C, acts as a nonessential, partial noncompetitive activator . The stimulatory effect of spermine seems to be due to its interaction with ribosomes . Further additions of spermine cause partial noncompetitive inhibition on the puromycin reaction . This result suggests that complex C possesses a second binding site, responsible for the inhibitory effect of spermine . Both activator and inhibitor sites can be occupied by spermine at the same time. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 179 - 85 Elucidation of the thermal stability of the neutral proteinase II from Aspergillus oryzae; Tatsumi H et al.; The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds . NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C . We analyzed the thermal stability of wild-type NpII and apo NpII . The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis . To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system . The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation . Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 189 - 92 Refolding and release of tubulins by a functional immobilized groEL column; Phadtare S et al.; Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer . These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL . Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP . Similar results were obtained with glutamine synthetase . These data suggest that groEL can function while it is attached to a solid support system. Biochemistry, 1994 Sep 20, 33(37), 11391 - 403 A site-specific endonuclease derived from a mutant Trp repressor with altered DNA-binding specificity; Pfau J et al.; Site-directed mutagenesis was used to construct mutant Trp repressors with each of the 38 possible single amino acid changes of the first 2 amino acid residues (Ile79 and Ala80) in the second "recognition" alpha-helix of the helix-turn-helix DNA-binding motif . Eight of these mutant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepressors . Eleven mutant repressors have extended DNA-binding specificies in vivo, and bind operators which the wild-type repressor cannot . One mutant repressor, Lys79, has a classical altered specificity phenotype in vivo, and binds the wild-type trp operator less well than wild-type repressor, yet binds a mutant operator better than wild-type repressor . A site-specific nuclease was derived from Lys79 repressor by constructing a double-mutant protein with Lys79 and a sole cysteine residue, Cys49, and alkylating this cysteine with a 1,10-phenanthroline-copper adduct . This nuclease has an altered specificity of DNA binding in vitro . When activated by the addition of thiol and hydrogen peroxide, the Lys79 nuclease cleaves operator DNA within its new recognition sequence with high efficiency. Biochemistry, 1994 Sep 20, 33(37), 11307 - 14 Structure and function of Escherichia coli DnaB protein: role of the N-terminal domain in helicase activity; Biswas SB et al.; We have analyzed the contributions of specific domains of DnaB helicase to its quaternary structure and multienzyme activities . Highly purified tryptic fragments containing various domains of DnaB helicase were prepared . Fragment I lacks 14 amino acid (aa) residues from the N-terminal of DnaB helicase . Fragments II and III are 33-kDa C-terminal and 12-kDa N-terminal polypeptides, respectively, of fragment I . The single-stranded DNA-dependent ATPase and DNA helicase activities of DnaB helicase and its fragments were examined in detail . The ATPase activities of native DnaB helicase and fragment I were comparable; however, the ATPase activity of fragment II was somewhat diminished . Unlike the ATPase activity, the DNA helicase activity was totally abolished in fragment II and was not complemented by the addition of equimolar fragment III . Consequently, the N-terminal 17-kDa domain appeared to have an indispensable role in the DNA helicase action, but not in other enzymatic activities . Fragment I had a hexameric structure similar to that observed with DnaB helicase in both size exclusion HPLC (SE-HPLC) and chemical cross-linking studies . SE-HPLC analysis indicated that fragment II had an apparent hexameric form . However, a detailed chemical cross-linking analysis showed that it formed stable dimers but the formation of a stable hexamer was severely impaired . Thus, the N-terminal domain appeared to have a strong influence on the hexamer formation.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Sep 20, 33(37), 11189 - 99 The A-state of barnase; Sanz JM et al.; The acid-induced denaturation of barnase and its mutants has been analyzed to search for partly-folded intermediates . Differential scanning calorimetry of barnase deviates from two-state behavior below pH 4.0 at low ionic strength, with the maximum discrepancy at pH 2.7 . Addition of 200 mM KCl apparently restores the two-state transitions . Thermograms of barnase mutants at pH 2.7 and low ionic strength fall into three classes: alpha, symmetric transitions which fit well to a two-state equilibrium; b, asymmetric transitions indicating deviation from two-state behavior; and c, transitions with an obvious second component . The most distorted thermograms are observed for mutants that had previously been engineered to accumulate at equilibrium the major kinetic folding intermediate state of barnase at neutral pH . Further analysis of these mutants show the existence of complex equilibria on thermal denaturation . Addition of KCl leads to the slow formation of soluble aggregated forms (A-state) which share some of the properties of the "molten globule" state, i.e., significant secondary structure, lack of fixed tertiary structure, and solvent-accessible hydrophobic patches . The far-UV CD spectrum of the A-state can be explained in terms of native-like secondary structure contributions . Kinetic and chemical cross-linking experiments show that dimerization of partly-folded molecules occurs in the transition region, and such dimerization is probably the rate-limiting step in the formation of the A-state in the presence of KCl . As the A-state has been observed clearly so far for only the mutants in which the folding intermediate has been designed to accumulate, we suggest that the A-state would be related to the main folding intermediate state of barnase . The intermediate would be highly stabilized at low pH, and it is prone to self-associate in these conditions. Biochemistry, 1994 Sep 20, 33(37), 11121 - 6 The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178; Olesen K et al.; A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure . We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position . The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues . There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178 . A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue . When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position . The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge . Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Sep 20, 33(37), 11087 - 96 Solution structure of a low molecular weight protein tyrosine phosphatase; Logan TM et al.; Protein tyrosine phosphatases (PTPs) are important enzymes involved in signal transduction, cell cycle regulation, and the control of differentiation . Despite the importance of this class of enzymes in the control of critical cell processes, very little structural information is available for this family of proteins . In this paper, we present the first solution structure of a protein tyrosine phosphatase . This protein is a low molecular weight cytosolic PTP that was initially isolated from bovine heart . The structure that was determined from 1747 NMR-derived restraints consists of a central four-stranded parallel beta-sheet surrounded by four alpha-helices and a short 3(10) helix . The phosphate binding site, identified by chemical shift changes upon the addition of the competitive inhibitors phosphate and vanadate, is in a loop region connecting the C-terminal end of the first beta-strand with the first alpha-helix . Residues in the second, fourth, and fifth alpha-helices and in some of the loop regions connecting the elements of regular secondary structure also contribute to the binding site . The structure determined here is consistent with previous mutagenesis and chemical modification studies conducted on this protein. Biochemistry, 1994 Sep 20, 33(37), 11254 - 63 Dynamic transitions of the transmembrane domain of diphtheria toxin: disulfide trapping and fluorescence proximity studies; Zhan H et al.; Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin's T (transmembrane) domain into the membrane . The T domain, consisting of nine alpha-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized . To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramolecular disulfide bridge linking the buried apolar hairpin (TH8-TH9) to one of the other two layers . Both disulfides inhibited exposure of the domain's apolar regions in solution at low pH, as determined by 2-p-toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers . Reduction of the bridges abolished these effects . Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers . Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions . The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenched excimer fluorescence, reflecting the physical separation of the TH8-TH9 hairpin from the other layers upon the T domain's interaction with the bilayer . The results indicate that (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin, (ii) the TH8-TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions. FEBS Lett, 1994 Sep 19, 352(1), 67 - 70 Observation of the Fe-O2 and FeIV=O stretching Raman bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli; Hirota S et al.; Reaction intermediates in dioxygen reduction by the E . coli cytochrome bo-type ubiquinol oxidase were studied by time-resolved resonance Raman spectroscopy using the artificial cardiovascular system . At 0-20 microseconds following photolysis of the enzyme-CO adduct in the presence of O2, we observed the Fe-O2 stretching Raman band at 568 cm-1 which shifted to 535 cm-1 with the 18O2 derivative . These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen-bound hemoglobin and aa3-type cytochrome c oxidase . In the later time range (20-40 microseconds), other oxygen-isotope-sensitive Raman bands were observed at 788 and 361 cm-1 . Since the 781 cm-1 band exhibited a downshift by 37 cm-1 upon 18O2 substitution, we assigned it to the FeIV=O stretching mode . This band is considered to arise from the ferryl intermediate, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (> 100 microseconds) . The 361 cm-1 band showed the 16O/18O isotopic frequency shift of 14 cm-1 similar to the case of bovine cytochrome c oxidase reaction. J Mol Biol, 1994 Sep 16, 242(2), 139 - 49 The activation of Escherichia coli aspartate transcarbamylase by ATP . Specific involvement of helix H2' at the hydrophobic interface between the two domains of the regulatory chains; Xi XG et al.; The regulatory chain of E . coli aspartate transcarbamylase (E.C . 