Biochemistry, 1996 Dec 17, 35(50), 16165 - 73 Effects of alpha-deuteration and of aza and thia analogs of L-tryptophan on formation of intermediates in the reaction of Escherichia coli tryptophan indole-lyase; Sloan MJ et al.; Tryptophan indole-lyase catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate . After the enzyme is mixed with L-tryptophan in the rapid-scanning stopped-flow spectrophotometer, there is an absorbance increase at 505 nm in the pre-steady state attributed to formation of a quinonoid intermediate, which occurs in at least three consecutive first-order phases . Reaction with {alpha-2H}-L-tryptophan results in significant primary kinetic isotope effects on the first two phases, and there is a significant isotope effect on the amplitude of the absorbance increase in the second phase . This result suggests that proton transfer to carbon to form the indolenine intermediate is relatively slow and is probably at least partially rate-determining . Reaction of L-tryptophan in the presence of benzimidazole results in a rapid increase in absorbance in the first phase, followed by a decrease in absorbance in the second phase, with rate constants very similar to those observed without benzimidazole . We have also examined aza and thia analogs of L-tryptophan, with the benzene ring of the indole replaced by pyridine or thiophene . Both 4,5-thiatryptophan and 6,7-thiatryptophan form quinonoid intermediates in the reaction with tryptophan indole-lyase; however, 6,7-thiatryptophan is a better substrate (kcat/K(m) = 32% of L-trp) for tryptophan indole-lyase than is 4,5-thiatryptophan (kcat/K(m) = 4% of L-trp) . Benzimidazole affects the pre-steady-state reaction of 6,7-thiatryptophan in a way similar to L-tryptophan, while benzimidazole does not affect the pre-steady-state reaction of 4,5-thiatryptophan . 4-Aza-, 5-aza-, 6-aza-, and 7-aza-L-tryptophan are all very slow substrates (kcat < 1% of L-trp) for Escherichia coli tryptophan indole-lyase . beta-Indazolyl-L-alanine is a relatively good substrate and exhibits a quinonoid intermediate in its reaction with tryptophan indole-lyase . 6-Aza- and 7-azatryptophan accumulate quinonoid intermediates in the reaction with tryptophan indole-lyase, whereas 4-aza- and 5-azatryptophans do not significantly accumulate quinonoid intermediates, and these latter compounds exhibit very high K(m) values . Addition of benzimidazole does not change the rapid-scanning stopped-flow spectra of 6-aza- and 7-azatryptophan . This suggests that the rate-determining step in the reaction changes depending on the position and type of heteroatom substitution . For 6-aza- and 7-azatryptophan, the very slow rates of elimination may be due to slow C-protonation of the azaindole, while for 4,5-thiatryptophan, the elimination of thienopyrrole is probably slow . Of all analogs examined, 6,7-thiatryptophan is most similar to tryptophan in its reaction with E . coli tryptophan indole-lyase. Biochemistry, 1996 Dec 17, 35(50), 16153 - 64 Rapid formation of the native 14-38 disulfide bond in the early stages of BPTI folding; Dadlez M et al.; Using recombinant variants of BPTI, we have determined the rate constants corresponding to formation of each of the fifteen possible disulfide bonds in BPTI, starting from the reduced, unfolded protein . The 14-38 disulfide forms faster than any of the other 14 possible disulfides . This faster rate results from significantly higher intrinsic chemical reactivities of Cys-14 and Cys-38, in addition to local structure in the reduced protein that facilitates formation of the 14-38 disulfide bond . This disulfide bond is found in native BPTI . Our results suggest that a significant flux of folding BPTI molecules proceed through the one-disulfide intermediate with the 14-38 disulfide bond, denoted {14-38}, that has recently been detected on the BPTI folding pathway . In addition to providing a detailed picture of the early events in the folding of BPTI, our results address quantitatively the effect of local structure in the unfolded state on folding kinetics. Biochemistry, 1996 Dec 17, 35(50), 16116 - 24 Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence . 1 . Tryptophanyl residues of the carrier can be mutated without impairing protein activity; Le Saux A et al.; During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier . To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts . The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus . A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange . The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined . Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity . The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses . When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed . Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al . {(1996) Biochemistry 35, 16125-16131}. Biochemistry, 1996 Dec 17, 35(50), 16077 - 84 Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding; Kiefer H et al.; An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST) . Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop . The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent . It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage . The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements . Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies. Biochemistry, 1996 Dec 17, 35(50), 16009 - 23 Dynamics of ribonuclease H: temperature dependence of motions on multiple time scales; Mandel AM et al.; The temperature dependence of the backbone motions in Escherichia coli ribonuclease HI was studied on multiple time scales by 15N nuclear magnetic spin relaxation . Laboratory frame relaxation data at 285, 300, and 310 K were analyzed using the model-free and reduced spectral density approaches . The temperature dependence of the order parameters was used to define a characteristic temperature for the motions of the backbone N-H bond vectors on picosecond to nanosecond time scales . The characteristic temperatures for secondary structure elements, loops, and the C-terminus are approximately 1000, approximately 300, and approximately 170 K, respectively . The observed variation in the characteristic temperature indicates that the energy landscape, and thus the configurational heat capacity, is markedly structure dependent in the folded protein . The effective correlation times for internal motions do not show significant temperature dependence . Conformational exchange was observed for a large number of residues forming a contiguous region of the protein that includes the coiled coil formed by helices alpha A and alpha D . Exchange broadening in the CPMG experiments decreased with increased temperature, directly demonstrating that the microscopic exchange rate is faster than the pulse repetition rate of 1.2 ms . The temperature dependence of the exchange contributions to the transverse relaxation rate constant shows approximately Arrhenius behavior over the studied temperature range with apparent activation enthalpies of approximately 20-50 kJ/mol . Numerical calculations suggest that these values underestimate the activation barriers by at most a factor of 2 . The present results obtained at 300 K are compared to those reported previously {Mandel, A . M., Akke, M., & Palmer, A . G., III (1995) J . Mol . Biol . 246, 144-163} to establish the reproducibility of the experimental techniques. Biochemistry, 1996 Dec 17, 35(50), 15949 - 61 Interactions of the RecBCD enzyme from Escherichia coli and its subunits with DNA, elucidated from the kinetics of ATP and DNA hydrolysis with oligothymidine substrates; Chamberlin M et al.; Oligothymidines eight nucleotides or longer stimulate ATP hydrolysis by the RecBC and RecBCD enzymes, and they are substrates for the ATP-stimulated nuclease activity of RecBCD . The steady-state kinetics of ATP hydrolysis by the RecBC enzyme are consistent with a single ATPase and DNA binding site . Results with RecBCD and RecBCD-K177Q {an enzyme with a Lys-to-Gln mutation in the ATP binding motif of the RecD subunit {Korangy, F., & Julin, D . A . (1992) J . Biol . Chem . 267, 1727-1732}} indicate that ATP hydrolysis by the RecB subunit is stimulated by pd(T)12 binding to a high-affinity site, while the RecD subunit hydrolyzes ATP stimulated by pd(T)12 binding to a low-affinity site . The site which stimulates RecB has about 50-fold greater affinity for DNA in either RecBCD or RecBCD-K177Q than does the corresponding site in RecBC . The rates of ATP hydrolysis observed for the RecBCD enzyme at low concentrations of pd(T)12 are best explained by a mechanism where the enzyme binds to the DNA and catalyzes multiple rounds of ATP hydrolysis before dissociating . Larger DNA molecules {pd(T)25-30 and poly(dT)} are bound more tightly by RecBCD, are hydrolyzed more rapidly, and are much more effective in stimulating ATP hydrolysis than is pd(T)12 . The results at low ATP concentrations where the nuclease activity is minimal (5 microM) suggest that ATP hydrolysis is stimulated by the DNA ends, but there is no evidence that the RecBCD enzyme moves along these DNA molecules in an ATP-dependent manner under these conditions. EMBO J, 1996 Dec 16, 15(24), 7178 - 87 Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription; Lanchy JM et al.; We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996) . Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes . Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex . Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation . As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme . This processivity change is reminiscent of the transition from initiation to elongation of transcription . Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme. EMBO J, 1996 Dec 16, 15(24), 6899 - 909 Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli; Missiakas D et al.; Heat shock response in Escherichia coli is autoregulated . Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures . A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin . We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin . One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon . HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome . HslU/ClpY is 50% identical to the ClpX protein of E . coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP . We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides . Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP. FEBS Lett, 1996 Dec 16, 399(3), 313 - 6 Mutations in the amino-terminal domain of the human poly(ADP-ribose) polymerase that affect its catalytic activity but not its DNA binding capacity; Trucco C et al.; Poly-ADP ribosylation of nuclear proteins is activated when poly(ADP-ribose) polymerase (PARP), a nuclear zinc-finger enzyme, binds to single-strand DNA breaks . To understand how the signal emerging from its DNA-binding domain (DBD) bound to such breaks is transduced to its catalytic domain, the structure-function relationship of the DBD was investigated . We have used mutagenesis by the polymerase chain reaction (PCR) to generate a random library of PARP mutants . In this work, we describe the identification of catalytically inactive mutants bearing single point mutations, located outside the two zinc fingers in the DBD, that have conserved their full capacity to bind DNA . The results obtained demonstrate that the DNA-dependent activation of PARP requires not only a capacity to bind DNA but also a number of crucial residues to maintain a conformation of the domain necessary to transfer an 'activation signal' to the catalytic domain. FEBS Lett, 1996 Dec 16, 399(3), 307 - 9 Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle; de Gier JW et al.; Targeting of the cytoplasmic membrane protein leader peptidase (Lep) and a Lep mutant (Lep-inv) that inserts with an inverted topology compared to the wild-type protein was studied in Escherichia coli strains that are conditional for the expression of either Ffh or 4.5S RNA, the two components of the E . coli SRP . Depletion of either component strongly affected the insertion of both Lep and Lep-inv into the cytoplasmic membrane . This indicates that SRP is required for the assembly of cytoplasmic membrane proteins in E . coli. FEBS Lett, 1996 Dec 16, 399(3), 283 - 9 Gene synthesis, high-level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin; Prytulla S et al.; A synthetic gene for apoleghemoglobin-a from soybean, optimized for expression in Escherichia coli has been designed and synthesized by a recursive polymerase chain reaction technique . The protein has been expressed with high efficiency and a purification protocol has been developed . The holoprotein is readily reconstituted by the addition of heme . 