Biochem Biophys Res Commun, 1993 Dec 15, 197(2), 606 - 11 Alcohol administration attenuates LPS-induced expression of inducible nitric oxide synthase in Kupffer and hepatic endothelial cells; Spolarics Z et al.; This study investigates the effects of in vivo ethanol (primed infusion, causing 170-190 mg% plasma alcohol for 12 hours) and/or LPS (12 hours after injection of E . coli LPS 1 mg/kg bw.) on the mRNA expression of inducible nitric oxide synthase (NOS II) in hepatic cells measured by competitive PCR technique, and on hepatic release of reactive nitrogen intermediates (RNI, NO2- + NO3-) . Perfused livers from alcohol- or saline-infused animals did not release measurable amounts of RNI . Under these conditions small amounts of NOS II mRNA were expressed in Kupffer and endothelial cells, while it was not detectable in parenchymal cells . LPS treatment along with markedly elevating hepatic RNI release increased NOS II mRNA levels by 35- and 200-fold, in endothelial and Kupffer cells, respectively . LPS injection and alcohol infusion to the same animal decreased hepatic RNI release by about 70% and almost completely inhibited the LPS-induced, elevated NOS II mRNA in Kupffer or endothelial cells . No similar changes were observed in the parenchymal cells . These data suggest that the primary target of in vivo LPS in upregulating hepatic NO release are the nonparenchymal cells . Furthermore, alcohol inhibits the LPS-induced response which may influence immune-related hepatic function. EMBO J, 1993 Dec 15, 12(13), 5201 - 8 Selection of a 'minimal' glutaminyl-tRNA synthetase and the evolution of class I synthetases; Schwob E et al.; The evolution of the aminoacyl-tRNA synthetases is intriguing in light of their elaborate relationship with tRNAs and their significance in the decoding process . Based on sequence motifs and structure determination, these enzymes have been assigned to two classes . The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS), a class I enzyme, complexed to tRNA(Gln) and ATP has been described . It is shown here that a 'minimal' GlnRS, i.e . a GlnRS from which domains interacting with the acceptor-end and the anticodon of the tRNA have been deleted, has enzymatic activity and can charge a tRNA(Tyr)-derived amber suppressor (supF) with glutamine . The catalytic core of GlnRS, which is structurally conserved in other class I synthetases, is therefore sufficient for the aminoacylation of tRNA substrates . Some of these truncated enzymes have lost their ability to discriminate against non-cognate tRNAs, implying a more specific role of the acceptor-end-binding domain in the recognition of tRNAs . Our results indicate that the catalytic and substrate recognition properties are carried by distinct domains of GlnRS, and support the notion that class I aminoacyl-tRNA synthetases evolved from a common ancestor, jointly with tRNAs and the genetic code, by the addition of non-catalytic domains conferring new recognition specificities. Biochem Pharmacol, 1993 Dec 14, 46(12), 2209 - 18 Varicella-zoster virus thymidine kinase . Characterization and substrate specificity; Roberts GB et al.; The varicella-zoster virus (VZV) thymidine kinase (TK) EC 2.7.2.21) catalyzes the phosphorylation of many anti-VZV nucleosides . Purified, bacterially expressed VZV TK was characterized with regard to N-terminal amino acid sequence, pI value, pH optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dTTP . Initial velocities of thymidine phosphorylation with variable MgATP concentrations fit a two-site model with apparent Km values for MgATP of 0.10 and 900 microM . dTTP was a noncompetitive inhibitor of thymidine phosphorylation but was competitive with MgATP . Phosphate donor and acceptor specificities of the bacterially expressed enzyme were indistinguishable from those of VZV TK purified from infected cells . Detailed studies of the nucleoside specificity with the bacterially expressed enzyme showed that, for a given sugar moiety, thymine nucleosides were the most efficient substrates followed by nucleosides of cytosine, uracil, adenine, and with some exceptions, guanine . For a given pyrimidine or purine (except guanine), 2'-deoxyribonucleosides were the most efficient substrates, followed by arabinosides, ribonucleosides, 2',3'-dideoxyribonucleosides, and the acyclic moiety of acyclovir. Biochim Biophys Acta, 1993 Dec 14, 1216(3), 360 - 8 Human dihydrolipoamide succinyltransferase: cDNA cloning and localization on chromosome 14q24.2-q24.3; Nakano K et al.; We isolated cDNA for dihydrolipoamide succinyltransferase from a human fibroblast cDNA library in lambda gt11 . The cDNA revealed that the human dihydrolipoamide succinyltransferase lacked a sequence motif of an E3 and/or E1 binding site . This suggests that the human dihydrolipoamide succinyltransferase possesses a unique structure consisting of two domains in contrast with the dihydrolipoamide acyltransferases of other alpha-keto acid dehydrogenase complexes . In addition, we found that the human dihydrolipoamide succinyltransferase gene is located on chromosome 14 at q24.2-q24.3 and that a sequence related to the dihydrolipoamide succinyltransferase gene is located on chromosome 1 at p31 . Interestingly, the gene for the dihydrolipoamide acyltransferase of the branched chain alpha-keto acid dehydrogenase complex is also located on chromosome 1p31 (Zneimer et al . (1991) Genomics 10, 740-747). Biochemistry, 1993 Dec 14, 32(49), 13673 - 80 Herpes simplex virus type 1 ribonucleotide reductase large subunit: regions of the protein essential for subunit interaction and dimerization; Conner J et al.; We have constructed a series of random N-terminal deletions of the large subunit (R1) of the herpes simplex virus type 1 ribonucleotide reductase . Deletions extended throughout the R1 gene open reading frame and, in total, 31 different truncated polypeptides were expressed in Escherichia coli using the T7 expression system . N-Terminal truncations were analyzed for their interaction with the small subunit (R2) of ribonucleotide reductase using a sensitive enzyme-linked immunosorbent assay (ELISA) method and for their ability to complement R2 in ribonucleotide reductase assays . Truncated proteins were also tested for homodimerization using gel-filtration chromatography . The results identified a region of R1 between amino acids 349 and 373 which was essential for subunit interaction . Proteins lacking up to 348 amino-terminal residues associated with R2 and complemented R2 in ribonucleotide reductase assays . Proteins commencing at amino acid 373 and beyond did not interact with R2 and were inactive in enzyme assays . Using a plasmid which expressed an N-terminal deleted protein commencing at amino acid 247, we constructed two defined C-terminal deletions to give proteins comprising amino acids 247-434 and 247-996 of R1 . Neither of these truncated proteins bound R2 and we concluded that a second region between amino acids 996 and 1137 (the C-terminus) is required for interaction with R2 . Gel-filtration studies indicated that deletion of the first 420 amino acids from R1 did not affect dimerization . However, deletions of 457 amino acids and larger gave proteins which existed as monomers.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Dec 14, 32(49), 13658 - 63 Mutational and kinetic analysis of a mobile loop in tyrosyl-tRNA synthetase; First EA et al.; The role of a mobile loop in tyrosyl-tRNA synthetase has been investigated by mutating each amino acid in the loop and kinetically analyzing the effect that each mutation has on the formation of the enzyme-bound tyrosyl adenylate intermediate . Kinetic analyses of mutations at three of the nine positions in the loop, K230, K233, and T234, have been reported elsewhere (Fersht et al., 1988; First & Fersht, 1993a,b) . In this paper, the kinetic analyses of mutants in the remaining six positions, as well as a mutant in which the entire loop is deleted, are reported . With the exception of E235, which stabilizes the E.-{Tyr-ATP}++ and E.Tyr.ATP complexes by 1.0 and 1.2 kcal/mol, respectively, none of the remaining amino acids appears to be directly involved in the catalytic mechanism of the enzyme . Instead, mutation of these residues results in small alterations in the stability of E.Tyr.ATP, E.{Tyr-ATP}++ and E.Tyr.AMP.PPi complexes . The precise amino acid residues which stabilize each state vary, suggesting that the loop adopts different conformations in each of the complexes with the most highly constrained conformation being in the E.{Tyr-ATP}++ complex . Deletion of the loop reveals that the net effect of the loop in catalysis is two-fold: (1) to destabilize the E.Tyr.ATP complex preceding formation of the E.{Tyr.ATP}++ complex and (2) to stabilize the E.{Tyr-ATP}++ complex, indicating that the involvement of the loop in catalysis occurs at the expense of ATP-binding energy.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Dec 14, 32(49), 13566 - 74 Collapse of parallel folding channels in dihydrofolate reductase from Escherichia coli by site-directed mutagenesis; Iwakura M et al.; The rate-limiting steps in the folding of dihydrofolate reductase from Escherichia coli have been shown to involve the conversion of a set of four intermediates to a corresponding set of native conformers via four parallel channels {Jennings et al . (1993) Biochemistry 32, 3783-3789} . Fluorescence and absorbance studies of the unfolding and refolding of the C85S/C152E double mutant at various final urea concentrations reveal two slow folding reactions, two fewer than observed in the wild-type protein . Refolding in the presence of substoichiometric levels of the inhibitor methotrexate shows that the two remaining slow reactions correspond to two parallel channels which lead to a pair of native conformers capable of binding the inhibitor . A combination of stopped-flow circular dichroism and cofactor binding studies confirms that the four parallel channels observed in the wild-type protein have collapsed into two channels in the mutant . Kinetic and equilibrium studies of the single cysteine mutants suggest that replacements of Cysteine-85 which perturb the hydrophobic core containing this side chain are responsible for the simplification of the kinetic mechanism . These results demonstrate that at least two of the parallel folding channels in dihydrofolate reductase arise when tertiary structure develops and are not dependent upon cis/trans isomerization at prolyl peptide bonds. Biochemistry, 1993 Dec 14, 32(49), 13393 - 8 DNA helicase associated with DNA polymerase alpha: isolation by a modified immunoaffinity chromatography; Biswas EE et al.; We have developed a novel immunoaffinity method for isolating a DNA polymerase alpha-associated DNA helicase from the yeast Saccharomyces cerevisiae . Earlier we have reported the characterization of a DNA helicase activity associated with the multiprotein DNA polymerase alpha complex from yeast {Biswas, E . E., Ewing, C . M., & Biswas, S . B . (1993) Biochemistry 32, 3030-3027} . We report here the isolation of the DNA helicase from the DNA polymerase alpha (pol alpha) complex bound to an anti-pol alpha immunoaffinity matrix . The DNA helicase activity eluted at approximately 0.35 M NaCl concentration . The eluted ATPase/helicase peak was further purified by size-exclusion high-performance liquid chromatography (HPLC) . At low ionic strength (50 mM NaCl), it remained associated with other proteins and eluted as a large polypeptide complex . At high ionic strength (500 mM NaCl), the helicase dissociated, and the eluted ATPase/helicase fraction contained 90-, 60-, and 50-kDa polypeptides . Photoaffinity cross-linking of helicase with ATP during the isolation process demonstrated a 90-kDa polypeptide to be the likely ATP binding component of the helicase protein . The DNA helicase has single-stranded DNA (ssDNA)-stimulated ATPase and dATPase activities . The ATPase activity was stimulated by yeast replication protein A (RPA) . The DNA helicase activity was stimulated by Escherichia coli ssDNA binding protein and RPA . The DNA helicase migrated on a DNA template in the 5'-->3' direction which is also the overall direction of migration of pol alpha on the lagging strand of the replication fork.