2.1.3.2) is folded into two domains . The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively . The zinc domain ensures the contact with the catalytic chains . The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain . This structural feature mediates the transmission of the ATP regulatory signal . In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process . The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP . The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP . A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains. J Mol Biol, 1994 Sep 16, 242(2), 116 - 29 DNA-binding parameters of the HU protein of Escherichia coli to cruciform DNA; Bonnefoy E et al.; We have previously studied the binding characteristics of the HU protein of Escherichia coli to different linear DNAs . In this work, using gel-retardation and footprint analysis, we studied the specific binding of HU protein to a synthetic cruciform DNA . We have quantified our results in order to precisely define the binding and cooperativity constants of HU protein towards cruciform DNA and compare them to those obtained with linear DNA . We used stringent high-salt conditions versus non-stringent low-salt conditions in order to differentiate the non-specific-protein HU-DNA complexes from the specific, high-salt-resistant complexes . We observe that HU-protein dimers bind specifically to the cruciform DNA with a binding constant K = 2.0 x 10(8) M-1 and a value for the cooperativity constant omega = 1 corresponding to a non-cooperative phenomenon . For the first time we observe a footprint pattern of HU protein bound to DNA using the hydroxyl-radical-footprinting technique on HU-protein-cruciform-DNA complexes . The residues protected by HU protein are localized at and near the junction point but interestingly they are mainly present in two of the four oligonucleotides which constitute the cruciform DNA . These two oligonucleotides are unpaired and opposite each other . These results support a model where two HU-protein dimers specifically bind to two equivalent angles present opposite each other in the four-way-junction-DNA structure with almost no dimer-dimer interactions. J Mol Biol, 1994 Sep 16, 242(2), 107 - 15 Functional map of the alpha subunit of Escherichia coli RNA polymerase . Deletion analysis of the amino-terminal assembly domain; Kimura M et al.; The alpha subunit of Escherichia coli RNA polymerase plays a major role in the assembly of the core enzyme . The amino-terminal two-thirds of the alpha subunit, as far as position 235, are involved in this assembly . To define the site(s) within this region required for core enzyme assembly, we constructed a set of amino-terminal and internal deletion mutants of the rpoA gene . The overexpressed alpha derivatives were purified to apparent homogeneity and examined for their abilities to assemble beta and beta' subunits into active core enzymes in vitro . Among a total of 22 alpha derivatives tested, only four mutants retained the activity form active core enzyme . These mutants had deletions of the extreme amino-terminal residues as far as amino acid residue 30 . The minimum fragment with full activity of the core assembly was alpha(21 to 235), with deletions of 20 amino-terminal and 94 carboxy-terminal amino acid residues . Most of the other mutants appeared to be defective in the formation of stable alpha dimers as analyzed by high-pressure liquid chromatography gel filtration, although some formed self-aggregates . These results, taken together, suggest that the amino-terminal region of the alpha subunit with the core assembly activity is highly structured, and any deletion within this domain disrupts its ordered conformation . Deletions of the extreme amino-terminal region did not affect transcription activation by CRP at the lacP1 promoter or by OmpR at the ompC promoter. Science, 1994 Sep 16, 265(5179), 1699 - 701 iaglu, a gene from Zea mays involved in conjugation of growth hormone indole-3-acetic acid; Szerszen JB et al.; Plants contain most of the growth hormone indole-3-acetic acid (IAA) in conjugated forms believed to be inactive in promoting growth . The iaglu gene, which controls the first step in the biosynthesis of the IAA conjugates of Zea mays, encodes (uridine 5'-diphosphate-glucose:indol-3-ylacetyl)-beta-D-glucosyl transferase . Protein synthesized by Escherichia coli that contained cloned 1-O-beta-D-indol-3-ylacetyl-glucose complementary DNA (cDNA) was catalytically active . The predicted amino acid sequence of the cDNA was confirmed by amino-terminal sequencing of the purified enzyme . Homologous nucleotide sequences were found in all plants tested . The blockage or enhancement of iaglu expression may permit regulation of plant growth. J Biol Chem, 1994 Sep 16, 269(37), 23171 - 6 Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit; Davis R et al.; Mammalian ribonucleotide reductase is a heterotetramer formed by the two non-identical homodimers proteins R1 and R2 . We have succeeded in expressing the 90-kDa mouse R1 protein in Escherichia coli in an active, soluble form using the T7 RNA polymerase pET vector system . To avoid inclusion bodies, the bacteria were grown at 15 degrees C with minimal concentration of the inducer isopropyl-1-thio-beta-D-galactopyranoside . After a rapid purification procedure, approximately 20 mg of pure R1 protein were obtained per liter of bacterial culture . The concentrated R1 protein solution had a pinkish red color . Spectroscopy in combination with iron and labile sulfur analyses demonstrated that the color originated from an iron-sulfur complex . However, all attempts to demonstrate a function of this complex have been inconclusive . A comparison of the recombinant R1 protein with the corresponding protein purified from calf thymus showed no evidence for glycosylation . Circular dichroism spectroscopy indicated an alpha-helical content of 50% . A flexible COOH-terminal tail of 7 residues in the R2 protein was earlier shown to be essential for binding to the R1 protein . Using a peptide protection assay and photoaffinity labeling, we now show that the R2 protein tail interacts with a region close to the carboxyl terminus of the R1 protein. J Biol Chem, 1994 Sep 16, 269(37), 23150 - 5 The catalytic subunit of bovine brain platelet-activating factor acetylhydrolase is a novel type of serine esterase; Hattori M et al.; Platelet-activating factor (PAF) acetylhydrolase is the key enzyme in PAF inactivation . We recently purified one isoform of the enzyme from a bovine brain soluble fraction and revealed it as a heterotrimeric enzyme consisting of 29-, 30-, and 45-kDa subunits . Among them, the 29-kDa subunit possesses an active serine residue since diisopropyl fluorophosphate (DFP), an inhibitor of the enzyme, labeled only this subunit (Hattori, M., Arai, H., and Inoue, K . (1993) J . Biol . Chem . 268, 18748-18753) . In the current study, we cloned the cDNA for the 29-kDa catalytic subunit . The predicted sequence of 232 amino acids is unique and is not homologous with those of any other proteins reported so far . When transfected into either Escherichia coli or COS7 cells, the cDNA produced PAF acetylhydrolase activity in both types of cells, indicating that this subunit alone is enough for catalysis . The recombinant 29-kDa protein was also inhibited and labeled by DFP . Furthermore, we isolated and sequenced the {3H}DFP-labeled peptide fragment, revealing that Ser47 is the active serine residue . The sequence surrounding it is different from the consensus sequence of the serine esterase family . Interestingly, the sequence of about 30 amino acids located 6 residues downstream from the active serine site exhibits significant homology to the first transmembrane region of the PAF receptor . These data demonstrate that the catalytic subunit of brain PAF acetylhydrolase is a novel type of serine esterase. J Biol Chem, 1994 Sep 16, 269(37), 23025 - 31 Arg-242 is necessary for allosteric coupling of cyclic AMP-binding sites A and B of RI subunit of cyclic AMP-dependent protein kinase; Symcox MM et al.; The functional consequences of Arg-242 to Ser or Lys substitutions in type I alpha regulatory (R) subunits of cAMP-dependent protein kinase were analyzed by using recombinant murine R subunits expressed in Escherichia coli . These mutations arose in cAMP-resistant mutants to S49 mouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding sites . Binding of cAMP to site A of the mutant R subunits could be detected by cAMP-dependent quenching of endogenous tryptophan fluorescence, {3H}cAMP binding to mutant R subunits with the Arg-242 mutations without or with an inactivating mutation in site B, or biphasic dissociation of {3H}cAMP from the mutant subunits at low temperature . The mutations reduced site A affinities by about 25-fold, and the reductions were attributable to accelerated rates of cAMP dissociation . While the presence of cAMP in site A retards dissociation of {3H}cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of {3H}cAMP from site B of the mutant subunits . The predominant effect of the mutations, therefore, was loss of allosteric coupling between the two cAMP-binding sites . A second allosteric interaction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutations at Arg-242. J Biol Chem, 1994 Sep 16, 269(37), 23018 - 24 Cloning and characterization of novel rat and mouse low molecular weight Ca(2+)-dependent phospholipase A2s containing 16 cysteines; Chen J et al.; A novel rat 4.4-kilobase (kb) cDNA encoding a low molecular weight Ca(2+)-dependent phospholipase A2 (PLA2) and its murine homologue were cloned . The rat and mouse cDNA predict a mature protein of 130 amino acids (M(r) = 14, 763) preceded by a 28-amino acid prepro-peptide . The deduced amino acid sequences encode a protein containing 16 cysteines which distinguishes them from both mammalian group I and II PLA2s and the recently described group of mammalian