15N- and 15N,13C-labeled samples were produced and backbone 15N and 13C assignments were determined by 2D and 3D NMR spectroscopy . Comparison of the chemical shifts of 13C(alpha) and 13CO with random coil shifts revealed a pattern of secondary structure which correlates well with the one previously derived from homonuclear NMR data and low-resolution X-ray crystallography. FEBS Lett, 1996 Dec 16, 399(3), 232 - 6 Biosynthetically lipid-modified human scFv fragments from phage display libraries as targeting molecules for immunoliposomes; de Kruif J et al.; A human anti-CD22 single chain (sc) Fv antibody fragment from a synthetic phage antibody display library was biosynthetically lipid-tagged by using Escherichia coli lipoprotein sequences . The purified anti-CD22 scFv lipoprotein was incorporated into liposomes by detergent dilution . Anti-CD22 immunoliposomes were shown to bind specifically in a dose- and time-dependent manner to CD22+ cell lines and CD22+ B-lymphocytes present in freshly isolated samples of blood mononuclear cells . The immunoliposomes were demonstrated to accumulate in intracellular compartments . Biosynthetically lipid-tagged human scFv antibody fragments isolated from phage display libraries may facilitate the construction of immunoliposomes with improved properties. FEBS Lett, 1996 Dec 16, 399(3), 223 - 6 Interaction of 10Sa RNA with ribosomes in Escherichia coli; Tadaki T et al.; 10Sa RNA is a bacterial small stable RNA, in which the 5'- and 3'-end sequences are folded into a tRNA-like structure . The RNA accepts alanine in vitro, and interacts with 70S ribosomes in the cells . In this study, we examined the ribosome-binding properties of Escherichia coli 10Sa RNA in vivo, and found that the aminoacylation ability of 10Sa RNA with alanine is necessary for the binding to 70S ribosomes . 10Sa RNA was also found to bind only to 70S monosomes and not to polysomes . Recently, E . coli 10Sa RNA was suggested to be used as mRNA for tag peptides, which were found to attach to the C-termini of truncated peptides synthesized in vivo . The present results are consistent with the 'trans-translation' model, which has been proposed for tag-peptide synthesis. FEBS Lett, 1996 Dec 16, 399(3), 215 - 9 Expression of active, human lysyl oxidase in Escherichia coli; Ouzzine M et al.; Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin . Human LO was expressed in Escherichia coli . At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates . Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees {3H}lysine-labeled elastin substrate . LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide . The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile . Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria. Eur J Biochem, 1996 Dec 15, 242(3), 712 - 9 Cloning, sequencing, mapping and hyperexpression of the ribC gene coding for riboflavin synthase of Escherichia coli; Eberhardt S et al.; The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6-kb fragment that has been sequenced . The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 213 amino acids with a mass of 23.4 kDa . It was mapped to a position at 37.5 min on the physical map of the E . coli chromosome . The 3' end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane-fatty-acid synthase . This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively . The gene ydhC is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein . The protein specified by ydhB shows sequence similarity to a large family of DNA-binding proteins and probably represents a helix-turn-helix protein . The ydhB gene is directly adjacent to the regulatory gene purR . A 288-bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min . The ribC gene was hyperexpressed in recombinant E . coli strains to a level of about 30% of cellular protein . The protein was purified to homogeneity by chromatography . The specific activity was 26000 nmol.mg-1.h-1 . The protein sediments at a velocity of S20 = 3.8 S . Sedimentation-equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure . The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the C-terminal and N-terminal parts suggesting two structurally similar folding domains. Eur J Biochem, 1996 Dec 15, 242(3), 648 - 56 Two potential indole-3-acetaldehyde dehydrogenases in the phytopathogenic fungus Ustilago maydis; Basse CW et al.; The phytopathogenic basidiomycetc Ustilago maydis produces indole-3-acetic acid (IndCH2COOH) and indole-3-pyruvic acid (Ind-Prv) from tryptophan . Indole-3-acetaldehyde (IndCH2CH2O) is the common intermediate in the conversion of Ind-Prv and tryptamine to IndCH2COOH . We purified an enzyme (Iad1) from U . maydis that catalyzes the NAD(+)-dependent conversion of IndCH2CH2O to IndCH2COOH and isolated corresponding cDNA and genomic clones . The identity of the cDNA clone was confirmed by expression in Escherichia coli and demonstration of enzymatic activity . In U . maydis, iad1-null mutants were generated by gene replacement . The ability to convert IndCH2CH2O to IndCH2COOH was at least 100-fold reduced in U . maydis iad1-null mutants grown in medium with glucose as carbon source . However, the iad1-null mutants were not diminished in their capacity to produce IndCH2COOH from tryptophan, indicating that IndCH2COOH formation from tryptophan apparently proceeds in the absence of IndCH2CH2O dehydrogenase activity under these conditions . Iad1 expression was strongly induced during growth on ethanol while under these conditions iad1-null mutants were unable to grow . This reveals that iad1 is primarily engaged in the conversion of ethanol to acetate . In iad1-null mutants we detected an additional NAD(+)-dependent IndCH2CH2O dehydrogenase activity that was induced during growth on L-arabinose but repressed in the presence of D-glucose . In arabinose-containing medium the conversion of tryptophan to IndCH2COOH was approximately 5-fold reduced in wild-type strains but 10-15-fold reduced in iad1-null mutant strains compared to IndCH2COOH formation in glucose-containing medium . In addition, the formation of Ind-Prv from tryptophan was abolished in wild-type and iad1-null mutant strains . During growth on arabinose, the conversion of tryptamine to IndCH2COOH was strongly favored suggesting that the glucose-repressible IndCH2CH2O dehydrogenase is required to convert IndCH2CH2O derived from tryptamine to IndCH2COOH. Eur J Biochem, 1996 Dec 15, 242(3), 585 - 91 Purification, characterization and subcellular localization of a type-1 ribosome-inactivating protein from the sarcocarp of Cucurbita pepo; Yoshinari S et al.; The flesh of the fruit of Cucurbita pepo contains a type-1 ribosome-inactivating protein (RIP), which we named pepocin . Pepocin was purified to apparent homogeneity by acid fractionation, ion-exchange chromatography and adsorption chromatography . The protein was found to have a molecular mass of 26 kDa and a pI of about 9.9 . It does not contain glycosidic linkages . The protein inhibits protein synthesis in a rabbit-reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 15.4 pM, and depurinates 28S rRNA in the ribosomes of the lysate in a manner identical to that of ricin A-chain and other RIP . The enzyme is also active on wheat-germ ribosomes and on Escherichia coli ribosomes . The sequence of the N-terminal 20 amino acids of the protein reveals a close relationship to other RIP . Immunoelectron-microscopic localization of pepocin in the sarcocarp shows that the protein is predominantly localized in intercellular spaces . In addition, the immunolocalized signals are observed in leaf intercellular spaces. Eur J Biochem, 1996 Dec 15, 242(3), 567 - 75 NMR studies of the Escherichia coli Trp repressor.trpRs operator complex; Evans PD et al.; To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy . We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor . Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor . Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence . The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules . We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex . The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA . This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator. Eur J Biochem, 1996 Dec 15, 242(3), 519 - 28 The recombinant alpha isoform of protein kinase CK1 from Xenopus laevis can phosphorylate tyrosine in synthetic substrates; Pulgar V et al.; The cDNA coding for protein kinase CK1 alpha has been cloned from a Xenopus laevis cDNA library . The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38874 Da . The sequence is identical to that of the human CK1 alpha and to the bovine CK1 alpha, except that it is 12 amino acids longer than the latter protein . Southern blotting with a 264-bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DNA with EcoR1 and Hind3, suggesting that X . laevis possesses a family of related CK1 genes . CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of its characteristics were determined . The recombinant GT-CK1 alpha fusion protein was found to have apparent Km values for ATP (12 microM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 microM) which are similar to those of the rat liver CK1 enzyme . The recombinant CK1 alpha activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80:Tyr20) . This inhibition is competitive and shows an approximate K1 of 5 microM . CK1 alpha can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE . This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine. Eur J Biochem, 1996 Dec 15, 242(3), 499 - 505 Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins; Yoshida N et al.; Fructosyl amino acid oxidase (FAOD), which is active toward model compounds of the glycated proteins in blood, N epsilon-fructosyl N sigma-Z-lysine and N-fructosyl valine, was purified to homogeneity from Aspergillus terreus GP1 . Though the enzyme did not use glycated proteins directly as its substrate, it used glycated human serum albumin (HSA) when HSA was treated with a protease . Linear relationships between both the concentration and the increase in absorbance and the glycation rate of glycated HSA and the increase in absorbance were observed . cDNAs coding for FAODs were cloned from cDNA libraries of A . terreus GP1 and Penicillium janthinellum AKU 3413 . The coding region for both fungal FAODs consisted of 1314 bp encoding 437 amino acids . The sequence of a dinucleotide-binding motif, GXGXXG, was in the deduced N-terminal region and a similar sequence to that the active site of bacterial sarcosine oxidases was found