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Dec 14, 32(49), 13516 - 22 Purification and characterization of the cytoplasmic domain of human receptor-like protein tyrosine phosphatase RPTP mu; Gebbink MF et al.; RPTP mu is a recently described receptor-like protein tyrosine phosphatase (PTP), the ectodomain of which mediates homophilic cell-cell adhesion . The cytoplasmic part contains two homologous PTP-like domains and a juxtamembrane region that is about twice as large as in other receptor-like PTPs . The entire 80-kDa cytoplasmic part of human RPTP mu was expressed in insect Sf9 cells and its enzymatic activity was characterized after purification to electrophoretic homogeneity . In addition, the effects of deletion and point mutations were analyzed following expression in Escherichia coli cells . The purified cytoplasmic part of RPTP mu displays high activity toward tyrosine-phosphorylated, modified lysozyme (Vmax 4500 nmol min-1 mg-1) and myelin basic protein (Vmax 8500 nmol min-1 mg-1) but negligible activity toward tyrosine-phosphorylated angiotensin or the nonapeptide, EDNDpYINASL, that serves as a good substrate for protein tyrosine phosphatase PTP1B . This suggests that RPTP mu and PTP1B have distinct substrate specificities . Catalytic activity is independent of Ca2+ (up to 1 mM) but is strongly inhibited by Zn2+, Mn2+, vanadate, phenylarsenic oxide, and heparin . The first of the two catalytic domains is 5-10 times less active than the expressed catalytic region containing both domains . Mutation of Cys 1095 to Ser in the first catalytic domain abolishes enzymatic activity when analyzed following expression in either E . coli or mammalian COS cells . Deletion of the first 53 amino acids from the juxtamembrane region reduces catalytic activity about 2-fold. Biochemistry, 1993 Dec 14, 32(49), 13414 - 8 Inhibition of the activity of protein tyrosine phosphate 1C by its SH2 domains; Townley R et al.; Full-length protein tyrosine phosphatase 1C (PTP1C), the catalytic domain of PTP1C (delta PTP1C), and the N-terminal SH2 domain truncated PTP1C (delta NPTP1C) were overexpressed in Escherichia coli and purified to near homogeneity . Various phosphorylated states of the synthetic phosphotyrosyl peptide TRDIYETDYYRK (IRP), corresponding to the major insulin receptor autophosphorylation sites, were used as substrates for the PTPs . There was no indication for selective dephosphorylation of any of the three phosphotyrosyl residues from the triphosphotyrosyl IRP . Kinetic studies were carried out using all seven different phosphotyrosyl IRPs . Saturation kinetics were observed for PTP1C using the triphosphotyrosyl IRP only . In contrast, for delta PTP1C, saturation was achieved for all seven phosphotyrosyl IRPs . The best substrate for delta PTP1C was the triphosphotyrosyl IRP possessing a Km of approximately 1.6 microM, about 3-4-fold lower than either the mono- or diphosphotyrosyl IRPs . However, in contrast to delta PTP1C, PTP1C had a 22-fold lower affinity for triphosphotyrosyl IRP . Furthermore, deletion of a single N-terminal SH2 domain increased the affinity of the enzyme for the triphosphotyrosyl IRP to a value similar to that obtained with delta PTP1C . The pH optima for all three enzyme constructs were very similar and could not account for the observed change in substrate affinity between the three enzymes . These results suggest that the SH2 domain of PTP1C exerts an inhibitory effect on its PTP activity. FEBS Lett, 1993 Dec 13, 335(3), 299 - 304 Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen; Dolecek C et al.; Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy . Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library . The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C-terminal portion of group I grass pollen allergens from different species . A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins . Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues . The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE-binding capacity as the natural allergen. Biochim Biophys Acta, 1993 Dec 12, 1153(2), 345 - 7 An Escherichia coli gene showing a potential ancestral relationship to the genes for the mitochondrial import site proteins ISP42 and MOM38; Cartwright P et al.; An ORF (OrfT) of 1911 base pairs, upstream of the hip operon in Escherichia coli at map position 33.82 has been identified . The protein encoded by this sequence is predicted to have a molecular mass of 68,249 Da and the carboxyterminal 276 residues shows 26.8% and 25.4% identity with the import site proteins ISP42 and MOM38 from the mitochondrial outer membrane of Saccharomyces cerevisiae and Neurospora crassa, respectively . These mitochondrial membrane proteins have been shown to be essential components of the protein translocation apparatus in yeast . These similarities raise the possibility that OrfT might represent the bacterial gene from which these eukaryotic genes evolved. Nucleic Acids Res, 1993 Dec 11, 21(24), 5661 - 6 The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp super-repressors in vivo; Shapiro M et al.; The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challenge-phage assay, an assay based on a positive genetic selection for DNA binding . These proteins, which differ by single amino acid changes from the wild type (Glu13-->Lys, Glu18-->Lys, Glu49-->Lys and Ala77-->Val), require less L-tryptophan than wild-type repressor for activation in vivo, and are super-aporepressors . However, none of the four mutant repressors binds DNA in a corepressor-independent manner . Three of the four mutant repressors (with Glu-->Lys changes) are more active when complexed with tryptophan, and are superholorepressors . Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding studies in vitro . Challenge-phage assays with limiting tryptophan reveal additional phenotypic differences among the mutant proteins . These results show that the challenge-phage assay is a robust assay for measuring the relative affinities of specific protein-DNA interactions in vivo. Nucleic Acids Res, 1993 Dec 11, 21(24), 5561 - 9 Purification and characterization of human 3-methyladenine-DNA glycosylase; O'Connor TR; A human cDNA coding sequence for a 3-methyladenine-DNA glycosylase was expressed in Escherichia coli . In addition to the full-length 3-methyladenine-DNA glycosylase coding sequence, two other sequences (resulting from differential RNA splicing and the truncated anpg cDNA) derived from that sequence were also expressed . All three proteins were purified to physical homogeneity and their N-terminal amino acid sequences are identical to those predicted by the nucleic acid sequences . The full-length protein has 293 amino acids coding for a protein with a molecular mass of 32 kDa . Polyclonal antibodies against one of the proteins react with the other two proteins, and a murine 3-methyladenine-DNA glycosylase, but not with several other E . coli DNA repair proteins . All three proteins excise 3-methyl-adenine, 7-methylguanine, and 3-methylguanine as well as ethylated bases from DNA . The activities of the proteins with respect to ionic strength (optimum 100 mM KCl), pH (optimum 7.6), and kinetics for 3-methyladenine and 7-methylguanine excision (average values: 3-methyladenine: Km 9 nM and kcat 10 min-1, 7-methylguanine: Km 29 nM and kcat 0.38 min-1) are comparable . In contrast to these results, however, the thermal stability of the full-length and splicing variant proteins at 50 degrees C is less than that of the truncated protein. Vet Rec, 1993 Dec 11, 133(24), 585 - 90 A hypertonic infusion in the treatment of experimental shock in calves and clinical shock in dogs and cats; Dupe R et al.; A novel composite hypertonic solution for intravenous use was tested in two experimental models, one of endotoxic shock and one of shock linked with dehydration, both in anaesthetised calves . Endotoxic shock was induced with Escherichia coli lipopolysaccharide and was characterised by a low cardiac output, hypoxaemia, acidosis and anuria . Treatment with a small volume of the solution increased cardiac output, improved oxygen carriage, corrected acidosis and stimulated renal function . Experimental dehydration in calves was induced by intraperitoneal mannitol and frusemide diuresis, and was characterised by reduced circulating plasma volume, acidosis and poor peripheral perfusion . Treatment with the new solution corrected the acidosis and stimulated peripheral circulation significantly better than treatment with hypertonic or isotonic saline alone, and also expanded the calves' plasma volume . The new solution was also compared with conventional fluid therapy in clinical small animal practice . Twenty cats and dogs with clinical shock were treated with either small volumes of the hypertonic solution or large volumes of isotonic fluids . The animals treated with small volumes of the hypertonic solution responded better than the animals treated with large volumes of isotonic fluid. Science, 1993 Dec 10, 262(5140), 1744 - 7 Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli; Derman AI et al.; Disulfide bonds are rarely found in cytoplasmic proteins . Mutations were selected for in Escherichia coli that allow disulfide bond formation in the cytoplasm . In the presence of these mutations, export-defective versions of alkaline phosphatase and mouse urokinase were able to fold into their enzymatically active conformations in the cytoplasm because their disulfide bonds were formed . The mutations were mapped to the gene for thioredoxin reductase and diminish or eliminate the activity of this enzyme . Thioredoxin itself was found to be unnecessary for this disulfide bond formation . Thioredoxin reductase, but not thioredoxin, is thus implicated in keeping cysteines reduced in cytoplasmic proteins. Biochim Biophys Acta, 1993 Dec 8, 1203(2), 175 - 83 Association of the mcrD gene product with methyl coenzyme M reductase in Methanococcus vannielii; Stroup D et al.; The mcrD gene, subcloned from the methyl coenzyme M reductase (MR) encoding mcrBDCGA operon in Methanococcus vannielii, has been expressed at a high level in Escherichia coli . Rabbit antibodies, raised against the product of this gene (rgpmcrD, recombinant gene product of mcrD) purified from E . coli, have been used to quantitate gpmcrD in M . vannielii and to follow its fate during MR purification . The molar ratio of gpmcrD to MR was found to be approx . 1:15 in cells of M . vannielii taken from batch cultures at all stages of growth . Sedimentation of lysates of M . vannielii cells through sucrose gradients and analyses of the fractions obtained by Western blotting and immunoprecipitation have demonstrated the presence of a macromolecular complex containing both gpmcrD and MR . Addition of mcrD antibodies or removal of gpmcrD from lysates of M . vannielii cells by immunoprecipitation decreased the rates of methanogenesis in vitro by approx . 