PLA2s containing 12 cysteines . A rat RNA blot hybridized with the rat cDNA exhibited an abundant 2.3-kb and a less abundant 5-kb transcript in testis . When the rat cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium . Conditioned medium optimally hydrolyzed the phospholipids of {1-14C}oleate-labeled Escherichia coli at neutral to alkaline pH with 1-7 mM Ca2+ . In assays with individual substrates, L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol was hydrolyzed more efficiently than L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, or L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine. J Biol Chem, 1994 Sep 16, 269(37), 22964 - 8 Sequence requirements for the biotinylation of carboxyl-terminal fragments of human propionyl-CoA carboxylase alpha subunit expressed in Escherichia coli; Leon-Del-Rio A et al.; Biotin-dependent enzymes play an essential role in the metabolism of all organisms . Their biotinylation is catalyzed by holoenzyme synthetases, which attach a biotin molecule to a specific lysine residue on the apoenzymes . The sequence flanking the biotin binding site is highly conserved among biotin-dependent enzymes . This sequence conservation might be related to the extensive cross-species activity showed by holoenzyme synthetases . In this study, we have expressed carboxyl-terminal fragments of the alpha subunit of human propionyl-CoA carboxylase (PCC-alpha) in Escherichia coli and used site-directed mutagenesis to determine the sequence requirements for biotinylation by the bacterial holoenzyme synthetase . We show that the carboxyl-terminal 67 amino acids of PCC-alpha act as an independent domain in the biotinylation reaction . Mutations that affect several conserved Gly residues and a Pro-Met-Pro sequence near the biotin binding site are critical for biotinylation . Substitution of the amino acids that flank the biotin acceptor Lys residue or elimination of the last 3 amino acids of the PCC-alpha peptides had little or no effect on their biotinylation despite their high conservation in biotin enzymes. J Biol Chem, 1994 Sep 16, 269(37), 22958 - 63 A novel candidate metastasis-associated gene, mta1, differentially expressed in highly metastatic mammary adenocarcinoma cell lines . cDNA cloning, expression, and protein analyses; Toh Y et al.; To understand the genes involved in breast cancer invasion and metastasis, we analyzed a novel candidate metastasis-associated gene, mta1, which was isolated by differential cDNA library screening using the 13762NF rat mammary adenocarcinoma metastatic system . Northern blot analyses showed that the mRNA expression level of the mta1 gene was 4-fold higher in the highly metastatic cell line MTLn3 than in the nonmetastatic cell line MTC.4 . The mta1 gene was expressed in various normal rat organs, especially in the testis, suggesting its essential normal function . The mRNA expression levels of the human homologue of this gene also correlated with the metastatic potential in two human breast cancer metastatic systems . The full-length mta1 cDNA sequence contained an open reading frame encoding a protein of 703 amino acid residues, and sequence analysis by data base homology search indicated that mta1 is a novel gene . The Mta1 protein contained several possible phosphorylation sites, and a proline-rich amino acid stretch at the carboxyl-terminal end completely matched the consensus sequence for the src homology 3 domain-binding motif . Using antibodies raised against glutathione S-transferase-Mta1 fusion protein or a synthetic oligopeptide, Western blots showed that the molecular mass of the Mta1 protein was approximately 80 kDa, and the levels of the Mta1 protein also correlated with the metastatic potential, results similar to those obtained from the Northern analyses . Thus, the novel gene mta1 may encode a molecule that is functional in normal cells as well as in breast cancer invasion and metastasis. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 866 - 73 A modified form of the cytochrome P450scc precursor: a new approach in studies on protein import into mitochondria; Novikova LA et al.; Recombinant DNA was constructed providing hypersynthesis of a hybrid protein with a MRGSH6GIR sequence preceding the NH2-terminus of the bovine cytochrome P450scc precursor (6His-pP450scc) in Escherichia coli cells . A large-scale procedure for isolation and purification of this protein was elaborated . 6His-pP450scc was imported into isolated rat liver mitochondria and processed to the mature-sized form . As a similar procedure can be applied to other proteins the results of this work offer new opportunities in studies of protein import into mitochondria. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 768 - 72 Xenopus laevis ribosomal protein S11: cloning and sequencing of the cDNA and primary structure of the protein; Annesi F et al.; We have cloned a Xenopus laevis cDNA coding for the 40S subunit cytoplasmic ribosomal protein S11 . The nucleotide sequence was determined and the derived amino acid sequence reveals that the protein has 158 amino acid residues and a calculated molecular mass of 18,424 Da . Amino acid sequence comparison with the homologous counterparts from very diverse groups of organisms representing animals (human and rat), fungi (yeast) and plants (maize and Arabidopsis thaliana), shows that this protein is very conserved during evolution . Furthermore, ribosomal protein S11 also shares a significant sequence homology to a set of related proteins: plastid ribosomal protein CS17 from different plants, Escherichia coli ribosomal protein S17 and Halobacterium marismortui ribosomal protein S14. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 1152 - 9 Requirement of the human immunodeficiency virus type 1 env gene sequence for TAR-independent trans activation by Tat from the major immediate-early promoter of murine cytomegalovirus; Kim YS; Previously it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Tat protein mediates induction of the HIV-1 env expression through a TAR-independent manner in heterologous and homologous promoter systems (Kim and Risser, 1993, J . Virol . 67, 239; Kim and Panganiban, 1993, J . Virol . 67, 3739) . To further explore the transactivation of HIV-1 env gene, I examined expression of the env, the bacterial CAT, and the firefly luciferase genes from a heterologous promoter, the major immediate-early promoter (MIEP) of murine cytomegalovirus (MCMV) . Here we show that Tat augments gene expression from the MCMV MIEP only when linked to the env gene . Surprisingly, in contrast to the expression from an HIV-1 LTR lacking the TAR element, TAR-independent transactivation of env gene expression from MCMV MIEP did not require the full length Tat protein . In addition, deletion of the previously identified cis-acting Tat-responsive element in env did not affect Tat transactivation of the env gene expression . Thus, there are multiple distinct elements that mediate Tat responsiveness in the absence TAR. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 1140 - 5 Stoichiometry of Sulfolobus ribosomal protein L12e in 50S subunits determined by quantification of immunoblots; Casiano CA et al.; A monoclonal antibody reactive with Sulfolobus solfataricus acidic ribosomal protein SsoL12e was prepared and employed to determine the stoichiometry of this protein in 50S ribosomal subunits by quantification of chloronaphthol-stained protein bands from immunoblots . Approximately four copies of SsoL12e were detected per 50S ribosome . This finding extends previous studies demonstrating the involvement of this protein in a multimeric protein complex in the ribosomal factor binding domain of Sulfolobus and strengthens the concept that this structural motif is a highly conserved and presumably critical feature of the ribosome. Gene, 1994 Sep 15, 147(1), 153 - 4 Isolation and sequencing of a cDNA encoding the hypoxanthine-guanine phosphoribosyltransferase from Toxoplasma gondii; Vasanthakumar G et al.; A hypoxanthine-guanine phosphoribosyltransferase-encoding gene (HGPRT) from Toxoplasma gondii has been isolated and sequenced . The identity of the enzyme was determined by sequence comparison and complementation in a mutant Escherichia coli. Gene, 1994 Sep 15, 147(1), 149 - 50 Purification of the Escherichia coli integration host factor (IHF) in one chromatographic step; Filutowicz M et al.; The integration host factor (IHF) participates in a diverse array of DNA transactions such as replication, recombination and gene expression . We describe a fast and very efficient isolation procedure which yields highly purified IHF in one chromatographic step. Blood, 1994 Sep 15, 84(6), 1887 - 95 Sustained high levels of circulating chaperoned interleukin-6 after active specific cancer immunotherapy; May LT et al.; In a phase 1 study of recombinant interleukin-6 (rIL-6) in patients with advanced solid tumors (n = 15), we discovered that the endogenous IL-6 levels, in pretreatment plasma or serum samples, were distributed into two groups . One set of patients (designated "type 1"; n = 9) was characterized by low plasma IL-6 levels (48 to 1,700 pg/mL) as measured using enzyme-linked immunosorbent assays (ELISA) for IL-6 . In the second set of patients (designated "type 2"; n = 6), IL-6 ELISAs showed high levels of plasma IL-6 (50 to 600 ng/mL) . Neither group had detectable B9 hybridoma cell growth factor activity associated with the IL-6 in their pretreatment plasma or serum . Plasma C-reactive protein (CRP) levels were markedly elevated in type II patients suggesting that the circulating IL-6 was biologically active in vivo . In both groups of patients there was a small but significant increase in B9 activity in the plasma within three hours after rIL-6 administration (n = 5) . Gel filtration profiles showed that circulating IL-6 in type 1 patients, 15 to 120 minutes after rIL-6 administration was of approximate mass 20 to 40 kD, whereas in type 2 patients, the IL-6 before and after exogenous rIL-6 administration was indistinguishable and was of an approximate mass of 200 kD . IL-6 immunoaffinity purification of the 200 kD complexes showed these to contain multiple isoforms of IL-6 (14 to 31 kD) and the soluble IL-6 receptor (sIL-6R; 50 to 55 kD) . A distinguishing clinical history was that all of the type 2 patients had been actively immunized with an anti-idiotypic monoclonal antibody (MoAb) (MK2-23) 3 to 12 months before initiation of this study for advanced melanoma . An analysis of the plasma IL-6 content in other