near the C-terminal region of FAOD . The of C-terminal tripeptides SKL and AKL of FAODs from A . terreus and P . janthinellum, respectively, represent typical peroxisomal-targeting signals . Finally, FAOD protein was produced in Escherichia coli transformants in an active form, and at the same level as in the original fungi. Nucleic Acids Res, 1996 Dec 15, 24(24), 4933 - 9 Interaction of the RecA protein of Escherichia coli with single-stranded oligodeoxyribonucleotides; Bianco PR et al.; The RecA protein of Escherichia coli performs a number of ATP-dependent, in vitro reactions and is a DNA-dependent ATPase . Small oligodeoxyribonucleotides were used as DNA cofactors in a kinetic analysis of the ATPase reaction . Polymers of deoxythymidilic acid as well as oligonucleotides of mixed base composition stimulated the RecA ATPase activity in a length-dependent fashion . Both the initial rate and the extent of the reaction were affected by chain length . Full activity was seen with chain lengths > or = 30 nt . Partial activity was seen with chain lengths of 15-30 nt . The lower activity of shorter oligonucleotides was not simply due to a reduced affinity for DNA, since effects of chain length on KmATP and the Hill coefficient for ATP hydrolysis were also observed . The results also suggested that single-stranded DNA secondary structure frequently affects the ATPase activity of RecA protein with oligodeoxyribonucleotides. Nucleic Acids Res, 1996 Dec 15, 24(24), 4845 - 52 Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis; Mizrahi V et al.; The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag . The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity . In accordance with recent predictions {Tabor, S . and Richardson, C.C . (1995) Proc . Natl . Acad . Sci . USA, 92, 6339-6343}, both recombinant forms of the M . tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis . This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design . A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated . Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues. J Physiol, 1996 Dec 15, 497 ( Pt 3), 773 - 9 Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms; Doring F et al.; l . Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport . PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes . In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity . In addition, both transporters show differences in pH dependence and substrate specificity . 2 . As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1 . Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique . 3 . Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters . We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region. Biochem J, 1996 Dec 15, 320 ( Pt 3), 879 - 84 Competitive inhibition of calcineurin phosphatase activity by its autoinhibitory domain; Sagoo JK et al.; Calcineurin (protein phosphatase 2B), a calmodulin- and calcium-dependent serine/threonine phosphatase, appears to be regulated by a C-terminal autoinhibitory domain . A 25 amino acid peptide derived from this domain inhibits calcineurin phosphatase activity in vitro . Here we show that a 97 amino acid fragment of the calcineurin A alpha C-terminus is approx . 8-fold more potent than the shorter peptide in calcineurin inhibition experiments . Mutation of an evolutionarily conserved Asp to Asn, previously shown to disrupt calcium-dependent signalling and calcineurin regulation in T-lymphocytes, greatly reduced inhibition by the autoinhibitory domain in vitro . Kinetic analysis of wild-type and mutated autoinhibitory domains show that both are competitive inhibitors of calcineurin phosphatase activity with Ki values of 5.0 +/- 0.2 microM and 36.0 +/- 3.7 microM respectively . This suggests intrasteric regulation of calcineurin, with the autoinhibitory domains interacting at the active site of the enzyme . The competitive behaviour of the autoinhibitory domains contrasts with the mechanism of calcineurin inhibition by immunosuppressant-immunophilin complexes, which have been shown to bind to calcineurin at a region removed from the active site. Biochem J, 1996 Dec 15, 320 ( Pt 3), 801 - 6 Biochemical characterization and deletion analysis of recombinant human protein phosphatase 2C alpha; Marley AE et al.; The use of protein phosphatase inhibitors has been instrumental in defining the intracellular roles of protein phosphatase 1 (PP1), PP2A and PP2B . Identification of the role of PP2C in vivo has been hampered, in part, by the unavailability of specific inhibitors . In order to facilitate the identification of novel and specific inhibitors of PP2C by random screening of compounds, and to further characterize this enzyme at the molecular level by site-directed mutagenesis and X-ray crystallography, we have expressed active recombinant human PP2C alpha (rPP2C alpha) in Escherichia coli . Biochemical characterization of rPP2C alpha showed that it could hydrolyse p-nitrophenyl phosphate (pNPP) although, in contrast with native PP2C, this was not stimulated by Mg2+ . As with native PP2C, okadaic acid failed to inhibit rPP2C alpha, whereas 50 mM NaF dramatically inhibited its activity . An alignment of the amino acid sequence of AMP-activated protein kinase (AMPK) with those of other serine/threonine protein kinases around the regulatory phosphorylation site (subdomains VII-VIII) revealed a high degree of conservation . Phosphopeptides derived from this region of AMPK and containing the almost invariant threonine (Thr172 in AMPK) were found to be good substrates for rPP2C alpha . We also showed that rPP2C alpha can inactivate AMPK, but only in the presence of Mg2+ . To define the regions of PP2C alpha important for catalytic activity, we expressed a number of truncated proteins based on the sequence and proposed domain structure of the PP2C alpha homologue from Paramecium tetraurelia . Deletion of 75 residues (9 kDa) from the C-terminus appeared to have little effect on the catalytic activity using pNPP, phosphopeptides or AMPK as substrates . This suggests that the residues important in catalysis lie elsewhere in the protein . A further deletion of the C-terminus led to a completely inactive and very poorly soluble protein. Biochem J, 1996 Dec 15, 320 ( Pt 3), 713 - 6 The Cu,Zn superoxide dismutase from Escherichia coli retains monomeric structure at high protein concentration . Evidence for altered subunit interaction in all the bacteriocupreins; Battistoni A et al.; Gel-filtration chromatography experiments performed at high protein concentrations demonstrate that the Cu,Zn superoxide dismutase from Escherichia coli is monomeric irrespective of the buffer and of ionic strength . The catalytic activity of the recombinant enzyme is comparable with that of eukaryotic isoenzymes, indicating that the dimeric structure commonly found in Cu,Zn superoxide dismutases is not necessary to ensure efficient catalysis . The analysis of the amino acid sequences suggests that an altered interaction between subunits occurs in all bacterial Cu,Zn superoxide dismutases . The substitution of hydrophobic residues with charged ones at positions located at the dimer interface of all known Cu,Zn superoxide dismutases could be specifically responsible for the monomeric structure of the E . coli enzyme. Structure, 1996 Dec 15, 4(12), 1401 - 12 The UmuD' protein filament and its potential role in damage induced mutagenesis; Peat TS et al.; BACKGROUND: Damage induced 'SOS mutagenesis' may occur transiently as part of the global SOS response to DNA damage in bacteria . A key participant in this process is the UmuD protein, which is produced in an inactive from but converted to the active form, UmuD', by a RecA-mediated self-cleavage reaction . UmuD', together with UmuC and activated RecA (RecA*), enables the DNA polymerase III holoenzyme to replicate across chemical and UV induced lesions . The efficiency of this reaction depends on several intricate protein-protein interactions . RESULTS: Recent X-ray crystallographic analysis shows that in addition to forming molecular dimers, the N- and C-terminal tails of UmuD' extend from a globular beta structure to associate and produce crystallized filaments . We have investigated this phenomenon and find that these filaments appear to relate to biological activity . Higher order oligomers are found in solution with UmuD', but not with UmuD nor with a mutant of UmuD' lacking the extended N terminus . Deletion of the N terminus of UmuD' does not affect its ability to form molecular dimers but does severely compromise its ability to interact with a RecA-DNA filament and to participate in mutagenesis . Mutations in the C terminus of UmuD' result in both gain and loss of function for mutagenesis . CONCLUSIONS: The activation of UmuD to UmuD' appears to cause a large conformational change in the protein which allows it to form oligomers in solution at physiologically relevant concentrations . Properties of these oligomers are consistent with the filament structures seen in crystals of UmuD'. Experientia, 1996 Dec 15, 52(12), 1069 - 76 Regulated protein degradation in mitochondria; Langer T et al.; Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis . The matrix-localized PIM1 protease, a homologue of the Escherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity . Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria . The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, the m- and the i-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins . In addition to its proteolytic activity, the m-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes . It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes. J Clin Invest, 1996 Dec 15, 98(12), 2683 - 7 Proliferation induced by keratinocyte growth factor enhances in vivo retroviral-mediated gene transfer to mouse hepatocytes; Bosch A et al.; Retroviral gene transfer to liver without prior injury has not yet been accomplished . We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors . This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers . When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced . Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division. FEMS Microbiol Lett, 1996 Dec 15, 145(3), 451 - 5 Allelic divergence at B alpha 1 pheromone receptor genes of Schizophyllum commune; Wendland J et al.; The multiallelic mating type locus B alpha 1 of Schizophyllum commune encodes a pheromone receptor and putative pheromone genes . A comparison of two alleles encoding receptors that share the same specificity B alpha 1 was performed using strains of different geographic origin . The amino acid sequence alignment revealed strong conservation of the largest part of the receptor . Only in the distal C-terminus major amino acid was divergence encountered . This C-terminal region of 117 of the total of 639 amino acids was shown to be unnecessary for function in vivo by transformation experiments. FEMS Microbiol Lett, 1996 Dec 15, 145(3), 315 - 24 A new role for RNase II in mRNA decay: striking differences between RNase II mutants and similarities with a strain deficient in RNase E; Cruz AA et al.; The effect of Escherichia coli ribonuclease II and polynucleotide phosphorylase was analysed on the degradation of Desulfovibrio vulgaris cytochrome c3 (cyc) mRNA . In the absence of these exoribonucleolytic activities, cyc mRNA was stabilised but the two enzymes had a different role in its decay . Surprisingly, a temperature-sensitive mutation in ribonuclease II gave a degradation pattern similar to what had been observed in the absence of endoribonuclease E activity . In an RNase II deletion mutant this was not observed . We propose and verify a model in which the temperature-sensitive ribonuclease II interferes with the action of ribonuclease E. FEMS Microbiol Lett, 1996 Dec 15, 145(3), 309 - 14 Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo; Aron L et al.; A clone containing a 6.4 kb Borrelia burgdorferi chromosomal DNA insert reacted only with sera from patients with Lyme disease and not with any normal human or rabbit sera . Restriction enzyme analysis indicated that this DNA fragment was located on the B . burgdorferi chromosomal map between rpoB and p22A; its direction of transcription was towards p22A . Sequence analysis suggests that LA006 encodes six proteins: three previously described immunodominant lipoproteins of the 39 kDa Bmp protein family, BmpA, BmpB and BmpC; a 51 kDa MgtE magnesium transporter protein; a 16 kDa protein kinase C inhibitor; and a 56 kDa protein with similarity to an uncharacterized Escherichia coli chromosomal open reading frame. Genomics, 1996 Dec 15, 38(3), 264 - 72 Isolation and characterization of GT335, a novel human gene conserved in Escherichia coli and mapping to 21q22.3; Lafreniere RG et al.; As part of efforts to identify candidate genes for disorders mapped to 21q22.3, we have constructed a 405-kb cosmid contig encompassing five tightly linked markers mapping to this region . A subset of these cosmids was used to identify cDNA fragments by the method of hybrid selection . We present here the cDNA sequence of one such gene (GT335) mapping to this region . The gene is expressed as a 1.7-kb transcript predominantly in heart and skeletal muscle, potentially displays alternate splicing, and is predicted to encode a protein 268 amino acids in length . GT335 spans an estimated 13 kb of genomic DNA and is split into seven exons . Five of the six introns conform to the GT . . . AG consensus for intronic splice junctions; the sixth contains nonconventional (AT . . . AC) intronic junctions . We screened this gene for single-basepair mutations using single-strand conformation polymorphism and sequence analysis of both cDNA and genomic DNA from a number of unrelated individuals and have identified several sequence variations, two of which cause conservative amino acid substitutions . This gene is well conserved evolutionarily, with homologs identified in zebrafish and Escherichia coli, suggesting that it plays an important role in basic cellular metabolism. Cancer Res, 1996 Dec 15, 56(24), 5571 - 5 Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine; Edara S et al.; Inactivation of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine renders tumor cells more sensitive to killing by methylating and chloroethylating agents, and O6-benzylguanine is currently undergoing clinical trials for development as an agent to enhance chemotherapy . It has been reported recently that a polymorphism in the human O6-alkylguanine-DNA alkyltransferase gene exists, with about 15% of the population studied having arginine at codon 160 instead of glycine (Y . Imai et al., Carcinogenesis (Lond.), 16: 2441-2445, 1995) . We have studied the effects of mutations of this glycine to arginine, tryptophan, or alanine on the interaction of human alkyltransferase with O6-benzylguanine using direct determination of the amount of activity remaining after incubation with various concentrations of the inhibitor and measurement of the rate of production of {8-3H}guanine from O6-benzyl{8-3H}guanine as assays . These mutations had little effect on the alkyltransferase activity in repairing O6-methylguanine in methylated DNA . Alteration of glycine 160 to tryptophan or alanine slightly increased the sensitivity to O6-benzylguanine (by up to 4-fold) . However, alteration of glycine 160 to arginine drastically reduced the inactivation by O6-benzylguanine with at least a 20-fold increase in the ED50 value and a similar reduction in the production of guanine whether inactivation was carried out in the absence or presence of DNA . These results raise the possibility that a subpopulation of patients may be resistant to O6-benzylguanine and that higher doses or additional alkyltransferase inhibitors capable of inactivating this form of the alkyltransferase will be necessary. Arch Biochem Biophys, 1996 Dec 15, 336(2), 309 - 15 The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli; Rehman A et al.; Isocitrate lyase from Escherichia coli has been expressed in transformed E . coli JE10 cells lacking the isocitrate lyase (icl) gene . After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells . Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively . Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R . Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes . The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis . The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes . Plots of pKm vs pH were different for the wt and H356K variant . Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4 . The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage. Anal Biochem, 1996 Dec 15, 243(2), 234 - 44 Real-time fluorescence assay system for gene transcription: simultaneous observation of protein/DNA binding, localized DNA melting, and mRNA production; Dunkak KS et al.; This article describes the development of an in vitro multicolor fluorescence assay system for studying protein/DNA complex formation, transcription bubble formation, and mRNA production . These studies were accomplished using three different fluorescent spectroscopic probes: rhodamine-labeled DNA (at the 5' position) to monitor protein/DNA complex formation, DNA internally labeled with the base analog 2-aminopurine in place of adenine to monitor transcription bubble formation, and gamma-fluorophore-labeled UTP nucleotide to measure mRNA transcription rates . Combining these three assay systems allows the simultaneous determination of protein/DNA binding, localized DNA melting transitions, and mRNA production at physiological concentrations of reagents (pM-nM) and millisecond timing resolution . The application of this multicolor fluorescence assay to Escherichia coli RNA polymerase reactions (binding, open complex formation, and mRNA production) demonstrates the importance of kinetically coupled events in gene transcription. Mol Gen Genet, 1996 Dec 13, 253(3), 397 - 400 A RecA/RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only; Rashid N et al.; A gene encoding a RecA/RAD51 homologue from a hyperthermophilic archaeon, Pyrococcus sp . KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli . The deduced 210-amino acid sequence was compared to homologues from bacteria (RecA), eukaryotes (RAD51, DMC1) and archaea (RadA) . The entire protein from Pk (Pk-REC) basically corresponds to the essential central domain of its counterparts and lacks the two smaller RecA subdomains at the N- and C-termini . The sequence comparison suggests that Pk-REC represents a common prototype of RecA, RAD51, DMC1 and RadA, with higher enzymatic activity . Recombinant Pk-REC was fully active and complemented the ultraviolet light sensitivity of an E . coli recA mutant strain. Mol Gen Genet, 1996 Dec 13, 253(3), 342 - 52 Isolation of a carbon source-regulated gene from Ustilago maydis; Bottin A et al.; We have isolated a carbon source-regulated gene from the phytopathogenic fungus Ustilago maydis by use of a promoter-probe vector . This gene, called crg1, is strongly induced by L-arabinose and efficiently repressed by D-glucose and D-xylose . The predicted 36.5-kDa mature crg1 gene product lacks similarity to known proteins but is likely to be secreted . Sequences required for regulated expression of a reporter gene are contained within a 3.6-kb fragment upstream of the crg1 gene . The promoter of crg1 fulfils requirements for an efficient controllable gene expression system in U . maydis Mol Gen Genet, 1996 Dec 13, 253(3), 303 - 14 Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei; Ilmen M et al.; Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter . An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of beta-galactosidase expression of a large number of transformants . A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter . Removal of sequences upstream of nucleotide -500 in relation to the initiator ATG abolished glucose repression . Mutation of a single hexanucleotide sequence 5'GTGGGG at nucleotide -720 was sufficient for derepression . This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi . Removal of the glucose repressor site did not affect sophorose induction . Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position -161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box. Mol Gen Genet, 1996 Dec 13, 253(3), 297 - 302 Increase in synthesis and stability of sigma 32 on treatment with inhibitors of DNA gyrase in Escherichia coli; Mizushima T et al.; We report here that in Escherichia coli, the anti-bacterial agent nalidixic acid induces transient stabilization and increased synthesis of sigma 32, accompanied by the induction of heat shock proteins (Dnak and GroEL proteins) . The induction of heat shock proteins, increased synthesis of sigma 32, and stabilization of sigma 32 observed on treatment of wild-type cells with nalidixic acid were not observed in a nalA26 mutant, a strain that is resistant to nalidixic acid as the result of a mutation in the gyrA gene . Not only oxolinic acid, but also novobiocin, whose targets are the A and B subunits of DNA gyrase, respectively, also induced stabilization and increased synthesis of sigma 32 . Thus, inhibition of the activity of DNA gyrase may cause stabilization and increased synthesis of sigma 32, resulting in turn in induction of heat shock proteins. Biochim Biophys Acta, 1996 Dec 13, 1304(3), 245 - 53 Association of lipid A disaccharide synthase with aerobic glycerol-3-phosphate dehydrogenase in extracts of Escherichia coli; Milla ME et al.; Variants of the Escherichia coli UDP-GlcNAc O-acyltransferase (LpxA) and of the lipid A disaccharide synthase (LpxB) containing affinity chromatography tags (C-terminal histidine 8 {H8} tails) were constructed in order to investigate whether or not these enzymes interact with other E . coli proteins . These variants (LpxA-H8 and LpxB-H8) had specific activities in vitro that were similar to wild-type enzymes . Crude extracts made from E . coli cells expressing LpxA-H8 or LpxB-H8 were chromatographed over Ni(2+)-NTA-Agarose, and proteins purifying with the tagged proteins were identified by SDS-PAGE, followed by blotting and N-terminal microsequencing . At high levels of LpxB-H8 expression, two heat-shock proteins (DnaK and GroEL) were associated with the disaccharide synthase, but not with the acyltransferase . Another major protein recovered with LpxB-H8 (both at low and high levels of expression) was the aerobic glycerol-3-phosphate dehydrogenase (GlpD) . The latter interaction was specific, since GlpD did not bind the affinity resin when the affinity tag was present on the UDP-GlcNAc O-acyltransferase (LpxA-H8) . Velocity centrifugation experiments indicated that both wild-type LpxB and GlpD sedimented together under some conditions, but these aggregates were smaller than and distinct from inner membranes . Our findings suggest a possible new mechanism by which the biosynthetic pathways for lipid A and glycerophospholipids may be coordinated. J Mol Biol, 1996 Dec 13, 264(4), 713 - 21 The RecG branch migration protein of Escherichia coli dissociates R-loops; Vincent SD et al.; The RuvAB and RecG proteins of Escherichia coli promote branch migration of Holliday junction intermediates in genetic recombination . Both are