20% . Addition of purified rgpmcrD to these lysates did not stimulate methanogenesis. Biochemistry, 1993 Dec 7, 32(48), 13362 - 8 Reaction of the ArsA adenosinetriphosphatase with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid; Ksenzenko MY et al.; The oxyanion-translocating ATPase encoded by the plasmid-borne ars operon catalyzes extrusion of antimonials and arsenicals from cells of Escherichia coli, thus providing resistance to those toxic oxyanions . The purified catalytic subunit of the ATPase, the ArsA protein, exhibits oxyanion-stimulated ATPase activity . The nature of the oxyanion binding site was probed by reaction with the fluorescent sulfhydryl probe 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) . Our results indicate that MIANS reacts with the ArsA protein in an antimonite-dependent manner . After the protein had been modified with MIANS, two of four cysteines in the ArsA protein had reacted with the probe in the absence of the oxyanionic substrate, and three in the presence of antimonite . The quantum yield of the MIANS-ArsA protein adduct was significantly higher if modification of the protein had occurred in the presence of oxyanionic substrates . Thus binding of the anionic substrate of the pump produces a conformational change in the ArsA protein such that a single additional cysteinyl residue reacts more readily with the sulfhydryl probe. Biochemistry, 1993 Dec 7, 32(48), 13254 - 61 Spectroscopic characterization of mutants supports the assignment of histidine-419 as the axial ligand of heme o in the binuclear center of the cytochrome bo ubiquinol oxidase from Escherichia coli; Calhoun MW et al.; The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of heme-copper oxidases which also includes the aa3-type cytochrome c oxidases . The oxygen-binding binuclear center of cytochrome bo is located in subunit I and consists of a heme (heme o; heme a3 in the aa3-type oxidases) and a copper (Cu(B)) . Previous spectroscopic studies have shown that heme o is bound to the protein via a single histidine residue . Site-directed mutagenesis of conserved histidine residues in subunit I has identified two residues (H284 and H419) which are candidates for the ligand of heme o, while spectroscopic studies of mutants at H284 definitively demonstrated that this residue cannot be the axial ligand . Consequently, the single remaining conserved histidine in subunit I (H419) was assigned as the ligand for the heme of the binuclear center . In this paper, this assignment is tested by characterization of additional mutants in which the putative heme o axial ligand, H419, is replaced by other amino acids . All mutations at H419 result in the loss of enzyme activity . Analyses via UV-visible and Fourier transform infrared spectroscopies reveal that substantial perturbation has occurred at the binuclear center as a result of the amino acid substitutions . In contrast with the wild-type enzyme, the mutant enzymes bind very little carbon monoxide . Three other amino acid residues which are potential ligands for heme o are shown tob e nonessential for enzyme activity . Mutations in these residues do not perturb the UV-visible or FTIR spectroscopic characteristics of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Dec 7, 32(48), 13237 - 44 Site-directed mutagenesis of tyrosine residues at nicotinamide nucleotide binding sites of Escherichia coli transhydrogenase; Olausson T et al.; Nicotinamide nucleotide transhydrogenase (E.C.1.6.1.1) from Escherichia coli was investigated with respect to the role of specific conserved tyrosine residues of putative substrate-binding regions . The enzyme from E . coli is made up of two subunits, alpha (510 residues) and beta (462 residues) . The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region corresponds to the alpha subunit and whose C-terminal region corresponds to the beta subunit . Tyrosines 245 and 1006 of the mitochondrial enzyme have been shown to react selectively with 5'-(p-fluorosulfonylbenzoyl)adenosine with inactivation of the enzyme . In E . coli these residues correspond to tyrosine 226 of the alpha subunit and tyrosine 431 of the beta subunit . In addition, tyrosine 315 of the beta subunit is of interest since mutation of an adjacent residue (glycine 314) leads to inactivation {Ahmad, S., Glavas, N . A., & Bragg, P . D . (1992) Eur . J . Biochem . 207, 733-739} . In order to assess the role of the aforementioned conserved tyrosine residues in the mechanism and structure of transhydrogenases, these were replaced by site-specific mutagenesis, using the cloned and overexpressed E . coli transhydrogenase genes {Clarke, D . M., & Bragg, P . D . (1985) J . Bacteriol . 162, 367-373} . Phenylalanine mutants of all three tyrosine residues showed approximately 50% activity or more with regard to catalytic activity assayed as reduction of 3-acetylpyridine-NAD+ by NADPH . These mutants were also active in proton pumping assayed as quenching of 9-methoxy-6-chloro-2-aminoacridine or quinacrine fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Dec 7, 32(48), 13231 - 6 Identification of lysine 153 as a functionally important residue in UDP-galactose 4-epimerase from Escherichia coli; Swanson BA et al.; The role of lysine 153 in the action of UDP-galactose 4-epimerase from Escherichia coli has been investigated by site specific mutagenesis and kinetic and spectrophotometric analysis of the mutant enzymes . The crystal structure of UDP-galactose 4-epimerase shows that the binding of NAD+ to the coenzyme site includes the hydrogen bonded interaction of the epsilon-ammonium group of lysine 153 with the 2'- and 3'-hydroxyl groups of the nicotinamide riboside . Mutation of this residue to methionine or alanine decreases the catalytic activity of the enzyme by a factor of more than 10(3) . The NAD+ associated with the wild type enzyme is subject to UMP-dependent reduction by sugars such as glucose and arabinose, but the mutant proteins K153M and K153A are not reduced by sugars in the presence or absence of UMP . NAD+ associated with the wild type enzyme is also subject to UMP-dependent reduction by sodium cyanoborohydride . However, although the mutant proteins bind UMP very well, the rate at which NAD+ associated with them is reduced by sodium cyanoborohydride is almost insensitive to the presence of UMP . The purified wild type enzyme contains significant amounts of NADH bound to the coenzyme site; however, the purified mutants K153M and K153A contain very little NADH . We conclude that lysine 153 plays an important role in increasing the chemical reactivity of enzyme-bound NAD+ in the uridine nucleotide-dependent conformational change associated with reductive inactivation and the catalytic activity of UDP-galactose 4-epimerase. Biochemistry, 1993 Dec 7, 32(48), 13220 - 30 The importance of binding energy in catalysis of hydride transfer by UDP-galactose 4-epimerase: a 13C and 15N NMR and kinetic study; Burke JR et al.; UDP-galactose 4-epimerase contains NAD+ irreversibly but noncovalently bound to the active site . Uridine nucleotides bind to the substrate site and induce a protein conformational change that increases the chemical reactivity of NAD+ at the coenzyme site . Activation of NAD+ by uridine nucleotides perturbs the 15N and 13C NMR chemical shifts of selectively enriched NAD+ bound to the coenzyme site . The proton-decoupled 15N NMR signal for enzyme-bound {carboxamide-15N}NAD+ does not change upon addition of UDP, indicating that activation is not brought about by a change in the binding of the carboxamide group . The 15N NMR signal of enzyme-bound {nicotinamide-1-15N}NAD+ is shifted upfield 3.0 ppm and the 13C NMR signal for {nicotinamide-4-13C}NAD+ is shifted downfield 3.4 ppm downfield by the binding of UDP at the substrate site . These changes are consistent with the induction of a distortion into the nicotinamide ring, in which positive charge is transferred from N-1 to C-4 . The kinetic and thermodynamic effects of these perturbations are significant, as indicated by the nonenzymatic chemical reactivities of a series of N-alkyl nicotinamides differing in the inductive electron withdrawing effects of the alkyl substituents . A downfield change of 3.4 ppm in the 4-13C chemical shifts brought about by electron withdrawal in the model compounds corresponds to a 3200-fold increase in the rate of reduction by NaBH3CN in water, a 15,000-fold increase in 86% ethanol, and a 152 mV more positive reduction potential in this series . The distortion of NAD+ by the binding of UDP is a long-range effect that is transmitted from the substrate binding site to the coenzyme through the protein conformational change . This apparently distorts the pi-electron distribution in the nicotinamide ring and reduces the activation energy for its reduction . Activation of enzyme-bound NAD+ toward reduction apparently arises from a destabilization in the nicotinamide ring structure rather than from a stabilization of the transition state through attractive interactions between the nicotinamide ring and the enzyme. Biochemistry, 1993 Dec 7, 32(48), 13190 - 7 SecA, the peripheral subunit of the Escherichia coli precursor protein translocase, is functional as a dimer; Driessen AJ; SecA, the peripheral ATPase domain of the Escherichia coli precursor protein translocase, was denatured in 6 M guanidine hydrochloride . Circular dichroism and intrinsic tryptophan fluorescence spectra revealed that the protein is transformed into a random-coil configuration . Upon dilution of the chaotropic agent, SecA refolds into its native, functional conformation as a homodimer . As structural criteria, the native dimeric state was assayed by size-exclusion chromatography, chemical cross-linking, tryptophan fluorescence, and circular dichroism . Functional SecA heterodimers were formed of which the individual subunits were tagged with fluorescent dyes to allow measurements of the association state of the monomers by resonance energy transfer using steady-state and time-resolved fluorescence spectroscopy . SecA retained its dimeric structure during translocation, while energy transfer was abolished only by denaturation . The "half-of-the-sites activity" was investigated by constructing heterodimers formed from native and 8-azido-ATP-inactivated SecA . Heterodimers have lost the ability to support translocation of the precursor protein proOmpA in an in vitro translocation system . It is concluded that the dimeric structure is maintained during translocation and required for functionality. Biochemistry, 1993 Dec 7, 32(48), 13146 - 55 Interactions of three strands in joints made by RecA protein; Chiu SK et al.; RecA protein from Escherichia coli has been used to form a triple-stranded DNA structure from either single-stranded M13 DNA or a single-stranded oligonucleotide plus a duplex oligonucleotide with a hairpin loop . The secondary structure of purified deproteinized triplex was examined by probing with DNase I, P1 nuclease, potassium permanganate, and diethyl pyrocarbonate . The two strands destined to form heteroduplex DNA showed the same patterns of chemical modification and enzymatic digestion as control duplex DNA, indicating that they formed a normal duplex substructure . However, the nascent outgoing strand showed properties consistent with a novel triplex structure: most of its purine residues, especially adenines, were hyperreactive to all probes . The patterns of digestion by DNase I and P1 nuclease indicated that the nascent outgoing strand was not a freely mobile or single-stranded branch but rather was still interacting with the newly formed heteroduplex DNA . On the basis of the planar base triads proposed previously (Rao et al., 1993) and energy minimization of a third strand in the major groove of B-form DNA, we derived a model that helps to rationalize the properties revealed by chemical and enzymatic probing. Biochemistry, 1993 Dec 7, 32(48), 13081 - 8 Secondary structure of the MutT enzyme as determined by NMR; Weber DJ et al.; The MutT enzyme (129 amino acids) catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleotides (NMP) and pyrophosphate by nucleophilic substitution at the rarely attacked beta-phosphorus of NTP {Weber, D . J., Bhatnagar, S . K., Bullions, L . L., Bessman, M . J., & Mildvan, A . S . (1992) J . Biol . Chem . 267, 16939-16942} . Backbone NMR assignments for the H alpha, 13C alpha, HN, 15N, and carbonyl 13C' resonances, based on heteronuclear methods have been reported for MutT {Abeygunawardana, C., Weber, D . J., Frick, D . N . Bessman, M . J., & Mildvan, A . S . (1993) Biochemistry (preceding paper in this issue)} . Here, we report the secondary structure of MutT in solution on the basis of these assignments, NOE data derived from 2D and 3D homonuclear and heteronuclear NMR spectra, and amide NH exchange data . Consistent with near neighbor NOEs, H alpha and C alpha chemical shifts, and amide exchange rates, MutT contains two alpha-helices spanning residues 47-59 (helix 1) and residues 119-128 (helix 2), respectively . The helical content predicted from NMR (17.8 +/- 1.0%) is consistent with that predicted by circular dichroism spectroscopy (20.9 +/- 5.4%) . A mixed parallel and antiparallel beta-sheet with five beta-strands (A-E) consists of residues A, 3-13; B, 18-24; C, 70-74; D, 79-87; and E, 102-106.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Dec 7, 32(48), 13071 - 80 Sequence-specific assignments of the backbone 1H, 13C, and 15N resonances of the MutT enzyme by heteronuclear multidimensional NMR; Abeygunawardana C et al.; The MutT protein, a 129-residue enzyme from Escherichia coli which prevents A.T-->C.G mutations, catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleoside monophosphates (NMP) and pyrophosphate {Bhatnagar, S . K., Bullions, L . C., & Bessman, M . J . (1991) J . Biol . Chem . 266, 9050-9054}, by a mechanism involving nucleophilic substitution at the rarely attacked beta-phosphorus of NTP {Weber, D . J., Bhatnagar, S . K., Bullions, L . C., Bessman, M . J., & Mildvan, A . S . (1992a) J . Biol . Chem . 