melanoma patients (n = 16) during antiidiotypic MoAb immunization indicated that marked (up to 600 ng/mL) and sustained (several months) elevations of circulating "chaperoned" IL-6 were induced by active immunization regimens. Nature, 1994 Sep 15, 371(6494), 264 - 7 Context is a major determinant of beta-sheet propensity; Minor DL Jr et al.; Residues in beta-sheets occur in two distinct tertiary contexts: central strands, bordered on both sides by other beta-strands, and edge strands, bordered on only a single side by another beta-strand . The delta delta G values for beta-sheet formation measured at an edge beta-strand of the IgG-binding domain of protein G(GB1) are quite different from those obtained previously at a central position in the same protein . In particular, there is no correlation at the edge position with statistically determined beta-sheet-forming preferences . The differences between beta-sheet propensities measured at central and edge beta-strands, delta delta delta G values, correlate with the values of water/octanol transfer free energies and side-chain non-polar surface area for the amino acids . These results strongly suggest that, unlike alpha-helix formation, beta-sheet formation is determined in large part by tertiary context, even at solvent-accessible sites, and not by intrinsic secondary structure preferences. Carbohydr Res, 1994 Sep 15, 262(2), 323 - 34 Structural elucidation of the capsular polysaccharide produced by Escherichia coli O20 : K84 : H26; Whittaker DV et al.; The primary structure of the acidic capsular antigen produced by E . coli K84 was shown by glycose and methylation analysis, and by 1D and 2D 1H and 13C NMR studies of the polysaccharide and an oligosaccharide produced by lithium-ethylenediamine degradation of the polysaccharide, to be comprised of linear hexasaccharide repeating units of the following structure: -->4)alpha-D-GaI pA-(1-->3)-alpha-D-Man p-(1-->4)-beta-D-GIc p-(1-->3)-alpha- D-Glc pNAc-(1-->2)-beta-D-Man p-(1-->3)-beta-D-Man pNAc-(1-->. Biochem Pharmacol, 1994 Sep 15, 48(6), 1105 - 12 Enzymatic phosphorylation and pyrophosphorylation of 2',3'-dideoxyadenosine-5'-monophosphate, a key metabolite in the pathway for activation of the anti-HIV (human immunodeficiency virus) agent 2',3'-dideoxyinosine; Nave JF et al.; 2',3'-Dideoxyadenosine-5'-monophosphate (ddAMP) is a key intermediate in the metabolic pathway involved in the activation of the anti-retroviral agent 2',3'-dideoxyinosine (ddI) to 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) . The potential phosphorylation of ddAMP by adenylate kinase (myokinase) and pyrophosphorylation by the reverse reaction of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase were investigated . Using ATP as phosphate donor, ddAMP was phosphorylated by adenylate kinase with an efficiency of 8.8% of that for AMP, as estimated from the Vmax/Km ratios . In the presence of PRPP, Escherichia coli and rat PRPP synthetases catalysed the pyrophosphorylation of ddAMP with efficiencies of 52 and 35% of that determined for AMP, respectively . Two carbocyclic phosphonate analogues of ddAMP were not substrates of adenylate kinase . Yet, they were pyrophosphorylated by both PRPP synthetases, albeit less efficiently than ddAMP . In vivo, the usual function of PRPP synthetase is to synthesize PRPP from ribose-5-phosphate and ATP . In the forward reaction ddATP proved to be a substrate as efficient as ATP for rat PRPP synthetase . ddATP was also studied as a potential phosphate donor in the reaction catalysed by adenylate kinase with AMP as phosphate acceptor and found to be as efficient as ATP . The relevance of these in vitro results to the in vivo situation is discussed. Biochem J, 1994 Sep 15, 302 ( Pt 3), 881 - 7 Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase; Chapman-Smith A et al.; A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E . coli . The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E . coli biotin ligase . The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin . We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with {3H}biotin, which greatly facilitated development of a purification procedure . The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid . The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase . The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s . The domain was readily produced and purified in sufficient quantities for n.m.r . structural analysis. Biochem J, 1994 Sep 15, 302 ( Pt 3), 837 - 44 Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein; Warren MJ et al.; The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology . Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein . However, this truncated protein was not able to complement E . coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem . Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site . When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin . The CysGA protein on the other hand showed no such coenzyme-dependent activity . Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation . The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation. Biochem J, 1994 Sep 15, 302 ( Pt 3), 813 - 20 Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli; Bauminger ER et al.; Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores . Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation . Mossbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin . Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN) . A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III) . Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex . Mossbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins . Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components . This provides further evidence that the ferritin protein shell can influence the structure of its iron core. Eur J Biochem, 1994 Sep 15, 224(3), 983 - 90 Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius; Gleissner M et al.; The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius . In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol . The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S . acidocaldarius . With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50 degrees C . Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (delta psi, interior negative), and transmembrane pH gradient (delta pH, interior alkaline) at temperatures up to 70 degrees C . The successful functional reconstitution of the cytochrome aa3-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon. Eur J Biochem, 1994 Sep 15, 224(3), 893 - 9 Naturally occurring human glutathione S-transferase GSTP1-1 isoforms with isoleucine and valine in position 104 differ in enzymic properties; Zimniak P et al.; Glutathione S-transferase P1-1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta {Ahmad, H., Wilson, D . E., Fritz, R . R., Singh, S . V., Medh, R . D., Nagle, G . T., Awasthi, Y . C . & Kurosky, A . (1990) Arch . Biochem . Biophys . 278, 398-408} . In the present report, the enzymic properties of these two proteins are compared . {I104}glutathione S-transferase P1-1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated to replace Ile104 by Val104, and {V104}glutathione S-transferase P1-1 was expressed and isolated as described for {I104}glutathione S-transferase P1-1 . The two enzymes differed in their specific activity and affinity for electrophilic substrates (KM values for 1-chloro-2,4-dinitrobenzene were 0.8 mM and 3.0 mM for {I-104}glutathione S-transferase P1-1 and {V-104}glutathione S-transferase P1-1, respectively), but were identical in their affinity for glutathione . In addition, the two enzymes were distinguishable by their heat stability, with half-lives at 45 degrees C of 19 min and 51 min, respectively . The resistance to heat denaturation was differentially modulated by the presence of substrates . These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate-binding site, and may also influence activity by interacting with residues directly involved in substrate binding. Eur J Biochem, 1994 Sep 15, 224(3), 797 - 802 Elongation on the amino-terminal part of stefin B decreases inhibition of cathepsin H; Jerala R et al.; Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino-terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily . The mutant proteins were expressed in Escherichia coli and purified to homogeneity . Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H . Inhibition of both amino-terminal elongated stefin B mutants was decreased particularly for cathepsin H . A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed . The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases . Variable regions were selected from fragments of other proteins from the protein data base . We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes . This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H . We conclude that the amino-terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H. EMBO J, 1994 Sep 15, 13(18), 4412 - 20 Active site of the replication protein of the rolling circle plasmid pC194; Noirot-Gros MF et al.; Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids . The effect of mutations was examined by an in vivo activity test . Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays . We conclude that all three amino acids have a catalytic role . Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases . We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate . The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step. Nature, 1994 Sep 15, 371(6494), 261 - 4 Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy; Chen S et al.; Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis . Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4) . GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity . Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations . Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown . Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL . Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees . GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate. J Physiol, 1994 Sep 15, 479 ( Pt 3), 441 - 9 Alteration of the physiological responses to indomethacin by endotoxin tolerance in the rat: a possible role for central vasopressin; Wilkinson MF et al.; 1 . Previous studies suggest that arginine vasopressin (AVP) is released into the ventral septal area (VSA) of the rat brain during the antipyresis induced by the cyclo-oxygenase inhibitor indomethacin . In addition, there is evidence for increased AVP transmission in the VSA of animals having a reduced pyretic response following three intravenous injections of bacterial endotoxin (LPS) (endotoxin tolerant) . Since ventral septal AVP receptors can also become 'sensitized' following exposure to AVP, we questioned whether the antipyretic action of indomethacin would increase, via an action involving central AVP, if this drug were administered into LPS-tolerant rats . 