structure-specific helicases that unwind and rewind DNA at the junction point . The helicase activities of these proteins were investigated using RNA:DNA hybrid molecules . RuvAB catalyses the unwinding of RNA:DNA partial duplexes of at least 218 bp in a reaction that requires both RuvA and RuvB, ATP and Mg2+ . RecG failed to unwind these substrates even when the duplex region was reduced to 35 bp . In contrast, RecG rapidly removes a 218 nt RNA from an R-loop substrate, whereas RuvAB does not . RecG's ability to dissociate R-loops is correlated with an ability to reduce the copy number of pUC plasmids and other constructs based on the ColE1 replicon . Copy number is reduced severely when the plasmid carries recG+ . RecG is assumed to reduce copy number by interfering with RNA II's ability to form an R-loop at the plasmid origin of replication and prime DNA synthesis . The dissociation of R-loops by RecG is discussed in terms of the functions needed to promote recombination and to prime DNA replication at D-loops formed during the early stages of RecA-mediated recombination. J Mol Biol, 1996 Dec 13, 264(4), 696 - 712 Characterization of a mutant RecA protein that facilitates homologous genetic recombination but not recombinational DNA repair: RecA423; Ishimori K et al.; A recA mutant (recA423; Arg169-->His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated . The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro . The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red- gam- phage as wild-type cells . At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain . The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair . In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity . The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed . These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein . The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal . However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting. Cell, 1996 Dec 13, 87(6), 1123 - 34 Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase; Niu W et al.; At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase . We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain . We further show that the two interactions affect different steps in transcription initiation . Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 612 - 7 Molecular cloning of a novel 120-kDa TBP-interacting protein; Yogosawa S et al.; TATA-binding protein (TBP) is a central component for transcriptional regulation and is a target for various transcription regulators . Using histidine-tagged TBP as a ligand for affinity-purification of proteins bound to TBP, we purified a 120-kD protein, termed TBP-interacting protein 120 (TIP120), from rat liver nuclear extracts . The entire cDNA sequence of TIP120 contained an open reading frame encoding a novel polypeptide of 1230 amino acids . The recombinant TIP120 interacted directly with TBP under a physiological condition in vitro . Immunoprecipitation analysis indicated that TIP120 was associated with TBP in nuclear extracts . Interestingly, the N-terminal region of TIP120 exhibited sequence similarity to that of Drosophila TAF80, which was shown to bind directly to TBP . This novel TBP-binding protein is considered to participate in transcription regulation through the interaction with TBP. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 531 - 5 Poly-L-lysine activates both peptide and ATP hydrolysis by the ATP-dependent HslVU protease in Escherichia coli; Yoo SJ et al.; Hs1VU in E . coli is a new type of ATP-dependent protease composed of two heat shock proteins, the HslU ATPase and the HslV peptidase related to certain beta-type subunits of the 20S proteasome . Here we show that the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin by the HslVU protease can be markedly stimulated by poly-L-lysine, that is known to activate the casein-degrading activity of the 20S proteasome . However, poly-L-lysine showed little or no effect on the peptidase activity of HslV itself . Instead, it stimulated the hydrolysis of ATP by HslU several-fold . Histone that could stimulate the ATPase activity of HslU also increased the rate of the ATP-dependent peptide hydrolysis by HslV, although to a much lesser extent than by poly-L-lysine . Thus, the poly-L-lysine-mediated increase in the ATPase activity of HslU appears to be responsible for the dramatic activation of the ATP-dependent peptide hydrolysis by HslV . These results suggest that, in the reconstituted HslVU complex, the peptide hydrolysis by HslV occurs in a tightly coupled process with the cleavage of ATP by HslU. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 412 - 8 Evidence for GroES acting as a transcriptional regulator; Legname G et al.; Cochaperonins (cpn10) assist chaperonins (cpn60) in promoting folding and assembly of other proteins . Upon expression of Mycobacterium tuberculosis cpn10 in Escherichia coli we have purified a polypeptide which, through amino acid sequencing, was identified as the endogenous E . coli 10K-S protein . Subsequent studies showed that its expression was specifically upregulated upon cloning of different members of the cpn10 family, including GroES, the E . coli cpn10 . Pulse-chase experiments demonstrated that 10K-S is but one of several proteins whose expression is modulated upon cloning of cpn10 . Up-regulation of 10K-S was also observed after exposure of normal cells, but not of groES- mutants, to elevated temperatures (42 degrees C) . This allowed us to define 10K-S as a heat-shock protein (hsp) whose expression is dependent on the production of another hsp, GroES . Northern blot experiments showed that enhanced expression of 10K-S was consequent to increased accumulation of transcripts and that groES- mutants were devoid even of baseline levels of transcripts both at 37 degrees C and after temperature upshift . These results show that GroES, in addition to its established role in assisting protein folding may act as a transcriptional regulator and is likely to play an important role in modulating gene expression particularly in those conditions, like the stress response, in which its production is greatly enhanced. J Biol Chem, 1996 Dec 13, 271(50), 32306 - 14 B cell antigen receptor signaling induces the formation of complexes containing the Crk adapter proteins; Ingham RJ et al.; Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes . We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosine-phosphorylated proteins . We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement . BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk.Cas and Crk.Cbl complexes in the particulate fraction . We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes . Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains . In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G . Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR. J Biol Chem, 1996 Dec 13, 271(50), 32288 - 92 Topological analysis of NhaA, a Na+/H+ antiporter from Escherichia coli; Rothman A et al.; Analysis of the hydropathic profile of the amino acid sequence of NhaA, a Na+/H+ antiporter from Escherichia coli has previously suggested the existence of 11 putative transmembrane segments (Taglicht, D., Padan, E., and Schuldiner, S . (1991) J . Biol . Chem . 266, 11289-11294) . In the present work to test the location of the C terminus, right-side-out and inside-out membrane vesicles were digested with carboxypeptidase B and probed with an antibody raised against a synthetic peptide whose sequence was based on the C terminus sequence . The results demonstrate that the C terminus is facing the cell interior because it is available for digestion only from the inside . Previous evidence from an NhaA-beta-galactosidase fusion to loop 5 of NhaA indicated that this loop is also facing the cytoplasm (Karpel, R., Alon, T., Glaser, G., Schuldiner, S., and Padan, E . (1991) J . Biol . Chem . 266, 21753-21759) and therefore was not consistent with the position of the C terminus in an 11-transmembrane segment model . Therefore, the model was re-evaluated . For this purpose, 10 nhaA'-'phoA gene fusions were constructed and assayed for alkaline phosphatase activity . The results support a 12-transmembrane segment model with the N and C termini located in the cytoplasm . The evidence indicates that two very short segments, 14 and 16 amino acids long, must cross the membrane in an unknown conformation. Gene, 1996 Dec 12, 183(1-2), 259 - 63 A single vector cloning, mutagenesis and expression system; Towler EM et al.; We describe a multipurpose Escherichia coli vector, pOTSf1blue, that can be utilized for high efficiency subcloning, epitope-tagged protein overexpression, authentic protein overexpression and efficient mutagenesis. Gene, 1996 Dec 12, 183(1-2), 137 - 42 Inducible retroviral vectors regulated by lac repressor in mammalian cells; Chang BD et al.; We have developed lac repressor-regulated retroviral expression vectors that are induced by beta-galactosides upon stable transduction in mammalian cells . These vectors, derived from a Moloney-virus-based vector LNCX, contain an internal Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter, coupled with 2 to 4 lac operator sequences and placed in anti orientation relative to the retroviral long terminal repeat (LTR) . Three different vectors were tested in stably infected mass populations of mouse and human cells expressing the lac repressor, in parallel with the constitutively expressed LNCX vector . The highest expression levels from these vectors ranged from 1-4% to 25-33% of the LNCX level, and the induction by beta-galactosides ranged from 6-11-fold to 29-54-fold . These vectors should be suitable for studies requiring efficient gene transfer and regulated expression in mass populations of stably transduced mammalian cells. Mutat Res, 1996 Dec 12, 361(2-3), 165 - 72 A plasmid rescue to investigate mutagenesis in transgenic D . melanogaster; Hersberger M et al.; We present a plasmid rescue from transgenic Drosophila to study spontaneous and mutagen-induced mutations in vivo . Transgenic Drosophila lines were established by transformation with a shuttle vector containing the bacterial lacZ gene as a target for mutagenesis . The target gene can be recovered into bacteria by restriction endonuclease treatment of total genomic DNA, followed by ligation of the recircularized shuttle vectors . The resulting circular plasmids are then transformed back into E . coli lacZ- mutants, where the activity of the lacZ genes is scored on the induction substrate X-Gal . The number of inactivated versus intact lacZ genes directly indicates the mutation frequency . By the described target gene rescue procedure up to 5000 lacZ gene copies can be rescued from one fly routinely . Spontaneous background mutation rates using this system are 2.6 +/- 0.6 x 10(-4) . Treatment of larvae with ethylnitrosourea (ENU) resulted in a dose-dependent increase of the mutation frequency to 4.8 +/- 0.6 x 10(-4) for 0.5 mM and 6.9 +/- 1.2 x 10(-4) for 1 mM ENU, respectively. Biochim Biophys Acta, 1996 Dec 11, 1309(3), 194 - 6 Molecular cloning of a cDNA encoding an antigen which is salt-stably attached to centrosomes; Angiolillo A et al.; A monoclonal antibody (MAB 2A8) was used for expression-cloning of a complete cDNA (1133/5) to a mRNA (3 kb) encoding a murine 76 kDa polypeptide . The N-terminal section of the polypeptide is composed of domains capable to form alpha-helical coiled-coils . Its C-terminus is proline-rich and has characteristics of the Src homology region 3 (SH3) . Affinity-purified antibodies to a recombinant section of the protein show that the antigen is salt-stably associated with the centrosome throughout the cell cycle. J Biotechnol, 1996 Dec 10, 52(2), 127 - 33 Method for increasing the yield of properly folded recombinant human gamma interferon from inclusion bodies; Arora D et al.; A strategy is described for improved refolding and purification of recombinant human gamma interferon (rh-IFN gamma), which could warrant a higher yield and specific activity than reported previously . The optimal conditions of refolding are obtained by addition of a labilizing agent, L-arginine, in the refolding buffer . A 10-fold increase in the yield was observed with 0.5 M L-arginine, compared with renaturation in its absence . By varying renaturation parameters, the conditions that allow functional refolding of approximately 25-30% of the recombinant protein have been standardized . A simple process is also described for the purification of rh-IFN gamma . The purification involves a single-column chromatography on S-Sepharose, after refolding of rh-IFN gamma in arginine containing buffer . This procedure has consistently produced rh-IFN gamma having a purity of at least 97%, the rest being the aggregated form of gamma interferon . The purified protein is a dimer under non-denaturing conditions and has a specific activity of 2 x 10(8) IU mg-1 protein, as measured by viral cytopathic assay. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14509 - 14 Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli; Jayakumar A et al.; We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously . In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site . The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion . The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography . As expected from the coding capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein . The maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells . Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (> 90%) and minimal amounts of stearic and arachidic acids . Similarly, a human FAS cDNA encoding domain I (beta-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and beta-hydroxyacyl dehydratase) was cloned and expressed in E . coli using pMAL-c2 . The expressed fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity (M(r) 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the beta-hydroxyacyl dehydratase . In addition, a human FAS cDNA encoding domains II and III (enoyl and beta-ketoacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E . coli as a fusion protein with thioredoxin and six in-frame histidine residues . The recombinant fusion protein, thioredoxin-human FAS domains II and III, that was purified from E . coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and beta-ketoacyl reductases and the thioesterase . Both the MBP and the thioredoxin-His-tags do not appear to interfere with the catalytic activity of human FAS or its partial activities. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14498 - 502 A general method for determining helix packing in membrane proteins in situ: helices I and II are close to helix VII in the lactose permease of Escherichia coli; Wu J et al.; It was previously shown that coexpression of the lactose permease of Escherichia coli in two contiguous fragments leads to functional complementation . We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximity in situ without the necessity of purifying the permease . After coexpression of the six N-terminal (N6) and six C-terminal (C6) transmembrane helices, each with a single Cys residue, crosslinking was carried out in native membranes and assessed by the mobility of anti-C-terminal-reactive polypeptides on immunoblots . A Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 28 or 29 (helix I), but not with a Cys residue at position 27, which is on the opposite face of helix I, thereby indicating that the face of helix I containing Pro-28 and Phe-29 is close to helix VII . Similarly, a Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 52 or 53 (helix II), but not with a Cys residue at position 54 . Furthermore, low-efficiency crosslinking is observed between a Cys residue at position 52 or 53 and a Cys residue at position 361 (helix XI) . The results indicate that helix VII lies in close proximity to both helices I and II and that helix II is also close to helix XI . The method should be applicable to a number of different polytopic membrane proteins. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14486 - 91 Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases; Srinivasula SM et al.; The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death . The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin . Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the pre-cursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401 . We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA . This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA . These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease . On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14468 - 73 Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination; Hegde SP et al.; The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis . Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO . These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins . Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded . Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins . These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein . Finally, we found that interactions of RecF with RecO are lost in the presence of ATP . We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14452 - 5 Dominant negative inhibition by fragments of a monomeric enzyme; Michaels JE et al.; Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired . By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway . Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose . We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase . Inhibition is fragment-specific, as not all fragments cause inhibition . An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation . This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive . Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14391 - 5 Phosphohistidyl active sites in polyphosphate kinase of Escherichia coli; Kumble KD et al.; In the synthesis of inorganic polyphosphate (polyP) from ATP by polyphosphate kinase (PPK; EC 2.7.4.1) of Escherichia coli, an N-P-linked phosphoenzyme was previously identified as the intermediate . The phosphate is presumed to be linked to N3 of the histidine residue because of its chemical stabilities and its resemblance to other enzymes known to contain N3-phosphohistidine . Tryptic digests of {32P}PPK contain a predominant 32P-labeled peptide that includes His-441 . Of the 16 histidine residues in PPK of E . coli, 4 are conserved among several bacterial species . Mutagenesis of these 4 histidines shows that two (His-430 and His-598) are unaffected in function when mutated to glutamine, whereas two others (His-441 and His-460) mutated to glutamine or alanine fail to be phosphorylated, show no enzymatic activities, and fail to support polyP accumulation in cells bearing these mutant enzymes. Biochemistry, 1996 Dec 10, 35(49), 15896 - 9 Determination of a transmembrane segment using cysteine-scanning mutants of transposon Tn10-encoded metal-tetracycline/H+ antiporter; Kimura T et al.; The metal-tetracycline/H+ antiporter is a bacterial plasma membrane protein belonging to the 12-transmembrane transporter family . The boundaries of membrane-embedded domains can be clearly determined by means of a method involving cysteine-scanning mutants and their reactivity to N-ethylmaleimide . The results for 37 cysteine-scanning mutants around putative transmembrane helix 9 indicate that the residues from 266 to 275 and from 296 to 298 form water extruding loops, while residues from 276 to 295 and from 299 to 302 are embedded in the membrane . This method is generally useful for determination of the transmembrane regions of a polytopic membrane protein. Biochemistry, 1996 Dec 10, 35(49), 15831 - 8 Metal and pH dependence of heptapeptide catalysis by human matrilysin; Cha J et al.; Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refolding of the guanidine hydrochloride solubilized protein . Matrilysin autolytically removes its N-terminal tripeptide Met-Tyr-Ser during the refolding process . The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degrees C . It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala-Leu bond with a kcat of 3.1 s-1 and K(m) of 1.8 x 10(-5) M at pH 7.5, 37 degrees C, values closely similar to those for the matrilysin produced by activation of the Chinese hamster ovary and E . coli-expressed promatrilysin . The properties of this form of matrilysin demonstrate that the propeptide is not essential for proper folding or stability of the enzyme but likely determines the N-terminal amino acid of the mature enzyme . The pH dependence of kcat/K(m) for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0-9.0) and the pKa values obtained are 4.3 and 9.6 at 25 degrees C . The activity is inhibited by several metal binding agents including 1, 10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline . OP inhibits instantaneously by likely forming a transient ternary enzyme.metal.chelator complex . The zinc atom is then removed from the protein in a time-dependent manner . In agreement with the kinetic studies, dialysis in the presence of OP and CaCl2 removes only the catalytic zinc atom . The monozinc enzyme can be reactivated to 90%, 56%, 27%, and 17% of the native activity by addition of zinc, manganese, nickel, and cobalt, respectively . Cadmium, on the other hand, forms an inactive Cd/Zn hybrid . The differences in the chelator accessibility properties of the two zinc sites can thus be exploited to yield metallohybrids of matrilysin. Biochemistry, 1996 Dec 10, 35(49), 15753 - 9 Refined crystal structure of adenylosuccinate synthetase from Escherichia coli complexed with hydantocidin 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin; Poland BW et al.; A crystal structure of adenylosuccinate synthetase from Escherichia coli, complexed with 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin at 100 K, has been refined to an Rfactor of 0.195 against data to 2.6 A resolution . Bond lengths and angles deviate from expected values by 0.012 A and 1.86 degrees, respectively . Lys 16 and backbone amides 15-17 and 42 interact with the phosphates of GDP, while Ser 414, Asp 333, and backbone amides 331 and 416 interact with the base . Mg2+ is octahedrally coordinated . Oxygen atoms from GDP, phosphate, and hadacidin define the equatorial plane of coordination of the Mg2+, while backbone carbonyl 40 and the side chain of Asp 13 are the apical ligands . HPO4(2-) hydrogen bonds with Lys 16, His 41, backbone amides 13, 40, and 224, and the base moiety of the hydantocidin inhibitor . The carboxylate of hadacidin interacts with Arg 303 and Thr 301; its N-formyl group coordinates to Mg2+, and its hydroxyl group hydrogen bonds with Asp 13 . The 5'-phosphate of the hydantocidin inhibitor interacts with Asn 38, Thr 129, and Thr 239 but is approximately 3.5 A from Arg 143 (related by molecular 2-fold symmetry) . The base moiety of hydantocidin 5'-phosphate hydrogen bonds to Gln 224 and participates in a hydrogen-bonded network that includes the phosphate molecule, several water molecules, and Asp 13 . Hydantocidin 5'-phosphate, GDP, HPO4(2-), and Mg2+ may represent a set of synergistic inhibitors even more effective