267, 16939-16942} . The bacterial MutT gene was inserted into the plasmid pET-11b under control of the T7 promoter and overexpressed in minimal media, permitting labeling of MutT with 13C and/or 15N . The yield after purification of the soluble fraction was approximately 35 mg of homogeneous MutT/L with physical and enzymatic properties indistinguishable from those of the originally isolated enzyme . Essentially complete sequence-specific assignments of the backbone HN, N, C alpha, H alpha, and CO resonances of the free enzyme (1.5 mM) were made at pH 7.4 and 32 degrees C, by heteronuclear double- and triple-resonance experiments using a modified Bruker AM 600 NMR spectrometer . Specifically, 1H{15N}HSQC, 1H{15N}TOCSY-HMQC, and 1H{15N}NOESY-HMQC experiments were done with uniformly 15N-labeled enzyme . A 1H{15N} HSQC experiment was done with selective {alpha-15N}Lys-labeled enzyme . Also HNCA, HN(CO)CA, HNCO, constant time 1H{13C}HSQC, HCACO, and HCA(CO)N experiments were done with uniformly 13C- and 15N-labeled enzyme . Sequence-specific assignments were initiated from HN and 15N chemical shifts of Gly residues and of selectively labeled Lys residues in 1H{15N}HSQC experiments . They were confirmed by C alpha chemical shifts of Ala residues uniquely identified by residual coupling to C beta resonances in constant time 1H{13C}HSQC experiments . The sequence-specific assignments proceeded bidirectionally, terminating at Pro residues and at residues with undetectable NH signals, and the segments were linked to complete the backbone assignments . The backbone assignments reported here have permitted the interpretation of NOEs in the elucidation of the solution secondary structure of MutT, and the C alpha and H alpha chemical shifts have provided an independent approach to identifying secondary structural elements and to define their extent {Weber, D . J., Abeygunawardana, C., Bessman, M . J., & Mildvan, A . S . (1993) Biochemistry (following paper in this issue)}. Biochemistry, 1993 Dec 7, 32(48), 13032 - 9 Incision at DNA G.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.T binding reaction; Griffin S et al.; We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells . G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch . Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC . In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence . Thus, oligonucleotides containing either CpgGpT or 5meCpGGpT were efficiently incised, but not those containing GpGCpT, ApGTpT, or TpGApT sequences . Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction . The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts . The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision . The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine . In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli. Biochemistry, 1993 Dec 7, 32(48), 13026 - 31 C-terminal peptide appendix in a class I tRNA synthetase needed for acceptor-helix contacts and microhelix aminoacylation; Kim S et al.; The 10 class I tRNA synthetases have an N-terminal nucleotide-binding fold which contains the catalytic center . Insertions into the nucleotide-binding fold provide contacts for acceptor-helix interactions, which stabilize the amino acid acceptor end of the tRNA substrate in the active site . A separate and largely nonconserved C-terminal domain provides contacts with distal parts of the tRNA, including the anticodon . For Escherichia coli methionyl tRNA synthetase, whose structure is known, the C-terminal domain is predominantly alpha-helical and forms a loop which interacts with the anticodon trinucleotide located about 76 A from the amino acid attachment site . Fused to the end of this helical domain is a peptide which curls back into the N-terminal nucleotide-binding fold and region of the active site . We show here that mutations in this peptide appendix disrupt aminoacylation and binding of a 7 base pair microhelix substrate based on the acceptor stem of tRNA(fMet), without affecting interactions with ATP or methionine or with the tRNA(fMet) anticodon . The impairment of acceptor-helix interactions by mutation of the C-terminal peptide can offset favorable anticodon interactions and severely reduce aminoacylation of tRNA(fMet) . Thus, in addition to, or as an alternative to, acceptor-helix-binding insertions into the N-terminal nucleotide-binding fold, C-terminal peptide epitopes in some class I enzymes may provide a mechanism for facilitating RNA microhelix interactions with the catalytic site. FEBS Lett, 1993 Dec 6, 335(2), 155 - 60 The Drosophila ankyrin repeat protein cactus has a predominantly alpha-helical secondary structure; Gay NJ et al.; The cactus protein is the Drosophila homologue of the mammalian I kappa B family of cytoplasmic anchor proteins . We have expressed in E . coli and purified a cactus fusion protein, CACT-Bgl . CACT-Bgl protein contains the six ankyrin repeat sequences which are necessary for specific binding to the Drosophila rel family transcription factor dorsal . We show that the purified CACT-Bgl protein can bind specifically to dorsal and, using circular dichroism spectroscopy, that the protein adopts a largely alpha-helical secondary structure . A further analysis of the ankyrin repeat domains of cactus, using an improved secondary structure prediction program indicates that the N-terminal of the repeat will form into a loop structure and the C-terminal section into an interrupted, amphipathic alpha-helix . On the basis of these findings we propose that the ankyrin repeats of cactus fold together into helical bundles interconnected by diverged loops. J Mol Biol, 1993 Dec 5, 234(3), 910 - 2 Crystallization and preliminary X-ray analysis of the uracil-DNA glycosylase DNA repair enzyme from herpes simplex virus type 1; Savva R et al.; A 28.5 kDa catalytic fragment of the uracil-DNA glycosylase DNA repair enzyme from Herpes simplex virus type 1 (HSV-1) has been crystallized using protein from a highly expressing Escherichia coli clone of the Herpes simplex virus type 1 UL2 gene . The protein crystallizes at 12 mg/ml from 11% (w/v) polyethylene glycol 8000 at pH values in the range 6.8 to 7.0, in the presence of (NH4)2SO4 . Long trigonal rods (0.08 mm x 0.08 mm x > 0.5 mm) diffract beyond 3.0 A using a laboratory source . The enzyme crystallizes in P3(1) (or P3(2)) a = 65.3 A, c = 49.0 A with a single molecule in the asymmetric unit and an estimated solvent content of 41% by volume. J Mol Biol, 1993 Dec 5, 234(3), 700 - 21 Evolution of allosteric control in glycogen phosphorylase; Hudson JW et al.; In relation to the primary sequence and three-dimensional structure of rabbit muscle glycogen phosphorylase, we have carried out a comparative sequence analysis of phosphorylases from human, rat, Dictyostelium, yeast, potato and Escherichia coli . Based on sequence similarity, a large region of the protein is shared by these enzymes extending from alpha-helix-1 to the last alpha-helix-33 . Conserved residues are equally distributed between the N and C-terminal domains and occur primarily in buried residues . Phylogenetic analysis indicates that the two isozymes within either E . coli, potato or Dictyostelium are more closely related to each other than they are to other phosphorylases . Yeast phosphorylase is most closely related to the Dictyostelium isozymes . Mammalian muscle and brain isozymes are more closely related to each other than to the liver isozyme and the muscle isozyme is evolving at the slowest rate . All phosphorylases exhibit high conservation of active site and pyridoxal phosphate binding residues . Most phosphorylases also exhibit high conservation of sugar binding residues in the glycogen storage site . Phosphorylation and AMP binding site residues are poorly conserved in non-mammalian phosphorylases . In contrast, glucose-6-P binding residues are highly conserved in four of the seven non-mammalian enzymes . Analysis of interacting pairs of dimer contact residues indicates that they can be grouped into three relatively independent networks . One network contains phosphorylation and AMP binding residues and is poorly conserved in non-mammalian enzymes . A second network contains glucose-6-P binding residues and is highly conserved in enzymes containing a conserved glucose-6-P binding site . A third, conserved network contains residues within the tower helix and gate loop . A model for the evolution of allostery in phosphorylase is proposed, suggesting that glucose-6-P inhibition was an early control mechanism . The later creation of primarily distinct ligand binding sites for AMP/phosphorylation control may have allowed the establishment of a separate dimer contact network for propagating conformational changes leading to activation rather than inhibition of enzyme activity. J Mol Biol, 1993 Dec 5, 234(3), 542 - 53 Operator sequence context influences amino acid-base-pair interactions in 434 repressor-operator complexes; Bell AC et al.; The 434 repressor binds more tightly to OR1 than it does to OR3 . The repressor makes several specific contacts with the symmetrically arrayed outer four base-pairs of the 14 base-pair site, and no specific contacts to the central six base-pairs . The sequence of the outer base-pairs of OR1 and OR3 differs only by an A-->G substitution at position 4 in one half-site of OR3, while that of central bases is very different . As expected from sequence analysis of wild-type operators, the data show repressor prefers an A.T base-pair at position 4 . The magnitude of this preference depends on operator sequence context and solution conditions . Position 4 changes in the context of OR1 have a greater effect on operator affinity for 434 repressor than do similar changes in OR3 . Although OR1 and OR3 display different affinities for 434 repressor, their repressor-operator complexes are similarly insensitive to changes in salt concentration and temperature . By contrast, complexes formed between repressor and position 4 mutant OR1, bearing an A.T-->G.C change, and OR3, which bears a G.C-->A.T change, are affected greatly, and to similar extents, by changes in ionic strength and temperature . Nuclease protection experiments show that 434 repressor protects the DNA phosphate backbone of wild-type operators from cleavage more efficiently than those of mutant operators . These data show that the biochemical and structural properties of a repressor-operator complex, while affected by position 4 base sequence, are independent of the identity of this base . The ability of repressor to recognize the base at position 4 depends on the sequence context at operator positions 5 to 7 . Apparently there is an interplay between the bases at operator positions 4 to 7 which has a global effect on the structure of the repressor-operator complex. J Mol Biol, 1993 Dec 5, 234(3), 534 - 41 The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase; Bernard P et al.; DNA topoisomerases perform essential roles in DNA replication, gene transcription, and chromosome segregation . Recently, we identified a new type of topoisomerase II poison: the CcdB protein of plasmid F . When its action is not prevented by CcdA protein, the CcdB protein is a potent cytotoxin . In this paper, using purified CcdB, CcdA and gyrase, we show that CcdB protein efficiently traps gyrase in a cleavable complex . The CcdA protein not only prevents the gyrase poisoning activity of CcdB but also reverses its effect on gyrase . The mechanism by which the CcdB protein induces DNA strand breakage is closely related to the action of quinolone antibiotics . However, the ATP dependence of the CcdB cleavage process differentiates the CcdB mechanism from quinolone-dependent reactions because the quinolone antibiotics stimulate efficient DNA breakage, whether or not ATP is present . We previously showed that bacteria resistant to quinolone antibiotics are sensitive to CcdB and vice versa . Elucidation of the mechanism of action of CcdB protein may permit the design of drugs targeting gyrase so as to take advantage of this new poisoning mechanism. J Biol Chem, 1993 Dec 5, 268(34), 25958 - 64 Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria; Dietmeier K et al.; We analyzed the submitochondrial location and biogenesis pathway of the phosphate carrier (PiC), also termed p32, of Saccharomyces cerevisiae mitochondria, PiC/p32 was found to behave as an integral membrane protein that cofractionated with the ADP/ATP carrier of the inner membrane . Import of the precursor of PiC/p32 required a membrane potential across the inner membrane, supporting its localization to the inner membrane . This makes it unlikely that the major function of PiC/p32 is that of an import receptor on the surface of the mitochondrial outer membrane . Furthermore, we found that both receptors MOM72 and MOM19 were involved in the import pathway of the precursor of PiC/p32 with MOM72 being responsible for the bulk of import . Yeast PiC/p32 is thus not only structurally homologous to the ADP/ATP carrier, but has a similar targeting mechanism and submitochondrial location, supporting its classification as a member of the inner membrane carrier family. J Biol Chem, 1993 Dec 5, 268(34), 25811 - 7 Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency; Monno S et al.; We identified a new homozygous missense mutation His373-->Leu in the CYP17 gene of two sisters with 17 alpha-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction . Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the Leu373 mutant protein . COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17 alpha-hydroxylase and 17,20-lyase activities . Also, the cells were devoid of 11 beta-hydroxylase and aldosterone synthase activities . To examine the mechanism by which the single amino acid change His373-->Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu373 mutation in Escherichia coli and performed spectral studies . Membrane preparations from E . coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes . The 17 alpha-hydroxylase and 17,20-lyase activities were observed only in E . coli cells expressing the wild-type enzyme . These results show that the His373-->Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of His373 in heme binding. J Biol Chem, 1993 Dec 5, 268(34), 25780 - 9 A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential; MacDonald NJ et al.; We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes . Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate . Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions . An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and {32P}orthophosphate-labeled human breast carcinoma and murine melanoma Nm23 . Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation . The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction . No effect of cAMP was observed on Nm23 NDPK activity . Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro . The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells. J Biol Chem, 1993 Dec 5, 268(34), 25735 - 9 Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA . Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities; Leong LE et al.; Protease 3C (3Cpro) encoded by human rhinovirus type 14 was purified from recombinant Escherichia coli and shown to bind specifically to the 5'-terminal 126 nucleotides of the viral RNA (126 RNA) in addition to efficiently cleaving a synthetic peptide in trans . The binding of 3Cpro to the viral RNA may be required for the initiation of plus strand viral RNA synthesis, suggesting a second non-proteolytic function for 3Cpro . Single amino acid substitutions were generated in 3Cpro at residues that are highly conserved among picornaviruses or that lie within the putative catalytic triad . Conservative changes at Asp-85 (D85E and D85N) destroyed the ability of 3Cpro to bind specifically to the 126 RNA, whereas the D85N mutation resulted in almost wild-type levels of proteolytic activity . Conversely, substitutions at His-40, Glu-71, or Cys-146 (H40D, E71A, or C146S) gave proteolytically inactive mutants that bound to the 126 RNA . These results suggest that the highly conserved Asp-85 is essential for specific binding to the 126 RNA, but is unlikely to function in proteolysis as the acidic member of the catalytic triad . Moreover, 3Cpro appears to have different domains for the RNA binding and proteolytic activities. J Biol Chem, 1993 Dec 5, 268(34), 25664 - 70 Membrane assembly of the outer membrane protein OmpA of Escherichia coli; Klose M et al.; The membrane part (residues 1 to approximately 170) of the 325-residue Escherichia coli outer membrane protein OmpA is thought to exist in the membrane as an 8-stranded beta-barrel, subdividing this part into four segments . The influence of proline residues on membrane assembly of the protein has been studied . These were introduced, using site-directed mutagenesis, into each of seven of the antiparallel beta-strands . One important parameter for allowing or not allowing membrane assembly was the potential H beta (i) which is the potential to form an amphiphilic beta-strand . When H beta (i) remained unaltered, 2 prolines were tolerated . Lowering H beta (i) in most cases caused failure of assembly when 2 such residues were present . An insert of 10 residues, including 3 prolines, did not alter H beta(i) and was tolerated, but caused "looping out" of the strand to the outer face of the membrane; displacement to its inner side would not have allowed for an amphiphilic beta-strand . Thus, a beta-structured protein is as adaptable as it has been shown for an alpha-helix . The wild type segment order 1-2-3-4 has been changed to 1-3-3-4 and 1-4-3-4 . Since the proteins were found associated with the outer membrane but could not be incorporated into it, it appears that sorting is less sensitive to alterations than assembly . A regulatory circuit was affected (missense mutants of outer membrane proteins can cause inhibition of synthesis of other such proteins); expression of the two rearranged genes effected a strong inhibition of synthesis of the unrelated porins OmpC and F as well as that of the maltoporin LamB and wild type OmpA . Hence, outer membrane proteins are designed not only for efficient membrane assembly but also for proper regulation of their synthesis. J Biol Chem, 1993 Dec 5, 268(34), 25636 - 42 Glucuronidation of hyodeoxycholic acid in human liver . Evidence for a selective role of UDP-glucuronosyltransferase 2B4; Pillot T et al.; Monospecific polyclonal antibodies were raised against a variable amino-terminal domain (amino acids 14-150) of a human liver form of UDP-glucuronosyl-transferase conjugating bile acids, UGT2B4 (Jackson, M . R., McCarthy, L . R., Harding, D., Wilson, S., Coughtrie, M . W., and Burchell, B . (1987) Biochem . J . 242, 581-588), expressed as a fusion protein in Escherichia coli . The antibodies were able to recognize the protein, stably expressed in a genetically engineered eukaryotic V79 cell line, against which they were directed . The specificity of these antibodies allowed their use for analyzing the substrate specificity of this isoform in human liver, as well as for determining its contribution to the total hepatic and extra-hepatic glucuronidation of hyodeoxycholic acid . Western blot analysis of microsomal proteins demonstrated the presence of UGT2B4 exclusively in human liver and not in human kidney . In human liver microsomes, the antibodies were able to inhibit and precipitate up to 90% of the total hyodeoxycholic acid 6-O-glucuronidation activity, but had no effect on activities toward several other substrates, such as phenols, bilirubin, or other bile acids, especially hyocholic acid and the steroids 4-hydroxyesterone and estriol . Moreover, Western blot analysis and immunoinhibition studies of human liver microsomes from healthy patients and from patients presenting liver diseases revealed a good correlation between the glucuronidation rate of hyodeoxycholic acid and the UGT2B4 expression level . The absence of immunoinhibition of hyodeoxycholic acid conjugation with UDP sugars other than UDP-glucuronic acid suggests the involvement of different enzymatic systems in the glucosidation and xylosylation of hyodeoxycholic acid . Altogether, the results provided strong evidence for the specific and predominant involvement of UGT2B4 in the 6-O-glucuronidation of this bile acid via a UDP-glucuronic acid-dependent mechanism. J Biol Chem, 1993 Dec 5, 268(34), 25547 - 52 The inactivation of dihydroxy-acid dehydratase in Escherichia coli treated with hyperbaric oxygen occurs because of the destruction of its Fe-S cluster, but the enzyme remains in the cell in a form that can be reactivated; Flint DH et al.; The enzyme dihydroxy-acid dehydratase previously has been shown to be inactivated in vivo in Escherichia coli within minutes of exposure to hyperbaric O2 . In this paper, we show its inactivation is due to the destruction of its catalytically active {4Fe-4S} cluster . The inactivation is not followed by an appreciable decrease in the amount of dihydroxy-acid dehydratase protein as determined by Western blots . Thus, the protein from the inactivated enzyme remains unproteolyzed in the cells . Dihydroxy-acid dehydratase activity recovers after the cells treated with hyperbaric O2 are returned to ambient oxygen . Since this recovery in activity is not accompanied by a significant increase in dihydroxy-acid dehydratase protein and is not prevented by chloramphenicol, it appears primarily to be due to reactivation of the previously inactivated enzyme . The reactivation occurs by reconstitution of the enzyme's Fe-S cluster . These results demonstrate that this enzyme can cycle between forms in which the Fe-S cluster is either present or absent . The facile ability to cycle between these two forms would be compatible with a regulatory role in addition to a catalytic role for this enzyme. J Biol Chem, 1993 Dec 5, 268(34), 25542 - 6 Comparison of kinetic properties between two mammalian ras p21 GDP/GTP exchange proteins, ras guanine nucleotide-releasing factor and smg GDP dissociation stimulation; Orita S et al.; The mammalian counterpart of the yeast ras p21 GDP/GTP exchange protein CDC25, ras GRF, was expressed in Escherichia coli and purified, and its kinetic properties were compared with those of another mammalian ras p21 GDP/GTP exchange protein, smg GDS . ras GRF was active on Ki- and Ha-ras p21s but inactive on rap1A p21, rhoA p21, rac1 p21, and rab3A p25, whereas smg GDS was active on Ki-ras p21, rap1A p21, rhoA p21, and rac1 p21 but inactive on Ha-ras p21 and rab3A p25 . The Kcat values of ras GRF and smg GDS for Ki-ras p21 as a common substrate were calculated to be 1.2 and 0.37 nmol/min/nmol, respectively . The Km values of ras GRF and smg GDS for Ki-ras p21 were 680 and 220 nM, respectively . rasGRF was slightly active on post-translationally unprocessed Ki-ras p21 but much more effective on post-translationally processed Ki-ras p21 than on post-translationally unprocessed Ki-ras p21 . smg GDS was active on post-translationally processed Ki-ras p21 but inactive on post-translationally unprocessed Ki-ras p21 . Moreover, as described for smg GDS, ras GRF showed a potency to inhibit the binding of Ki-ras p21 to membrane and to induce the dissociation of prebound Ki-ras p21 from the membrane . These results indicate that ras GRF and smg GDS show apparently similar kinetic properties except for the different substrate specificities and the requirement of the post-translational processing of Ki-ras p21. J Biol Chem, 1993 Dec 5, 268(34), 25369 - 75 Four contiguous amino acids define the target for streptolydigin resistance in the beta subunit of Escherichia coli RNA polymerase; Heisler LM et al.; Streptolydigin (stl), a bacteriostatic inhibitor of transcription elongation, interacts with the beta subunit of Escherichia coli RNA polymerase . We have defined the target for stl resistance using chemical mutagenesis and mutagenic polymerase chain reaction . Mutations resulting in stl resistance are confined to a small cluster of contiguous amino acids, amino acids 543 to 546 . These stlr mutants differ from one another in their levels of resistance to stl in vivo and in vitro . We have analyzed two of the mutants, A543V and F545S, for their effects on elongation and termination in vivo and in vitro . Neither affected termination at rho-dependent or rho-independent terminators . These mutants were indistinguishable from wild type in a T7 in vitro elongation assay . F545S, however, did exhibit slower elongation kinetics in a lambda tR1 pausing assay . We conclude that mutations in the stlr region can influence transcription elongation, but that these amino acids are not directly involved in catalysis. J Biol Chem, 1993 Dec 5, 268(34), 25334 - 42 Crystal structure of adenylosuccinate synthetase from Escherichia coli . Evidence for convergent evolution of GTP-binding domains; Poland BW et al.; The structure of the P2(1) crystal form of adenylosuccinate synthetase from Escherichia coli has been determined to a resolution of 2.8 A . The refined model for the enzyme gives an R factor of 0.20 and a root-mean-square deviation from expected bond lengths and angles of 0.016 A and 2.27 degrees, respectively . The dominant structural element of each monomer of the homodimer is a central beta-sheet of 10 strands . The first nine strands of the sheet are mutually