2 . Intraperitoneal indomethacin (7.5 mg kg-1) was effectively antipyretic when administered 2 h after an intravenous challenge with LPS (50 micrograms kg-1) into conscious unrestrained rats . This dose of indomethacin had no effect on the core temperature of non-febrile rats given intravenous 0.9% pyrogen-free saline . 3 . Three intravenous injections of LPS over a period of 3 days resulted in rats that were tolerant to the pyrogenic effects of LPS . When indomethacin was administered 2 h following the third LPS injection, a dose-dependent hypothermia was observed . This effect was age dependent, as profound hypothermia was seen in 8 week but not 20 week old rats . 4 . A mortality rate of 41% (P = 0.02) was observed within 24 h of indomethacin treatment in 8 week old tolerant rats compared with 0% in 8 week old non-tolerant and 20 week old tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS) J Physiol, 1994 Sep 15, 479 ( Pt 3), 433 - 40 Segmental differences in the effects of guanylin and Escherichia coli heat-stable enterotoxin on Cl- secretion in human gut; Kuhn M et al.; 1 . Mucosally added synthetic guanylin and Escherichia coli heat-stable enterotoxin (STa) increased short-circuit current (ISC) across isolated muscle-stripped human intestine in vitro . 2 . Serosal bumetanide inhibited ISC responses indicating that guanylin and STa stimulate electrogenic chloride secretion . 3 . ISC responses were markedly greater in the colon than in the jejunum . 4 . Pretreatment with indomethacin did not significantly alter the effects of guanylin and STa . 5 . Both peptides induced concentration-dependent increases in the cyclic GMP content of human intestinal mucosa in vitro; cyclic AMP levels remained unchanged . 6 . In contrast to ISC responses, increases in cyclic GMP content induced by guanylin and STa were markedly greater in the jejunum than in the colon . 7 . Sodium nitroprusside (SNP) but not human alpha-atrial natriuratic peptide (CDD/ANP(99-126)) increased chloride secretion in human intestine; both agents induced small increases in intestinal cyclic GMP content . 8 . Guanylin, STa and the nitric oxide (NO) donor SNP increased electrogenic chloride secretion across human intestinal mucosa in vitro by stimulation of cyclic GMP . The discrepancy between the effects on chloride secretion and intracellular cyclic GMP content suggest different cellular action sites of guanylin and STa in human small and large intestine. Cytometry, 1994 Sep 15, 18(3), 147 - 55 Evaluation of flow cytometric methods for diagnosis of chronic granulomatous disease variants under routine laboratory conditions; Emmendorffer A et al.; Neutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10 chronic granulomatous disease (CGD)-patients (4 CGD+), and from 18 X-linked carriers for CGD have been tested for their production of H2O2 using staining with dihydrorhodamine 123 and subsequent flow cytometry . Additionally, neutrophils from three patients with myeloperoxidase deficiency were assessed . Cells were activated to produce H2O2 by the phorbol ester phorbol-myristate-acetate (PMA) and by phagocytosis of Escherichia coli bacteria . To evaluate the sensitivity of the method, H2O2-production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured . The results were compared to those from other methods (NBT-testing, cytochrome c-reduction, and especially chemiluminescence) . Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, x = 840 +/- 59 (S.D.), 97% < 890, for pediatric patients . Normal quantitative values also resulted from small blood samples of infants (< 1 year, n = 6, x = 830 +/- 52) . For CGD+ (n = 4) the results were clearly far below the normal range . In indicating decreased production of reactive oxygen intermediates the method was at least as sensitive as lucigenin enhanced chemiluminescence . Cytochrome b558-expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the monoclonal antibody 7D5 . The normal range was 97% > 485, 97% < 680, peak channel fluorescence . We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods. Structure, 1994 Sep 15, 2(9), 853 - 68 High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin; Jeng MF et al.; BACKGROUND: Thioredoxin participates in thiol-disulfide exchange reactions and both oxidized thioredoxin (disulfide form) and reduced thioredoxin (dithiol form) are found under physiological conditions . Previous structural studies suggested that the two forms were extremely similar, although significant functional and spectroscopic differences exist . We therefore undertook high-resolution solution structural studies of the two forms of Escherichia coli thioredoxin in order to detect subtle conformational differences . RESULTS: The solution structures of reduced and oxidized thioredoxin are extremely similar . Backbone structure is largely identical in the two forms, with slight differences in the region of the active site, which includes Cys32 and Cys35 . The side chain sulfur atom of Cys32 is tilted away from that of Cys35 in the reduced form of the protein to accommodate the increase in S-S distance that occurs upon reduction of the disulfide, but the chi 1 angles of the two cysteines remain the same in the two forms . CONCLUSIONS: Only subtle conformational changes occur upon changing the oxidation state of the active site cysteines, including the positions of some side chains and in hydrogen bonding patterns in the active site region . Functional differences between the two forms are probably therefore related to differences in local conformational flexibility in and near the active site loop. Structure, 1994 Sep 15, 2(9), 833 - 8 Use of a purified heterodimer to test negative cooperativity as the basis of substrate inactivation of Escherichia coli thymidylate synthase (Asn177-->Asp); Hardy LW et al.; BACKGROUND: Thymidylate synthase (TS) converts deoxyuridylate to thymidylate, an essential DNA precursor . Replacement of Asn177 with aspartate (Asn177-->Asp) in Escherichia coli TS creates a novel ability to methylate 2'-deoxycytidylate (dCMP) . The dCMP-methylase activity of TS(Asn177-->Asp) is transiently inactivated by reaction with deoxyuridylate and methylene-tetrahydrofolate, the methyl donor . We have tested the possibility that the inactivation is due to negative cooperativity, created in the TS dimer by the Asn177-->Asp mutation . RESULTS: A heterodimeric form of TS, containing one wild type and one Asn177-->Asp active site, was created to test for negative cooperativity . Substrate inactivation still occurred, even with the mutation present at only one active site . CONCLUSIONS: Inactivation of TS(Asn177-->Asp) by deoxyuridylate is not due to negative cooperativity created by the mutation . The 'artificial isozyme' method we have developed for purifying heterodimers away from the progenitor homodimers is generally applicable to other hetero-oligomeric proteins. Structure, 1994 Sep 15, 2(9), 793 - 6 The ribonucleotide reductase jigsaw puzzle: a large piece falls into place; Sjoberg BM; The three-dimensional structure of ribonucleotide reductase protein R1 from Escherichia coli reveals a novel 10-stranded alpha/beta barrel fold . A long loop penetrates the center cavity to assemble the active site cysteine triad. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 9067 - 71 Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli; Hwang DJ et al.; The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro . Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles . To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants . The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications . Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA . U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties . However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein) . If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked . In vivo, E . coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8994 - 8 Specific inhibition of herpes virus replication by receptor-mediated entry of an antiviral peptide linked to Escherichia coli enterotoxin B subunit; Marcello A et al.; Mimetic peptides capable of selectively disrupting protein-protein interactions represent potential therapeutic agents for inhibition of viral and cellular enzymes . This approach was first suggested by the observation that the peptide YAGAVVNDL, corresponding to the carboxyl-terminal 9 amino acids of the small subunit of ribonucleotide reductase of herpes simplex virus, specifically inhibited the viral enzyme in vitro . Evaluation and use of this peptide as a potential antiviral agent has, however, been thwarted by its failure to inhibit virus replication in vivo, presumably because the peptide is too large to enter eukaryotic cells unaided . Here, we show that the nontoxic B subunit of Escherichia coli heat-labile enterotoxin can be used as a recombinant carrier for the receptor-mediated delivery of YAGAVVNDL into virally infected cells . The resultant fusion protein specifically inhibited herpes simplex virus type 1 replication and ribonucleotide reductase activity in quiescent Vero cells . Preincubation of the fusion protein with soluble GM1 ganglioside abolished this antiviral effect, indicating that receptor-mediated binding to the target cell is necessary for its activity . This provides direct evidence of the usefulness of carrier-mediated delivery to evaluate the intracellular efficacy of a putative antiviral peptide. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8885 - 9 Interaction between the aphid transmission factor and virus particles is a part of the molecular mechanism of cauliflower mosaic virus aphid transmission; Schmidt I et al.; Cauliflower mosaic virus (CaMV) aphid transmission factor (ATF or P18) is presumed to interact with both virus particles and vector mouthparts, thereby mediating virus aphid transmission . We developed a protein-protein binding assay and our results clearly show that virus particles bind strongly and specifically to P18 whether P18 was obtained from plants, a baculovirus expression system, or the pGEX-3X Escherichia coli expression system . We overproduced, using the pGEX-3X expression system, various fragments of P18 and thereby demonstrated that the C-terminal 31 amino acid residues are responsible for the interaction . Using PCR-based mutagenesis, 2 amino acid residues essential for interaction were identified . Point substitutions (amino acids 157 from Ile to Asn or 159 from Gly to Ser) were sufficient to abolish the interaction, whereas another mutation (amino acid 158 from Ile to Ser) had no effect on P18 virus binding . We evaluated whether there was a correlation between the ability of P18 to interact with CaMV particles and its biological activity . Aphid transmission assays were carried out and we demonstrated that the loss of the virus binding capacity had a dramatic effect on the ability of P18 to mediate aphid transmission . Thus, our results suggest that binding between P18 and virus particles is likely to be one of the molecular mechanisms involved in CaMV aphid transmission. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8747 - 51 Betadoublet: de novo design, synthesis, and characterization of a beta-sandwich protein; Quinn TP et al.; How an amino acid sequence encodes the information necessary for a protein to adopt a unique tertiary structure remains unresolved . We are addressing this problem by designing "from scratch" protein molecules that will adopt predetermined three-dimensional structures . Based on this strategy, two identical four-stranded beta-sheets were designed to dimerize and form a beta-sandwich protein, called betadoublet . A synthetic gene encoding half the beta-sandwich protein was expressed in Escherichia coli, and the protein was purified to homogeneity . Biophysical characterization of betadoublet in aqueous solution demonstrated that the disulfide formed between the two sheets and that the dimer was a compact unaggregated globular protein, consisting predominantly of beta-sheet and stable to thermal denaturation . It has some backbone amide protons whose exchange is slow enough to be measured by NMR but binds more of the dye 1-anilinonaphthalene-8-sulfonate than a well-folded protein. Biochim Biophys Acta, 1994 Sep 13, 1219(1), 73 - 80 Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates; Wenz C et al.; According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein . However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis . Guided by molecular modelling we have replaced . Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil . We have purified and characterized the resulting N188Q mutant . The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200. Biochim Biophys Acta, 1994 Sep 13, 1219(1), 175 - 8 Cloning and sequencing of the groESL homologue from Porphyromonas gingivalis; Hotokezaka H et al.; The homologue of groESL from Porphyromonas gingivalis was cloned and sequenced . Nucleotide sequencing suggested an operon containing two open reading frames (ORFs) homologous to groESL operon of Escherichia coli . The upstream ORF consisted of 267 bp corresponding to 89 amino acid residues . The downstream ORF consisted of 1635 bp corresponding to 545 amino acid residues. Biochemistry, 1994 Sep 13, 33(36), 10977 - 84 Independent folding of the domains in the hydrophilic subunit IIABman of the mannose transporter of Escherichia coli; Markovic-Housley Z et al.; The active form of the hydrophilic subunit (IIABman) of the mannose transporter of Escherichia coli is a homodimer of two 35-kDa subunits . Each subunit consists of two distinct domains, IIA and IIB, which can be separated by limited trypsin digestion . Separation of tryptic fragments yields monomers of IIB and dimers of IIA, which are active and stable . To test whether the domains fold as independent units, the effects of guanidine hydrochloride (GuHCl) and temperature on the structural stability of the intact IIABman were compared with those of the isolated fragments . Equilibrium GuHCl-induced reversible unfolding, measured by circular dichroism and tryptophan fluorescence, showed a biphasic transition for intact IIABman and monophasic transitions for each isolated fragment . The midpoint transitions of the isolated IIB and IIA fragments (at 1.0 and 2.3 M GuHCl) coincide with the first and second transitions of intact IIABman . Analytical ultracentrifugation studies suggested that dissociation precedes the unfolding of IIA . Thermal unfolding of IIABman, monitored by differential scanning calorimetry, showed two well-separated transitions near 52 and 95 degrees C which corresponded to the midpoint transitions of the isolated IIB and IIA fragments . The combined results demonstrate an independent stepwise unfolding of the domains in IIABman as well as the absence of stabilizing interdomain interactions . The lack of interdomain interactions suggests an unrestricted domain motion . This may play an important role in the phosphoryl transfer reaction which is catalyzed by the binding of IIABman to a phosphoryl carrier protein HPr (via the IIA domain) and to the transmembrane subunits of the mannose transporter (via the IIB domain). Biochemistry, 1994 Sep 13, 33(36), 10944 - 50 Annexin V binding to the outer leaflet of small unilamellar vesicles leads to altered inner-leaflet properties: 31P- and 1H-NMR studies; Swairjo MA et al.; Calcium-dependent binding to phospholipid membranes is closely associated with annexin functional properties . In these studies, 31P- and 1H-nuclear magnetic resonance (NMR) experiments have been performed to study the effects of binding of recombinant rat annexin V to sonicated small unilamellar vesicles (SUVs) . High-resolution 31P-NMR spectra of SUVs containing mixtures of synthetic phosphatidic acid (PA) and phosphatidylcholine (PC) show resolvable resonances corresponding to the inner-leaflet PA, outer-leaflet PA, and PC phosphoryl groups . When annexin binding occurs, the outer-leaflet PA 31P resonance shifts while that of PC is unaffected, consistent with selective binding of the protein to the phosphoryl moiety of the PA component . Further, annexin V binding to membrane outer-leaflet phospholipids has a measurable effect on inner-leaflet phospholipids of intact vesicles . 1H-NMR T1 relaxation measurements of SUVs containing acyl-chain-perdeuterated PC show no effects on the PA hydrocarbon-chain segmental motions upon annexin binding . Circular dichroism measurements indicate that the protein does not undergo a significant conformational change upon binding to the vesicles . The observed NMR changes do not correspond to proton or calcium gradients, nor to lateral segregation of extended patches of homogeneous phospholipids . The combined evidence suggests that selective, peripheral annexin-membrane interactions influence the environment of the inner vesicular surface . The mechanism proposed is a protein-induced change in vesicle morphology that corresponds to reduced curvature. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 9022 - 6 An in vitro polysome display system for identifying ligands from very large peptide libraries; Mattheakis LC et al.; We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes . A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system . Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B . The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection . A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA . After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody . Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM . The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems. Biochemistry, 1994 Sep 13, 33(36), 11040 - 5 An RNA binding site in a tRNA synthetase with a reduced set of amino acids; Kim S et al.; A 30 amino acid helix-loop of known structure on the surface of the C-terminal domain of the class I Escherichia coli methionine tRNA synthetase is essential for methionine tRNA anticodon discrimination . Replacing this 30 amino acid peptide with a previously described sequence containing residues from the wild-type protein imbedded in a sequence matrix of mostly alanines and serines, we used a combinatorial mutagenesis and selection strategy to define residual wild-type residues that are not replaceable with alanine or serine, because they are needed for specific recognition of methionine tRNA . Four were identified, of which three have functional side chains (Asn, Arg, Lys) . These four and a fifth (Trp) that was previously identified are located at the end of the helix and within the loop, lie on the same side of the structure, and span a distance of about 20 A . We conclude that, within the alanine, serine sequence matrix, only a few non-alanine, non-serine residues in the specificity-determining part of the structure are essential. FEBS Lett, 1994 Sep 12, 351(3), 423 - 6 Functional and structural similarity between the X protein of hepatitis B virus and nucleoside diphosphate kinases; De-Medina T et al.; One of the four genes encoded by hepatitis B virus (HBV) is the regulatory 17 kDa protein called HBx (or pX) . HBx is a transcription transactivator of many cellular and viral regulatory elements . We report here that recombinant HBx supports transcription in vitro and has phosphotransfer enzymatic activity . In the presence of EDTA, a phosphoryl-HBx is formed that releases the phosphate residue upon the addition of Mg2+ . This two-step NTP hydrolysis reaction is characteristic of a group of enzymes termed nucleoside diphosphate kinases (NDPKs) . Remarkably, structural similarity between HBx and NDPKs is also evident . Our findings suggest that HBx has evolved from this group of enzymes but acquired additional activities that satisfy the viral needs. FEBS Lett, 1994 Sep 12, 351(3), 303 - 7 Mechanism of gene activation by the Escherichia coli positive regulator, OmpR: a mutant defective in transcriptional activation; Aiba H et al.; The OmpR protein is an activator specific for the E . coli ompC and ompF genes . This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase . In this study we isolated OmpR mutants which were suggested to be defective for transcriptional activation, but not for DNA binding . Two such mutants, that we isolated, have a single amino acid alteration at positions 131 {P131S}, and 179 {P179L}, respectively, of OmpR, comprising 239 amino acids . These altered amino acids in OmpR may be implicated, directly or indirectly, in the presumed RNA polymerase/OmpR interaction that is important for transcriptional activation. Nucleic Acids Res, 1994 Sep 11, 22(18), 3765 - 72 Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis; Devine SE et al.; We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing . A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed . Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles . A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids . Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process . Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure . We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors . Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts . Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis. Nucleic Acids Res, 1994 Sep 11, 22(18), 3793 - 800 Quantitative analysis of RNA cleavage during RNA-directed DNA synthesis by human immunodeficiency and avian myeloblastosis virus reverse transcriptases; DeStefano JJ et al.; We have determined the extent of RNA cleavage carried out during DNA synthesis by either human immunodeficiency virus (HIV) or avian myeloblastosis virus (AMV) reverse transcriptases (RTs) . Conditions were chosen that allowed the analysis of the cleavage and synthesis performed by the RT during one binding event on a given template-primer . The maximum quantity of ribonuclease H (RNase H) sensitive template RNA left after synthesis by the RTs was determined by treatment with Escherichia coli RNase H . RNA cleavage products that were expected to be too short to remain hybridized, less than 13 nucleotides in length, were quantitated . Results showed that HIV- and AMV-RT degraded about 80% and less than 20%, respectively, of the potentially degradable RNA to these short products . Survival of longer, hybridized RNA was not a result of synthesis by a population of RTs that had selectively lost RNase H activity . Using an assay that evaluated the proportion of primers extended versus RNA templates cleaved during primer-extension by the RTs, we determined that essentially each molecule of HIV- and AMV-RT with polymerase also has RNase H activity . The results indicate that although both HIV- and AMV-RTs cleave the RNA template during synthesis, the number of cleavages per nucleotide addition with HIV-RT is much greater . They also suggest that some hybridized RNA segments remain right after the passage of the RT making the first DNA strand . In vivo, these segments would have to be cleaved or displaced in later reactions before second strand DNA synthesis could be completed. Nucleic Acids Res, 1994 Sep 11, 22(18), 3681 - 4 The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516; Bakin A et al.; The number and location of pseudouridine residues in Escherichia coli 16S ribosomal RNA has been determined by a combination of direct and indirect methods . Only one residue was found, at position 516 . This site is at the 5'-end of one of the three most highly conserved long sequences of this RNA molecule . A number of experimental findings have strongly implicated this loop in the fidelity of codon recognition by A-site bound tRNA . By virtue of its location, we suggest that psi 516 may also play a role in maintaining the fidelity of protein synthesis. Cell, 1994 Sep 9, 78(5), 889 - 96 Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding; Blatter EE et al.; Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker . We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2) . Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet) . Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity . Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins. Cell, 1994 Sep 9, 78(5), 877 - 87 An explanation for lagging strand replication: polymerase hopping among DNA sliding clamps; Stukenberg PT et al.; The replicase of E . coli, DNA polymerase III holoenzyme, is tightly fastened to DNA by its ring-shaped beta sliding clamp . However, despite being clamped to DNA, the polymerase must rapidly cycle on and off DNA to synthesize thousands of Okazaki fragments on the lagging strand . This study shows that DNA polymerase III holoenzyme cycles from one DNA to another by a novel mechanism of partial disassembly of its multisubunit structure and then reassembly . Upon completing a template, the polymerase disengages from its beta clamp, hops off DNA, and reassociates with another beta clamp at a new primed site . The original beta clamp is left on DNA and may be harnessed by other machineries to coordinate their action with chromosome replication. Cell, 1994 Sep 9, 78(5), 835 - 43 SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion; Economou A et al.; SecA, the peripheral subunit of E . coli preprotein translocase, alternates between a membrane inserted and a deinserted state as part of the catalytic cycle of preprotein translocation . When SecA is complexed with SecY/E and preprotein, ATP drives a profound conformational change, leading to membrane insertion of a 30 kDa domain of SecA . The inserted domain is protease-inaccessible from the cytosolic side of the membrane, but becomes accessible upon membrane disruption . Concomitant with 30 kDa domain insertion, approximately 20 aminoacyl residues of the preprotein are translocated . Additional ATP, which may be hydrolyzed at the second ATP site of SecA, releases the translocated preprotein and allows the 30 kDa domain to deinsert, whence it can exchange with cytosolic SecA . Thus, SecA is the mobile subunit of an integral membrane transporter, consuming ATP during both the insertion and deinsertion phases of its catalytic cycle while guiding preprotein segments across the membrane. J Mol Biol, 1994 Sep 9, 242(1), 99 - 102 Crystallization of the malonyl coenzyme A-acyl carrier protein transacylase from Escherichia coli; Serre L et al.; The malonyl coenzyme A-acyl carrier protein transacylase, a single polypeptide chain of 358 amino acid residues and a molecular mass of 32 kDa, is a key component of the fatty acid synthase multienzyme complex . The elucidation of its three-dimensional structure will help in the understanding of the molecular basis of the biosynthesis of fatty acids, as well as of polyketides and related biologically active molecules . Three X-ray-quality crystal forms of the Escherichia coli fabD gene product encoding for malonyl coenzyme A-acyl carrier protein transacylase have been obtained using the hanging-drop method and ammonium sulfate as precipitant . Two are tetragonal and each contains two molecules in the asymmetric unit (form I: space group P4(3(1))2(1)2 with a = b = 83.9 A, c = 166.5 A and form II: space group P4 with a = b = 132.64 A, c = 38.85 A), whereas the third form belongs to the hexagonal system and contains one molecule in the asymmetric unit (space group P6(1(5)) with a = b = 68.52 A, c = 117.71 A) . In each case, the diffraction pattern extends to approximately 2.0 A resolution using CuK alpha radiation from a rotating anode source. J Biol Chem, 1994 Sep 9, 269(36), 22614 - 22 Holoenzyme interaction sites in the cAMP-dependent protein kinase . Histidine 87 in the catalytic subunit complements serine 99 in the type I regulatory subunit; Cox S et al.; Two mutations of the catalytic (C) subunit of the cAMP-dependent protein kinase where His87 was changed to Ala and Asp were expressed in Escherichia coli and purified . These mutants were phosphorylated at Thr197 and were catalytically active, although some changes in their kinetic parameters were observed . The most striking differences were in their interaction with the physiological inhibitors . Both mutants were inhibited by protein kinase inhibitor with Ki values below 50 nM . Both mutants were defective in their interaction with the type I regulatory (RI) subunit as measured by (i) the rate of holoenzyme formation with cAMP bound RI-subunit and (ii) the apparent Kd with the cAMP-free RI-subunit . The rate of holoenzyme formation was impaired both in the presence and absence of ATP with the His to Asp mutant showing the greatest effect . The mutant C-subunits were also combined with RI-subunits that contained mutations in the autoinhibitor sequence at Arg94 (P-3) and Ser99 (P + 2) . Complementarity between His87 and Ser99 was established, but not between His87 and Arg94 . Holoenzyme formation with a Ser99-->Lys mutant RI-subunit was less dependent on ATP when combined with either of the C-subunit mutants than when it was combined with the wild-type C-subunit . The apparent Kd values in the presence of ATP for the mutant combinations were also measured . The Ser99-->Lys mutant was compensated for by both His87 mutants . The His87-->Ala C-subunit mutant was unable to form an inhibited holoenzyme complex with a mutant RI-subunit which was defective in cAMP binding to the A-site . This indicated that this R-subunit was defective in C-subunit recognition as well as in cAMP binding . The roles of His87 on the C-subunit and Ser99 and Arg209 on the RI-subunit in R-C interactions are discussed. J Biol Chem, 1994 Sep 9, 269(36), 22586 - 92 Molecular cloning of a protein serine/threonine phosphatase containing a putative regulatory tetratricopeptide repeat domain; Becker W et al.; Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach . One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids) . The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT . Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids) . When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid . As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs . This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis . mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver . It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase. J Biol Chem, 1994 Sep 9, 269(36), 22581 - 5 Properties and structure of spermidine acetyltransferase in Escherichia coli; Fukuchi J et al.; Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold . The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits . The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine . The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively . The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH . By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained . Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E . coli chromosome . E . coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold . The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein . Thus, the predicted molecular mass was 21,756 Da . Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141 . The results suggest that the active center of SAT may be located in the COOH-terminal portion. J Biol Chem, 1994 Sep 9, 269(36), 22557 - 64 C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450; Uhlmann H et al.; The role of the C-terminal region of adrenodoxin was studied by analyzing deletion mutants 4-114 and 4-108 lacking amino acids 1-3 and 115-128 or 109-128, respectively . Absorption spectra of these mutants were found to be identical to that of wild type adrenodoxin . However, EPR and CD studies indicated that the structure of deletion mutants 4-114 and 4-108 differs from that of wild type adrenodoxin . Mutant 4-107, which in addition to residues 109-128 lacks the unique proline 108, showed no EPR spectrum . This indicates that proline 108 plays an essential role for the formation of the iron-sulfur cluster . Deletion of residues 115-128 or 109-128 did not essentially affect adrenodoxin reductase binding as shown by nearly unchanged cytochrome c reduction activity . In a CYP11A1 assay, mutants 4-108 and 4-114 exhibited 3.2- and 5-fold decreased Km values, respectively, whilst the Kd values for CYP11A1 decreased 3- and 1.9-fold, respectively . Additionally, in a CYP11B1 assay, mutants 4-108 and 4-114 showed decreased Km values . Furthermore, the first step of electron transfer to CYP11B1, but not to CYP11A1, was accelerated up to 4.5-fold by the adrenodoxin mutants . The results suggest that the C-terminal peptide of adrenodoxin, especially proline 108, affects the structural integrity of the iron-sulfur cluster and that electron donation from adrenodoxin to CYP11A1 and CYP11B1 is determined at least in part by different features of the cytochromes. J Chromatogr A, 1994 Sep 9, 679(1), 67 - 83 Purification of recombinant human granulocyte-macrophage colony-stimulating factor from the inclusion bodies produced by transformed Escherichia coli cells; Belew M et al.; Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), produced as inclusion bodies in genetically transformed Escherichia coli cells was purified to homogeneity by a three-step chromatographic procedure involving hydrophobic interaction, ion exchange and gel filtration . Each purification step is reproducible and well suited for process-scale operations . The purification process also leads to a significant decrease in DNA and endotoxin levels in the final product . Of the three gel media used, Phenyl Sepharose 6 FF (high sub) was most effective in reducing the DNA content (by a factor of ca . 2000) while Superdex 75 prep grade was more effective for removing endotoxins (reduction factor ca . 15) . The recovery of purified rhGM-CSF was 35% by enzyme-linked immunosorbent assay and 70% by a biological assay method . The overall purification factor obtained was about 4.6, which is in the range of those reported for recombinant proteins produced in E . coli as inclusion bodies . The purified rhGM-CSF is an acidic protein (pI = 5.4) and has a specific activity of ca . 3.3 x 10(7) units/mg, which is in excellent agreement with that reported for its natural counterpart . Its monomer molecular mass of 14,605, as determined by electrospray mass spectrometry, corresponds exactly to the mass calculated from its cDNA sequence . Its amino acid composition and partial NH2-terminal sequence (up to seventeen residues) are also identical with those reported for this protein . These and other results confirm the identity of the purified rhGM-CSF with its natural counterpart . However, the results also showed that it is apparently heterogeneous from its NH2-terminal side as it is composed of three polypeptides having Met, Ala and Pro as the NH2-terminal residues in which the intact Met analogue accounts for 60% for the mixture . This heterogeneity does not seem to have any biological significance since the specific activity of the purified rhGM-CSF is identical with that of its natural counterpart. J Biol Chem, 1994 Sep 9, 269(36), 22683 - 90 Opposing adenine nucleotide-dependent pathways regulate guanylyl cyclase C in rat intestine; Parkinson SJ et al.; Opposing adenine nucleotide-dependent pathways regulating guanylyl cyclase C (GC-C) in rat intestinal membranes have been identified and characterized . ATP analogues substituted in the 2-position were potent inhibitors of basal and Escherichia coli heat-stable enterotoxin (ST)-stimulated GC-C, independent of the metal cation cofactor present . Inhibition of GC-C was associated with large changes in Vmax but only small changes in the S0.5, suggesting a noncompetitive mechanism . Also, inhibition of GC-C was associated with a concentration-dependent shift from positive to negative cooperativity when manganese served as the cation cofactor . These data support the existence of a noncompetitive allo steric regulatory mechanism mediating adenine nucleotide-dependent inhibition of GC-C . Adenine nucleotides not substituted in the 2-position potentiated the activation of GC-C by ST in intestinal membranes . The potentiating and inhibitory pathways regulating GC-C enzyme activity were separate and distinct . A specific inhibitor (2-chloroadenosine 5'-triphosphate (2ClATP)), was without effect on the potency of a selective activator (adenosine 5'-O-thiomonophosphate (AMPS)) of GC-C . Similarly, AMPS was without effect on the potency of 2ClATP to inhibit GC-C . These data suggest that adenine nucleotide-dependent activation and inhibition are mediated by independent sites which may modulate the second messenger response of GC-C to ST. J Biol Chem, 1994 Sep 9, 269(36), 22524 - 32 Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain; Silverman JA et al.; Entry of the catalytic domain of diphtheria toxin into the cytoplasma of eukaryotic cells depends on insertion of the T (transmembrane) domain into the endosomal membrane, a process triggered by low pH . To probe the mechanism of insertion, we mutated ionizable residues within the helical hairpin region of the T domain . Only three mutations caused significant effects on cytotoxicity, D295K, E349K, and D352K . Each of these represents a substitution of a basic for an acidic residue at the tip of a helical hairpin . Substitution of Lys for Glu349 or Asp352, in the TH8/9 hairpin, reduced toxicity for Vero cells > 100-fold, whereas a Lys substitution for Asp295, one of 3 acidic residues in the TH5/6/7 hairpin, caused a less marked reduction . All three mutations also altered the pH-dependent formation, and/or ion conductance, of channels formed by the toxin in artificial bilayers or the plasma membrane . E349K or D352K did not alter the pH dependence of conformational changes in the toxin occurring near pH 5 . Our findings support the hypothesis that the TH8/9 hairpin inserts into the endosomal membrane after low pH-mediated partial unfolding of the T domain . A positive residue at the tip of this hairpin apparently inhibits insertion and blocks toxin action . The ion-conducting properties of channels formed by selected mutants, described elsewhere, are consistent with this model . The status of the TH5/6/7 hairpin in the integral membrane form of the T domain remains uncertain. Biochemistry, 1994 Sep 6, 33(35), 10815 - 24 Vaccinia virus expresses a novel profilin with a higher affinity for polyphosphoinositides than actin; Machesky LM et al.; We expressed in Escherichia coli the vaccinia virus gene for a protein similar to vertebrate profilins, purified the recombinant viral profilin, and characterized its interactions with actin and polyphosphoinositides . Compared with cellular profilins, this viral profilin has a low affinity (Kd > or = 35 microM) for human platelet actin monomers, a weak effect on the exchange of the nucleotide bound to the actin, and no detectable affinity for poly(L-proline) . Vaccinia profilin binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate in micelles and large unilamellar vesicles, but not to phosphatidylserine or phosphatidylcholine . Kinetic analysis by surface plasmon resonance showed that both vaccinia and amoeba profilins bind slowly to polyphosphoinositides, with association rate constants in the range of (1-4) x 10(4) M-1 s-1 . The higher affinity of vaccinia profilin for polyphosphoinositides (Kd = 0.2-8.5 microM) than for actin or poly(L-proline) and the concentration of vaccinia profilin expressed in infected HeLa cells (approximately 20 microM) suggest that vaccinia profilin binds preferentially to PIP and PIP2 in vivo . Consequently, vaccinia profilin is more likely to influence phosphoinositide metabolism than actin assembly . Expression of 7-105 microM vaccinia profilin in a Saccharomyces cerevisiae profilin null mutant did not rescue the null phenotype, so that the affinity of vaccinia profilin for phosphoinositides alone is insufficient for normal profilin function in yeast. Biochemistry, 1994 Sep 6, 33(35), 10809 - 14 Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition; Dunne PW et al.; The predicted protein kinase activity of the cloned gene product of the human myotonic dystrophy locus has been experimentally verified . Affinity-purified recombinant DM protein kinase became phosphorylated itself and transphosphorylated histone H1 . These activities were not present in the bacterial host cells and were exhibited by DMPK and DMPKH, recombinant proteins which contain the protein kinase domain but exhibit distinct sizes, 43 and 66 kDa, respectively . DMPKH was further purified by velocity sedimentation on sucrose gradients; both activities migrated with the recombinant protein at 41 S, consistent with discrete multimeric particles . Phosphoamino acid analysis showed that threonine (predominantly) and serine were phosphorylated in both DMPKH and histone H1 . Although PKA and PKC are the known types of protein kinase with closest sequence homology to the DM protein kinase domain, purified DMPKH was inhibited by 4 mM but not 0.04-0.4 mM H7 and H8, which inhibit PKA and PKC with Ki's of 0.4-15 microM . Specific inhibitors of other classes of multifunctional serine/threonine protein kinases such as casein kinases I (CKI-7) and II (heparin) and calcium/calmodulin-dependent protein kinase II (KN-62) did not inhibit DMPKH . DMPKH did not phosphorylate membrane-associated phosphoproteins such as acetylcholine receptor or spectrin which are known to be substrates for PKA, PKC, and CKI and -II, respectively . These experimental results suggest that the active center of the recombinant human myotonic dystrophy protein kinase may have properties distinct from the well-studied classes of serine/threonine protein kinases, in contrast to predictions based upon primary structure alone. Biochemistry, 1994 Sep 6, 33(35), 10731 - 42 Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using thre