than the combination of IMP, GDP, NO3-, and Mg2+. Biochemistry, 1996 Dec 10, 35(49), 15646 - 53 Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase; Renzoni DA et al.; The interaction of the Fyn SH3 domain with the p85 subunit of PI3-kinase is investigated using structural detail and thermodynamic data . The solution structure complex of the SH3 domain with a proline-rich peptide mimic of the binding site on the p85 subunit is described . This indicates that the peptide binds as a poly(L-proline) type II helix . Circular dichroism spectroscopic studies reveal that in the unbound state the peptide exhibits no structure . Thermodynamic data for the binding of this peptide to the SH3 domain suggest that the weak binding (approximately 31 microM) of this interaction is, in part, due to the entropically unfavorable effect of helix formation (delta S0 = -78 J.mol-1.K-1) . Binding of the SH3 domain to the intact p85 subunit (minus its own SH3 domain) is tighter, and the entropic and enthalpic contributions are very different from those given by the peptide interaction (delta S0 = +252 J.mol-1.K-1; delta H0 = +44 kJ.mol-1) . From these dramatically different thermodynamic measurements we are able to conclude that the interaction of the proline-rich peptide does not effectively mimic the interaction of the intact p85 subunit with the SH3 domain and suggest that other interactions could be important. Biochemistry, 1996 Dec 10, 35(49), 15618 - 25 The ATPase inhibitor protein from bovine heart mitochondria: the minimal inhibitory sequence; van Raaij MJ et al.; The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase . In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield . The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria . Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis . By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47 . The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable . At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution . Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase . However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region . The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit . The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase. FEBS Lett, 1996 Dec 9, 399(1-2), 135 - 9 Toxicity of expanded polyglutamine-domain proteins in Escherichia coli; Onodera O et al.; Five neurodegenerative diseases are caused by proteins with expanded polyglutamine domains . Toxicity of these proteins has been previously identified only in mammals, and no simple model systems are available . In this paper, we demonstrate in E . coli that long polyglutamine domains (59-81 residues) as GST-fusion proteins inhibit growth while smaller glutamine (10-35 residues) or polyalanine (61 residues) domains have no effect . Analogously in humans, polyglutamine repeats less than 35-40 glutamines produce a normal phenotype, while expansion greater than 40 glutamines is always associated with disease . Expression of polyglutamine proteins in E . coli may help identify the molecular mechanism of pathogenesis of CAG trinucleotide repeat diseases and be a useful screen to identify potential therapeutic compound. FEBS Lett, 1996 Dec 9, 399(1-2), 99 - 102 Escherichia coli inorganic pyrophosphatase: site-directed mutagenesis of the metal binding sites; Avaeva S et al.; Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine . Each mutation was found to decrease the k(app) value by approx . 2-3 orders of magnitude . At the same time, the Km values changed only slightly . Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis . All mutant variants have practically the same affinity to Mg2+ as the wild-type pyrophosphatase. FEBS Lett, 1996 Dec 9, 399(1-2), 26 - 8 Subunit a of proton ATPase F0 sector is a substrate of the FtsH protease in Escherichia coli; Akiyama Y et al.; Escherichia coli FtsH is a membrane-bound ATPase with a proteolytic activity against the SecY subunit of protein translocase . We now report that subunit a of the membrane-embedded Fo part of H+-ATPase is another substrate of FtsH . Pulse-chase experiments showed that subunit a is unstable when it alone (without Fo subunits b and c) was oversynthesized and that it is stabilized in the ftsH mutants . Selective and ATP-dependent degradation of subunit a by purified FtsH protein was demonstrated in vitro . These results suggest that FtsH serves as a quality-control mechanism to avoid potentially harmful accumulation of free subunit a in the membrane. FEBS Lett, 1996 Dec 9, 399(1-2), 21 - 5 The reaction of Escherichia coli cytochrome bo with H2O2: evidence for the formation of an oxyferryl species by two distinct routes; Brittain T et al.; We have re-examined the reaction of fast oxidised cytochrome bo with H202 in a stopped-flow spectrophotometer . Monitoring the reaction at 582 nm allows us to observe the formation and decay of a spectroscopically distinct intermediate which accumulates transiently prior to the formation of an oxyferryl species previously characterised in this laboratory (Watmough, N.J., Cheesman, M.R., Greenwood, C . and Thomson, A.J . (1994) Biochem . J . 300, 469-475 {1}) . The reaction shows three distinct phases of which the fast and intermediate phases are bimolecular and show a marked pH dependence . Initially these results appeared incompatible with the report that only one equivalent of H202 is required to generate the oxyferryl species (Moody, A.J . and Rich, P.R . (1994) Eur . J . Biochem . 226, 731-737 {21} . However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used. Biochim Biophys Acta, 1996 Dec 6, 1291(3), 189 - 94 Natural and synthetic betaines counter the effects of high NaCl and urea concentrations; Randall K et al.; Escherichia coli was used as a model system to evaluate a range of betaines for their ability to protect against salt and urea stresses . Betaine structure determined the salt and urea protective effects . Dimethylthetin conferred salt protection similar to glycine betaine, whereas dimethylsulfoniopropionate (DMSP) was less effective than either glycine betaine or dimethylthetin, but similar to propionobetaine (its nitrogen analogue) . Hydrophobic alpha-substituents altered salt tolerance . Valine betaine with an aliphatic side group conferred salt tolerance similar to glycine betaine . Betaines containing phenyl groups (phenylglycine, phenylalanine and N-phenylglycine betaines) did not confer salt protection, growth being similar to, or less than the control (no betaine) . Hydrophobic groups decreased the ability to protect against urea stresses; valine betaine conferred poor urea tolerance . The addition of an hydroxyl group increased the ability of a betaine to protect against urea denaturation . Proline betaine, an effective salt protector, conferred poor urea tolerance . Increasing the charge separation in the betaine molecule decreased the ability to confer urea tolerance . Thiolanium, pyridinium and triethylglycine betaines, with larger cationic functions, conferred no urea tolerance to E . coli. Neurosci Lett, 1996 Dec 6, 220(1), 66 - 8 Neural toxicity of retroviruses; Hurley MJ et al.; Recombinant retroviruses containing the cDNA for human tyrosine hydroxylase-1 and Escherichia coli lacZ gene were used to infect primary foetal ventral mesencephalon and cortical cultures from rat brain . Severe neuronal toxicity resulted 3-4 days after infection, glial cells seemed to be much more resistant . The toxicity was likely to have resulted from an agent present within the virus-containing medium itself, rather than from the retrovirus itself . The results of this study indicate that retroviruses are not suitable vectors for the introduction of tyrosine hydroxylase into primary neuronal cultures. J Mol Biol, 1996 Dec 6, 264(3), 472 - 83 The donor substrate site within the peptidyl transferase loop of 23 S rRNA and its putative interactions with the CCA-end of N-blocked aminoacyl-tRNA(Phe); Porse BT et al.; An RNA region associated with the donor substrate site, located at the base of the peptidyl transferase loop of 23 S rRNA, was subjected to a comprehensive single-site mutational study . Growth phenotypes of Escherichia coli cells were characterized on induction of synthesis of the mutated rRNAs and the mutated ribosomes were tested, selectively, for their capacity to generate peptide bonds under the conditions of the "fragment" assay . Most of the mutants exhibited dominant or recessive lethal growth phenotypes and, in general, defective growth correlated with low activities in peptide bond formation, although exceptions were observed with normal growth and low activities, and vice versa . All these phenotypes are consistent with defects occurring in the structure of the ribosomal donor site and/or the capacity of the donor substrate to enter or leave this site . A compensating base change approach was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA(Phe) and this region of the peptidyl-transferase loop . Single nucleotide substitutions were introduced into the -CCA end of tRNA(Phe) and the ability of the 3'-terminal pentanucleotide fragments to act as donor substrates was examined for ribosomes carrying the different mutated 23 S rRNAs . No evidence was found for the occurrence of Watson-Crick base-pairing interactions . However, the data are consistent with the formation of a Hoogsteen pair between the 3'-terminal adenosine base of the donor substrate and U2585 of the 23 S rRNA. J Mol Biol, 1996 Dec 6, 264(3), 412 - 25 A novel promoter motif for Caulobacter cell cycle-controlled DNA replication genes; Winzeler E et al.; Caulobacter crescentus contains a single chromosome that is replicated once during a defined period in the cell cycle . The onset of replication coincides with the stimulation of transcription of several genes involved in the replication process . Analysis of the C . crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, identified the dnaX transcription start site and showed that activity from the dnaX promoter is stimulated fourfold at the onset of DNA replication . We have identified a conserved sequence motif that is present in the promoter of dnaX and several other genes involved in the replication of DNA, all of which show an induction of transcription at the onset of chromosome replication . Independent mutations in the conserved sequence that lies between the -10 and -35 regions increased transcription, suggesting that a repressor may bind at this site . We propose that the coincident transcriptional activation of several dna genes at the swarmer to stalked cell transition occurs in response to cell cycle regulatory factors, in a manner analogous to the transient transcriptional regulation of flagellar and DNA methylation genes later in the cell cycle. J Mol Biol, 1996 Dec 6, 264(3), 407 - 11 Functional effects of mutating the closing GxA base-pair of a conserved hairpin loop in 23 S ribosomal RNA; Xu W et al.; Recently, Draper and co-workers solved the structure of a hexanucleotide hairpin loop that is conserved in large subunit ribosomal RNAs . (In Escherichia coli, the hexanucleotide consists of nucleotides 1093 to 1098, in the GTPase center of 23 S rRNA.) A major feature of that structure is a G1093xA1098 base-pair that closes the loop . Our laboratory reported previously the isolation of the mutation G1093A and its characterization as a suppressor of UGA mutations and a cause of temperature-conditional lethality . For the work reported here, we asked whether G1093A causes its phenotypes precisely because it is part of the G1093/A1098 base-pair . Using oligonucleotide-directed site-specific mutagenesis, we introduced base substitutions at nucleotides 1093 and 1098 into a plasmid-borne ribosomal RNA operon (rrnB) . Each mutant plasmid was then tested for the two mutant phenotypes, nonsense suppression and temperature-dependent growth inhibition . Our results indicate that mutations at 1093 do not cause the mutant phenotypes because G1093 is part of the G1093xA1098 base-pair . We discuss alternative avenues to the observed mutant phenotypes and, in particular, present a model in which a specific interaction of the loop is involved in peptide chain termination. J Biol Chem, 1996 Dec 6, 271(49), 31749 - 55 Expression in Escherichia coli and refolding of the malonyl-/acetyltransferase domain of the multifunctional animal fatty acid synthase; Rangan VS et al.; A cDNA encoding residues 429-815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant protein refolded in vitro as a catalytically active malonyl-/acetyltransferase . Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein . Replacement of the active site Ser-581 (full-length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity . The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibition was afforded by both malonyl- and acetyl-CoA . Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocarbonate . The rate of acylation of the active site serine residue was also reduced by several orders of magnitude in the His-683 --> Ala mutant . These results indicate that His-683 plays an essential role in catalysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity. J Biol Chem, 1996 Dec 6, 271(49), 31580 - 4 Separable ATPase and membrane insertion domains of the SecA subunit of preprotein translocase; Price A et al.; The SecA subunit of preprotein translocase drives ATP-dependent translocation of preproteins across the bacterial inner membrane concomitant with cycles of membrane insertion and de-insertion (Economou, A., and Wickner, W . (1994) Cell 78, 835-843) . We have identified the membrane-inserting region of SecA as a 30-kDa domain in the C-terminal third of the protein beginning at aminoacyl residue 610 . Limited proteolysis in the absence of translocation ligands indicates that the SecA monomer is composed of two primary structural domains, the 30-kDa membrane-inserting domain and an N-terminal 65-kDa ATPase domain . This limited protease treatment of SecA results in constitutive ATPase activity, indicating that intramolecular constraints between the two domains may play a role in the regulation of ATP hydrolysis by SecA. J Biol Chem, 1996 Dec 6, 271(49), 31549 - 55 Contrasting enzymatic activities of topoisomerase IV and DNA gyrase from Escherichia coli; Ullsperger C et al.; DNA gyrase and topoisomerase IV (Topo IV) have distinct roles as unlinking enzymes during DNA replication despite 40% sequence identity between them . DNA gyrase unlinks replicating DNA by introducing negative supercoils while Topo IV decatenates the two daughter molecules . For this study, we measured the rates of unlinking of various topoisomers of DNA by DNA gyrase and Topo IV . Each enzyme has marked preferences for certain strand-passage reactions . DNA gyrase is a relatively poor decatenase, catalyzing strand-passage events that result in supercoiling at rates several orders of magnitude faster than those causing decatenation . Topo IV, in contrast, decatenates linked circles 10-40 times more quickly than it removes the intramolecular crossings from supercoiled DNA . Supercoiled catenanes are unlinked at an even more increased rate by Topo IV . Thus, the supercoils augment decatenation rather than compete with catenane crossings for their removal . Knot crossings and the crossings of multiply interlinked catenanes are also preferentially removed by Topo IV . This ability of Topo IV to selectively unlink catenated molecules mirrors its key role in decatenation of replicating chromosomes in vivo. J Biol Chem, 1996 Dec 6, 271(49), 31227 - 33 Transit peptides play a major role in the preferential import of proteins into leucoplasts and chloroplasts; Wan J et al.; The in vitro import characteristics of six different precursors of plastid proteins were assessed to determine differences in the protein import pathways of leucoplasts and chloroplasts . Five of these precursor proteins are destined to different subchloroplast sites, and one is a leucoplast stromal precursor protein . The results indicate that some of these precursors can be imported equally into both plastid types and others preferentially into one type of plastid versus the other . The ability of plastids to import different proteins correlates with the in vivo steady state levels of these proteins . Additional differences were also observed in the intraorganellar portion of the translocation pathway for two thylakoidal proteins . The differences in import characteristics were found to be predominantly governed by information in the transit peptides, since attachment of the various transit peptides to different plastid and foreign proteins demonstrated that the import behavior of the proteins is transferable with the transit sequence . These results indicate that the import mechanisms of leucoplasts and chloroplasts are sufficiently different such that the plastids respond differently to the information present in the transit peptides. J Biol Chem, 1996 Dec 6, 271(49), 31196 - 201 FtsH (HflB) is an ATP-dependent protease selectively acting on SecY and some other membrane proteins; Akiyama Y et al.; The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins . SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein) . We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH . We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini . Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (A1) is essential for ATPase activity and in vivo functioning of FtsH . Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro . Mutant proteins without ATPase activity were inactive in proteolysis . A zinc binding motif mutant showed a decreased proteolytic activity . SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation . These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity . FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation. J Biol Chem, 1996 Dec 6, 271(49), 31172 - 8 Heterotetramers of human liver mitochondrial (class 2) aldehyde dehydrogenase expressed in Escherichia coli . A model to study the heterotetramers expected to be found in Oriental people; Wang X et al.; About 50% of the Oriental population have less liver mitochondrial aldehyde dehydrogenase (ALDH2) activity than do other people . It was found that they possessed an enzyme with a lysine at position 487 (E487K) instead of glutamate (Glu487) . We previously found that the Km for NAD of recombinant human and rat E487K enzymes increased more than 150-fold (Farres, J., Wang X., Takahashi, K., Cunningham, S . J . , Wang, T.T., and Weiner, H (1994) J . Biol . Chem . 269, 13854-13860) . Many aldehyde dehydrogenase-deficient people were found to be heterozygous when genotyped for ALDH2 . In this study liver tissue from heterozygous people was analyzed and found to possess mRNAs for both the glutamate and the lysine subunits . Western blot analysis showed that the glutamate subunit was present . The cDNAs for Glu487 and E487K were coexpressed on one plasmid in Escherichia coli, and the enzyme forms were separated from each other by isoelectric focusing to show that heterotetramers were formed . Only one Km value for NAD could be measured with the purified heterotetrameric enzyme that possessed just 16-18% activity of the glutamate homotetrameric enzyme . The E487K homotetramers had 8% specific activity of the Glu487 enzyme . There was no pre-steady state burst of NADH formation with the heterotetramer, a property found with the glutamate enzyme . Similar results were found for the coexpressed rat liver enzyme, except that a higher specific activity, 48%, was obtained . Thus, we conclude that presence of the lysine subunit altered the activity of the glutamate subunit in the heterotetramer to make it function more like an E487K enzyme. J Biol Chem, 1996 Dec 6, 271(49), 31160 - 5 Binding of an N-terminal rhodanese peptide to DnaJ and to ribosomes; Kudlicki W et al.; A peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese was fluorescently labeled with a coumarin derivative at its primary amino group(s) and then purified by high performance liquid chromatography . This peptide interacted with the molecular chaperone DnaJ in the absence of other chaperones and ATP . In the presence of ATP, the molecular chaperone DnaK bound to the DnaJ-peptide complex, but not to the peptide alone . The chaperone GrpE appeared to cause the release of the peptide bound to the ternary complex in the presence of ATP but not in the presence of ADP . This nucleotide apparently stabilized the complex . The peptide also bound to salt-washed Escherichia coli 70 S ribosomes, specifically to 50 S ribosomal subunits, not to 30 S subunits . DnaJ plus DnaK interacted with the peptide on the ribosome . GrpE caused dissociation of the peptide from the ribosome; ATP was required for this reaction . It was inhibited by ADP . A comparable series of chaperone-mediated reactions is assumed to occur with the N-terminal segment of the nascent polypeptide to facilitate its folding on ribosomes. J Biol Chem, 1996 Dec 6, 271(49), 31044 - 8 Negative dominance studies demonstrate the oligomeric structure of EmrE, a multidrug antiporter from Escherichia coli; Yerushalmi H et al.; EmrE, the smallest known ion-coupled transporter, is an Escherichia coli 12-kDa protein 80% helical and soluble in organic solvents . EmrE is a polyspecific antiporter that exchanges hydrogen ions with aromatic toxic cations such as methyl viologen . Since it is many times smaller than the classical consensus 12 transmembrane segments transporters, it was particularly interesting to determine its oligomeric state . For this purpose, a series of nonfunctional mutants has been generated and characterized to test their effect on the activity of the wild-type protein upon mixing . As opposed to the wild type, these mutants do not confer resistance to methyl viologen, ethidium bromide, or a series of other toxicants . Co-expression of each of the nonfunctional mutants with the wild-type protein results in a reduction in the ability of the functional transporter to confer resistance to several toxicants . To perform mixing experiments in vitro, all the mutants have been purified by extraction with organic solvents, reconstituted in proteoliposomes, and found to be inactive . When co-reconstituted with wild-type protein, they inhibit the activity of the latter in a dose-dependent form up to full inhibition . We assume that this inhibition is due to the formation of mixed oligomers in which the presence of one nonfunctional subunit causes full inactivation . A binomial analysis of the results based on the latter assumptions do not provide statistically significant answers but suggests that the oligomer is composed of three subunits . The results described provide the first in vitro demonstration of the functional oligomeric structure of an ion-coupled transporter. J Biol Chem, 1996 Dec 6, 271(49), 31033 - 6 Mutational analysis of the functional domains of the large subunit of the isozyme form of wheat initiation factor eIF4F; Metz AM et al.; The isozyme form of plant eukaryotic initiation factor 4F (eIF(iso)4F) contains two subunits: p28, a cap-binding protein, and p86 . To identify the functional domains of p86, truncations of the p86 cDNA were made, and the protein was expressed in Escherichia coli and purified