parallel with right-handed crossover connections between the strands . The 10th strand is antiparallel with respect to the first nine strands . In addition, the enzyme has two antiparallel beta-sheets, comprised of two strands and three strands each, 11 alpha-helices and two short 3/10-helices . The overall fold of the polypeptide chain has not been observed heretofore in any other protein structure . Residues tentatively assigned to the active site of the enzyme on the basis of chemical modification and directed mutation cluster in two separate regions . Gly12, Gly15, Gly17, Lys18, Ile19, and Lys331 lie at one end of a crevice that measures 12 A by 30 A by 12 A deep . Lys140 and Arg147 are not part of this crevice, but instead lie at the interface between monomers of the dimer . Lys140 makes a salt link with Asp231 of a monomer related by molecular symmetry and Arg147 binds to the carbonyl of the same Asp231 . Superposition of the p21 ras protein (Pai, E . F., Kabsch, W., Krengel, U., Holmes, K., John, J., and Wittinghofer, A . (1989) Nature 341, 209-214) onto the synthetase reveals significant correspondences between side chains of the two proteins . Residues which interact with GTP in the p21ras protein have structurally equivalent residues in the synthetase . The GTP molecule, when transformed to the coordinate frame of the synthetase, falls into the crevice defined by studies in directed mutation . We suggest that the similarities in the GTP-binding domains of the synthetase and the p21ras protein are an example of convergent evolution of two distinct families of GTP-binding proteins. J Biol Chem, 1993 Dec 5, 268(34), 25296 - 301 The interplay of the GrpE heat shock protein and Mg2+ in RepA monomerization by DnaJ and DnaK; Skowyra D et al.; Genetic and biochemical studies have established that the sole function of the Escherichia coli DnaJ, DnaK, and GrpE heat shock proteins in plasmid P1 DNA replication is to convert RepA dimers to monomers . Monomers bind avidly to oriP1 DNA and initiate DNA replication . However, with purified heat shock proteins, only DnaJ, DnaK, and ATP were required for the monomerization of RepA; GrpE was not required . We have found reaction conditions that mimic the physiological situation . GrpE function is absolutely necessary for RepA activation in vitro with DnaJ and DnaK when the free Mg2+ concentration is maintained at a level of approximately 1 microM by a metal ion buffer system . EDTA or physiological metabolites, including citrate, phosphate, pyrophosphate, and ATP, all elicit the GrpE requirement . With these metal ion-buffering systems, GrpE specifically lowers the concentration of Mg2+ required for the RepA activation reaction . The absence of Mg2+ blocks activation and high levels of Mg2+ in solution bypass the requirement for GrpE but not for the other two heat shock proteins . Our results imply that GrpE facilitates the utilization of Mg2+ for an essential step in RepA activation. J Biol Chem, 1993 Dec 5, 268(34), 25425 - 31 Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase . Different mechanisms for the same activity; Ziemienowicz A et al.; In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can . In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required . GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP . The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP . GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES . The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated . We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay . However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein . Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex. Biophys J, 1993 Dec, 65(6), 2578 - 85 Removal of the transducer protein from sensory rhodopsin I exposes sites of proton release and uptake during the receptor photocycle; Olson KD et al.; The phototaxis receptor sensory rhodopsin-I (SR-I) was genetically truncated in the COOH terminus which leads to overexpression in Halobacterium salinarium and was expressed in the presence and absence of its transducer, HtrI . Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH probe to show that proton release to the bulk phase results from the SR-I587 to S373 photoconversion, but only in the absence of transducer . The stoichiometry is 1 proton/S373 molecule formed . When SR-I is overexpressed in the presence of HtrI, the kinetics of the thermal return of S373 to SR-I587 is biphasic . A kinetic dissection indicates that overexpressed SR-I is present in two pools: one pool which generates an SR-I molecule possessing a normal (i.e., transducer-interacting) pH-independent rate of S373 decay, and a second pool which shows the pH-dependent kinetics of transducer-free S373 decay . The truncated SR-I receptor functions normally based on the following criteria: (i) Truncated SR-I restores phototaxis (attractant and repellent responses) when expressed in a strain lacking native SR-I, but containing HtrI . (ii) The absorption spectrum and the flash-induced absorption difference spectrum are indistinguishable from those of native SR-I . (iii) The rate of decay of S373 is pH-dependent in the absence of HtrI but not in the presence of HtrI . The data presented here indicate that a proton-conducting path exists between the protonated Schiff base nitrogen and the extramembranous environment in the transducer-free receptor, and transducer binding blocks this path. Photochem Photobiol, 1993 Dec, 58(6), 831 - 5 Photoreactivation in a phrB mutant of Escherichia coli K-12: evidence for the role of a second protein in photorepair; Dorrell N et al.; In Escherichia coli, the light-dependent repair of pyrimidine dimers in UV-irradiated DNA is now accepted as being due to enzymatic photoreactivation (PR) by a 50 kDa enzyme, photolyase (EC 4.1.99.3) . The gene for this enzyme has been mapped at 16.2 min and designated phr . This gene was earlier described as phrB, another locus phrA having been proposed in association with PR . The relevance of the putative phrA gene has now been placed in doubt . The recent report of the discovery of a photoreactivating enzyme in Drosophila melanogaster, which specifically repairs pyrimidine (6-4) pyrimidone photoproducts ({6-4} photoproducts), and that E . coli does possess a protein with specific affinity for the (6-4) photoproduct, has cast new light on the prospective role of phrA in PR . We have determined the nucleotide sequence of the putative phrA gene, which suggests it codes for a protein of 38 kDa . When the putative phrA gene was cloned into an expression vector and transformed into a phrA phrB mutant of E . coli, a level of photorepair was observed, which could correspond to repair of (6-4) photoproducts. Immunology, 1993 Dec, 80(4), 633 - 9 The role of the phosphatidylinositol turnover in 12-hydroxyeicosatetraenoic acid generation from human platelets by Escherichia coli alpha-haemolysin, thrombin and fluoride; Konig B et al.; Activation of human platelets with either the Escherichia coli alpha-haemolysin or thrombin, or with pharmacological agonists such as sodium fluoride (NaF), Ca-ionophore A23187 or phorbol myristate acetate (PMA), induced a similar pattern of serotonin release, unlike 12-hydroxyeicosatetraenoic acid (12-HETE) generation . In the presence of neomycin (0.1, 1, 10 mM), an inhibitor of the phosphatidylinositol-specific phospholipase C (PIP2-PLC), the E . coli alpha-haemolysin-induced 12-HETE generation was enhanced up to threefold in a dose-dependent manner . 12-HETE generation by NaF and thrombin was slightly inhibited at high neomycin concentrations (10 mM) . Treatment of human platelets with E . coli alpha-haemolysin induced a different activation pattern of PIP2-PLC and phosphatidylinositol4-kinase (PI4-kinase), compared to NaF and thrombin . Haemolysin treatment resulted in a down-regulation of PIP2-PLC and PI4-kinase enzymatic activities by 20 +/- 9/40 +/- 8% compared to unstimulated cells; the decrease in enzymatic activities was observed within 2 min of stimulation and was still apparent after 60 min of stimulation . In NaF- and thrombin-stimulated platelets, dose- and time-dependent increases in PIP2-PLC (by up to 21 +/- 10%, 34 +/- 11%, respectively) and PI4-kinase (by up to 71 +/- 18, 54 +/- 14) activities were measured . Maximal enzymatic activities were reached after 5-20 min of stimulation (NaF, thrombin) followed by a decline to baseline levels (thrombin) or below baseline levels (NaF) . Our results indicate that the phosphatidylinositolphosphate metabolism plays an important role in the regulation of 12-HETE release from human platelets by E . coli alpha-haemolysin. Curr Genet, 1993 Dec, 24(6), 520 - 4 Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library; Gems DH et al.; Autonomously-replicating, marker-less "helper" plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration . This resulted in as much as a 200-fold increase in transformation efficiency . Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency . Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency . Using helper plasmids and an A . nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate . In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo. Protein Sci, 1993 Dec, 2(12), 2198 - 205 P22 Arc repressor: enhanced expression of unstable mutants by addition of polar C-terminal sequences; Milla ME et al.; Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization . Here we examine the effects of several different C-terminal extension sequences on the expression and activity of a set of Arc mutants . We show that two tail sequences, KNQHE (st5) and H6KNQHE (st11), increase the expression levels of most mutants from 10- to 20-fold and, in some cases, result in restoration of biological activity in the cell . A third tail sequence, HHHHHH (st6), was not as effective in increasing mutant expression levels . All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild-type Arc . The properties of the st11 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel-chelate affinity chromatography . Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st11 background . The thermodynamic stability of the EA28 mutant (delta delta Gu approximately -0.4 kcal/mol) is reduced modestly compared to the st11 parent, whereas the RL31 mutant (delta delta Gu approximately -3.0 kcal/mol) and SA32 mutant (delta delta Gu approximately -3.3 kcal/mol) are substantially less stable. Protein Sci, 1993 Dec, 2(12), 2066 - 76 Characterization of the NR1, NR2A, and NR2C receptor proteins; Kopke AK et al.; N-Methyl-D-aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies . The antibodies were directed against NR1-A, NR2A-N1, and NR2C-N1, representing N-terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1-E, NR2A-C1, and NR2C-C1, derived from C-terminal sequences of these subunits . The anti-NR1-D antibody was raised against the putative internal loop of NR1 . A size of 118 kDa was found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti-NR1-D and -NR1-E, but not anti-NR1-A . With the anti-NR1-A antibody, a 125-kDa protein was discovered that may represent a glutamate receptor not yet characterized . NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively . Enzymatic N-deglycosylation generated a 97-kDa protein from NR1, a 105-kDa protein from the 125-kDa protein, a 162-kDa protein from NR2A, and a 127-kDa protein from NR2C . In contrast to the deglycosylation product of the NR2A, the 97- and 127-kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data . These products may represent truncated proteins . The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum . In contrast, NR2C was solely found in the cerebellum . The 125-kDa protein was highest in the cerebellum and cortex. Biophys Chem, 1993 Dec, 48(2), 281 - 91 Detection of intermediate protein conformations by room temperature tryptophan phosphorescence spectroscopy during denaturation of Escherichia coli alkaline phosphatase; Mersol JV et al.; The reversible denaturation of Escherichia coli alkaline phosphatase (AP) was followed by monitoring changes in enzymatic activity as well as by measurements of the time-resolved room temperature phosphorescence from Trp 109 . It is well known that the denaturants, ethylene diamine tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCl) inactive AP by different mechanisms as reflected by differences in the time dependence of inactivation . However, further information about structural changes that result during inactivation is obtained by measurement of the phosphorescence intensity and radiative decay rate . Time-resolved tryptophan phosphorescence is exquisitely sensitive to changes in the local environment of the emitting residue, unlike the steady state phosphorescence intensity which is a composite of both the lifetime and concentration of the emitting protein species . The results show that while inactivation in EDTA proceeds by loss of the zinc ion as expected, denaturation in acid or GdnHCl produces a heterogeneous population of AP molecules, detected by a distribution analysis of the phosphorescence lifetime, which may reflect multiple pathways to the final unfolded state . Time-resolved phosphorescence also demonstrates the existence of an enzymatically active but structurally less rigid intermediate state during unfolding . As the rigidity decreases, the susceptibility to further denaturation decreases at lower pH but increases with GdnHCl concentration . The experiments provide new insight into the mechanism of denaturation of AP and demonstrate the sensitivity of time-resolved room temperature phosphorescence to the structural details of intermediate states produced during unfolding of proteins. J Appl Bacteriol, 1993 Dec, 75(6), 574 - 82 Genetic diversity of Helicobacter pylori indexed with respect to clinical symptomatology, using a 16S rRNA and a species-specific DNA probe; Desai M et al.; DNA probes are described which identify group and fingerprint strains of the human gastric pathogen Helicobacter pylori, on the basis of well-defined band homologies . A 544 bp internal fragment of the 16S ribosomal RNA gene was generated by polymerase chain reaction (PCR) with primers derived from the Escherichia coli rRNA gene sequence . In genomic Southern blots this probe detected restriction site variation around these loci, generating simple but strain-specific molecular fingerprints . A small conserved chromosomal fragment of 1.2 kbp, Hps, species-specific for H . pylori, was obtained by cloning random HindIII fragments into pUC19 . It was useful for dot-blot identification, and also separated isolates into one major and two minor groups . When results for these two probes were combined, a baseline characterization of genotype was obtained . A band-matching database of molecular fingerprints for the type strain and 63 clinical isolates of H . pylori from asymptomatic, ulcer and gastritis contexts is presented . No significant association between the genotypes at this level of definition and the associated clinical symptomatology of the isolates was detected. Plant Physiol, 1993 Dec, 103(4), 1361 - 7 3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase; Tai H et al.; A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated . Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA . A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated . Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III . Northern analysis confirmed that it was expressed in both leaf and root . The clone contained a 1218-bp open reading frame coding for 405 amino acids . The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein . The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E . coli, respectively . It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals. Protein Expr Purif, 1993 Dec, 4(6), 570 - 9 Expression in Escherichia coli of the cloned polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus; Lavallee C et al.; Cloned cDNA of genomic segment 10 of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was placed downstream from the lambda PL promoter in expression plasmid pRC23 and expressed in Escherichia coli cells . A polypeptide of the same molecular weight (28 kDa) as natural polyhedrin was synthesized at the level of approximately 10% of total host cell protein . This polypeptide was identified as CPV polyhedrin (r-polyhedrin) after comparative studies . The r-polyhedrin did not form any crystalline structure in E . coli cells but instead accumulated in the form of an insoluble inclusion body, even though natural polyhedrin is known to form a crystalline matrix (polyhedra) in infected insect cells . The purified r-polyhedrin complex, like natural polyhedra, was not soluble in neutral or acidic buffer but soluble in alkaline buffer . Upon solubilization, the r-polyhedrin complex did not undergo proteolytic degradation, while natural polyhedra were digested into small peptides by the associated protease . Incubation of r-polyhedrin with natural polyhedra in alkaline buffer, however, degraded the r-polyhedrin, resulting in an identical profile of peptide products to that of natural polyhedra . These results indicate that even though r-polyhedrin molecules produced in E . coli cells are not in the natural conformation, the molecules can present the identical cleavage sites to the polyhedra-associated alkaline protease . Experiments showed that the alkaline protease was associated with the matrix of polyhedra and not with virus particles. Protein Expr Purif, 1993 Dec, 4(6), 547 - 51 Purification and characterization of recombinant polymeric hemoglobin P1 of Glycera dibranchiata; Zafar RS et al.; The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli . The expressed globin was reconstituted with heme and purified . The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem . Biophys . Acta, 1041, 117-123, 1990) . Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin . The molar ellipticity at 222 nm provided an estimate of 77% for the alpha-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II . Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4). Protein Expr Purif, 1993 Dec, 4(6), 539 - 46 One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase; Serra EC et al.; Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988) . A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase . When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences . The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione . It showed both transferase and reductase activities . Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous . The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host . The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells . The system is potentially useful for the purification of activity-impaired forms of the flavoprotein. Protein Expr Purif, 1993 Dec, 4(6), 503 - 11 Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly; Borukhov S et al.; New improved methods were developed for the purification to apparent homogeneity of alpha, beta, beta', and sigma subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains . The purified subunits were assembled into enzymatically active RNAP holoenzyme (alpha 2 beta beta' sigma) using the optimal subunit molar ratio (alpha:beta:beta':sigma = 2:8:4:1) at a total protein concentration of 0.5 mg/ml . The presence of sigma subunit and 10 microM ZnCl2 in the reconstitution mixture increased the yield of RNAP approximately 4 times . The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56% . The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of {3H}UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h. Microsc Res Tech, 1993 Dec 1, 26(5), 457 - 65 In vivo effects of endotoxin on DNA synthesis in rat nasal epithelium; Harkema JR et al.; Airway inflammation in bacterial infections is characterized by the presence of neutrophils and often epithelial injury and repair . Release of endotoxin from bacteria may contribute to these processes . The purpose of this study was to determine the in vivo effects of repeated endotoxin exposure on DNA synthesis in rat nasal epithelium in the presence and absence of neutrophilic influx . Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls) . Before the first and third instillations, half of the saline and endotoxin-instilled animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum . Rats were sacrificed 6 or 24 h after the last instillation . Two hours prior to sacrifice, rats were intraperitoneally injected with bromodeoxyuridine (BrdU), an analog of thymidine that is incorporated in the nucleus of cells in the S-phase of the cell cycle . Nasal tissues were processed for light microscopy and immunohistochemical detection of BrdU in nasal epithelial cells . The numbers of nasal epithelial cells, BrdU-labeled epithelial nuclei, and neutrophils per millimeter of basal lamina in the epithelium lining the nasal turbinates in the proximal nasal passages were determined by morphometric analysis . We did not observe a neutrophilic influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, the numbers of nasal epithelial cells and the BrdU-labeling index were significantly increased compared to saline-instilled controls.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Dis Child, 1993 Dec, 69(6), 644 - 9 Development of IgG responses to mycobacterial antigens; Pilkington C et al.; Recent studies link mycobacterial and human heat shock protein antigens with autoimmune diseases . Little is known about the development of antibody responses to these antigens in children . IgG responses to mycobacterial antigens were studied in children living in the UK (an environment low in mycobacteria) who had not received BCG vaccination . Age curves of IgG response to sonicates from different species of mycobacteria were similar suggesting that the greater part of the developing IgG response is to the common antigens shared by all mycobacteria . The major part of the IgG response was to carbohydrate antigens: lipoarabinomannan is a mycobacterial cell wall carbohydrate and was confirmed as a major immunodominant antigen . Infants showed a marked early response to the mycobacterial 65 kilodalton (kDa) and 70 kDa heat shock proteins, but not to the human 65 kDa heat shock protein . The early IgG response to heat shock proteins may reflect cross reactivity to proteins released by a wide variety of bacteria (possibly from breakdown in the gut) or recognition of other immunodominant antigens with high levels of cross reactivity to self. Jpn J Clin Oncol, 1993 Dec, 23(6), 384 - 8 Splenic abscess associated with colon cancer: a case report; Kawamoto K et al.; The present report describes a case of colon cancer which presented with a rare complication of splenic abscess . A 52-year-old Japanese man with diarrhea, fever and chills was admitted to our hospital . He complained of fever, with chills at night, and abdominal pain occurring during the last month . The origin of the fever was investigated, and Escherichia coli grew from a blood culture . Multilocular splenic abscesses and wall thickening of the descending colon were revealed by CT scan, magnetic resonance imaging and ultrasound . A cancer of the descending colon was found by barium enema and colonoscopy . A curative resection was performed and the pathological report revealed the splenic abscess to have developed from a direct extension of, and perforation by, the carcinoma of the descending colon. FEMS Microbiol Lett, 1993 Dec 1, 114(2), 235 - 41 Molecular characterization of the p100 gene of Borrelia burgdorferi strain PKo; Jauris-Heipke S et al.; The p100 gene coding for the p100 protein of Borrelia burgdorferi strain PKo has been cloned, sequenced and expressed in Escherichia coli . An open reading frame including upstream and downstream sequences with potential translation and transcription signals could be identified . The reading frame consists of 1989 nucleotides corresponding to a protein of 663 amino acids and a calculated molecular mass of 75.8 kDa . The protein has a leader peptide and is processed without modification at the N-terminus . A high percentage of amino acid sequence identity could be found to the high-molecular mass protein p83/p93 of B . burgdorferi strain B31. FEMS Microbiol Lett, 1993 Dec 1, 114(2), 179 - 84 Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120; Elhai J; The strengths of several promoters were assessed in the cyanobacterium Anabaena PCC 7120 by fusing them to luxAB, encoding bacterial luciferase . Two promoters, Ptac and PpsbA, with sequences nearly identical to consensus Escherichia coli sigma 70 promoters, gave as high or higher expression than the strong Anabaena promoter, Prbc . Pnpt, the natural promoter driving expression of the kanamycin-resistance determinant from Tn5, was poorly expressed in Anabaena . The Lac repressor partially repressed expression from Ptac, permitting regulated expression in Anabaena after induction with isopropyl thiogalactoside to a level 4-5-fold higher than without inducer. Am J Physiol, 1993 Dec, 265(6 Pt 1), E920 - 7 Effects of endotoxin on pigs prefed omega-3 vs . omega-6 fatty acid-enriched diets; Murray MJ et al.; We investigated in a porcine model whether omega-3 fatty acids modify the physiological response to sepsis . For 8 days, 16 male pigs were fed a diet containing 18% fat by weight enriched with either omega-3 or omega-6 fatty acids (FA) . A group of six pigs receiving their regular diet served as controls . The omega-3 FA-supplemented pigs had elevated levels of omega-3 FA in their serum-free FA, serum phospholipid (PL), and platelet PL levels compared with either of the other groups . On the ninth day, the unanesthetized pigs were injected with 0.3 mg/kg of endotoxin (Escherichia coli) intravenously . The animals had a significant decrease in their arterial O2 pressure (PaO2) {from 84.4 +/- 6.8 (SD) to 64 +/- 9.4, and from 83.1 +/- 7.2 to 55.9 +/- 6.3 mmHg in the omega-6 FA and regular diet groups, respectively} . The PaO2 did not decrease in the omega-3 FA pigs . The omega-3 FA group had significantly lower pulmonary vascular resistance (541 +/- 205 dyn.s.cm-5) 20 min after endotoxin compared with either the omega-6 FA or regular diet groups (797 +/- 233 and 1,102 +/- 552 dyn.s.cm-5, respectively) and more normal blood pressure compared with the other two groups . Plasma thromboxane (Tx) B2 and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) levels were lowest in the omega-3 FA diet group and highest in the regular diet group.(ABSTRACT TRUNCATED AT 250 WORDS) Radiat Res, 1993 Dec, 136(3), 397 - 403 A simple method to assess in vivo repair of ultraviolet radiation-induced lesions of specific DNA sequences of restriction sites; Kovacs A et al.; Based on the direct relationship between ultraviolet irradiation of DNA and its susceptibility to restriction endonucleases, we have devised a simple method to quantify in vivo damage and repair of selected restriction sites . The simplicity and power of the method were demonstrated with HindIII restriction endonuclease and pAc360-501-beta-gal plasmid in UV-irradiated Escherichia coli cells . The large number of available restriction endonucleases makes the method quite flexible . This method provides a simple and inexpensive means to screen mutagenic and antimutagenic drugs that interact with the DNA repair mechanism. J Parasitol, 1993 Dec, 79(6), 900 - 7 Localization of a low abundance membrane protein (Bm86) on the gut cells of the cattle tick Boophilus microplus by immunogold labeling; Gough JM et al.; A preembedding immunogold technique was used to locate Bm86, an antigen from the gut digest cells of the cattle tick Boophilus microplus . Gut from partially engorged female ticks was everted to expose the cells, lightly fixed in 4% paraformaldehyde, and then incubated in rabbit antisera against a recombinant form of Bm86 . Following incubation in a secondary antibody conjugated to 1-nm colloidal gold, Bm86 antigenic sites were visualized for both light and electron microscopy using silver enhancement . Bm86 was shown to be located predominantly on the microvilli of digest cells . Antiserum against a nonglycosylated Escherichia coli recombinant form of Bm86 was used to avoid cross-reactivity with carbohydrate epitopes of other digest cell proteins. J Gen Virol, 1993 Dec, 74 ( Pt 12), 2807 - 12 Characterization of vaccinia virus gene B12R; Banham AH et al.; We report the characterization of vaccinia virus gene B12R which is predicted to encode a 33K protein with 36% amino acid identity to the serine/threonine protein kinase encoded by vaccinia virus gene B1R . S1 nuclease protection experiments showed that gene B12R is transcribed early during infection from an initiation site 11 bp upstream of the open reading frame (ORF) . The gene encodes a 33K polypeptide that is not required for virus replication in tissue culture nor for virus virulence in a murine intranasal model . Expression of the B12R gene in Escherichia coli produced an abundant 33K polypeptide which lacked protein kinase activity under conditions in which the protein kinases encoded by vaccinia virus gene B1R and African swine fever virus gene j9L are active. J Gen Virol, 1993 Dec, 74 ( Pt 12), 2645 - 52 High level expression in Escherichia coli cells and purification of poliovirus protein 2Apro; Martinez-Abarca F et al.; The poliovirus protease 2Apro has been produced to high levels in Escherichia coli using the inducible system that utilizes T7 RNA polymerase . The protease coding sequences that contained an additional AUG to start translation were cloned in pET vectors . Synthesis of 2Apro was induced by IPTG or IPTG plus rifampicin, the levels of the protein made being higher when IPTG alone was used . The expression of the protein is not toxic for E . coli cells and can be readily visualized by Coomassie blue staining of total bacterial protein extracts separated in polyacrylamide gels . Centrifugation of the broken bacterial cells sediments more than 95% of the 2Apro synthesized at a 95% purity level after sarkosyl treatment . Antibodies raised against 2Apro in E . coli recognize a 16K protein in poliovirus-infected cells . In addition, 2Apro shows activity in trans as measured by the cleavage of p220 in HeLa cell extracts and by cleavage of a poliovirus protein substrate that contains the junction between the P1 and P2 polypeptides. Genes Dev, 1993 Dec, 7(12B), 2618 - 28 The activity of sigma E, an Escherichia coli heat-inducible sigma-factor, is modulated by expression of outer membrane proteins; Mecsas J et al.; sigma E and sigma 32 are two heat- and ethanol-inducible sigma-factors in Escherichia coli . The sigma 32 regulon is also induced by unfolded and misfolded proteins in the cytoplasm, and the function of many of the proteins in the sigma 32 regulon is to bind to cytoplasmic proteins and assist them in folding or unfolding . To further understand the function of the sigma E regulon, we searched for mutants that affected sigma E activity . Our results indicate that a signal generated by expression of outer membrane proteins modulates sigma E activity . Specifically, sigma E activity is induced by increased expression of OMPs and is reduced by decreased expression of OMPs . In addition, mutations that cause misfolded OMPs induce sigma E activity . This signal is generated after the fate of OMPs and periplasmic proteins diverge in the secretory pathway and is not the result of an accumulation of OMP precursors in the cytoplasm . Our results indicate that this effect of OMPs is specific to the sigma E regulon, because none of the above mutations affect sigma 32 activity . We propose that the sigma E regulon is involved in processes that occur in extracytoplasmic compartments and that these two heat-inducible regulons may have distinct but complementary roles of monitoring the state of proteins in the cytoplasm (sigma 32) and outer membrane (sigma E). Arch Biochem Biophys, 1993 Dec, 307(2), 405 - 10 Identification, cloning, and expression of the gene for adenylate kinase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius; Kath T et al.; An adenylate kinase gene from a member of the archaebacterial kingdom, the thermoacidophilic archaebacterium (archaeon) Sulfolobus acidocaldarius, has been cloned and sequenced for the first time . Two degenerate oligonucleotide probes, based on the N-terminal amino acid sequence information, led to the amplification of a gene-specific DNA fragment, used to screen subgenomic libraries . Comparing the DNA-derived amino acid sequence of total 194 residues with those of known procaryotic and eucaryotic adenylate kinases revealed only a low degree of similarity, except for a glycine-rich region close to the N-terminus, the so-called P-loop . Using inducible expression systems catalytically active S . acidocaldarius adenylate kinase was produced in large amounts . Although the total length of the protein and the results of alignment procedures suggest a closer relation to eucaryotic than to procaryotic sequences, the archaebacterial enzyme may represent a novel class of adenylate kinases . This is corroborated by the finding that an antiserum against this protein does not cross-react with Escherichia coli nor yeast or rabbit adenylate kinases for example. Arch Biochem Biophys, 1993 Dec, 307(2), 350 - 4 Site-specific mutagenesis of the metal binding sites of porcine fructose-1,6-bisphosphatase; Chen L et al.; Mutations in the metal binding sites of porcine fructose-1,6-bisphosphatase were carried out by site-specific mutagenesis based on the crystal structure of the enzyme . The mutant and wild-type enzymes have been characterized by circular dichroism spectrometry and initial-rate kinetics . One of the mutant forms of fructose-1,6-bisphosphatase (Glu280Gln) is associated with a single metal site, whereas two other mutants (Glu97Gln and Asp118Asn) ligate two metal ions . The mutant enzymes exhibit very large decreases in kcat relative to the wild-type enzyme; however, other kinetic parameters, such as Km values, are not greatly altered . Metal binding cooperativity and binding affinity is decreased in the mutants compared to wild-type fructose-1,6-bisphosphatase . Mutations in the metal binding sites greatly enhance the enzyme's affinity for AMP, a potent regulator of fructose-1,6-bisphosphatase activity . The results of these investigations are fully consistent with predictions made on the role of specific amino acid residues at the metal binding sites in porcine fructose-1,6-bisphosphatase from X-ray diffraction studies {Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W.N . (1993) Biochemistry, 32, 1844-1857}. Arch Biochem Biophys, 1993 Dec, 307(2), 342 - 9 Molecular cloning of a novel isotype of Mg(2+)-dependent protein phosphatase beta (type 2C beta) enriched in brain and heart; Terasawa T et al.; Two complementary DNA (cDNA) clones (pTK-1 and -2) encoding two distinct isotypes of mouse Mg(2+)-dependent protein phosphatase beta (MPP beta-1 and -2, respectively) were isolated from a melanocyte cDNA library . Although mouse pTK-1 is orthologous to the rat cDNA (JW5) reported previously {Wenk, J., Trompeter, H.I., Pettrich, K.G., Cohen, P.T.W., Campbell, D.G., and Mieskes, G . (1992) FEBS Lett . 297, 135-138}, pTK-2 is a novel cDNA clone . It was strongly suggested that the pTK-1 and -2 cDNAs are splicing variants of a single pre-mRNA . The difference in the amino acid sequences between MPP beta-1 and -2 was observed only at the carboxy-terminal regions . Both the recombinant MPP beta-1 and -2 expressed in Escherichia coli cells were immunoreactive to an anti-MPP beta antibody and exhibited Mg(2+)-dependent and okadaic acid-insensitive protein phosphatase activities with similar substrate specificities . Although the mRNA of MPP beta-1 was expressed ubiquitously in various mouse tissues, that of MPP beta-2 was expressed exclusively in brain and heart . These results suggest the difference in the physiological roles of these two enzyme isotypes. Arch Biochem Biophys, 1993 Dec, 307(2), 248 - 52 Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase; McMichael RW Jr et al.; Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation . When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with {gamma-32P}ATP the enzyme is phosphorylated by a copurifying protein kinase . In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography . Three distinct 32P-phosphopeptides were resolved . Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys . This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS {Klein, R . R., Crafts-Brandner, S . J., and Salvucci, M . E . (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W . P., MacRae, E., Krause, K.-P., and Stitt, M . (1993) Planta 189, 174-181} . Identification of the phosphoseryl residue was accomplished by manual Edman sequencing . The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced . An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS . Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158 . These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS. J Environ Sci Health B, 1993 Dec, 28(6), 763 - 77 Aluminum interaction with human brain tau protein phosphorylation by various kinases; el-Sebae AH et al.; Phosphorylation is an indispensable process for energy and signal transduction in biological systems . AlCl3 at 10 nM to 10 microM range activated in-vitro {gamma-32P}ATP phosphorylation of the brain (tau) tau protein in both normal human or E . coli expressed tau forms; in the presence of the kinases P34, PKP, and PKC . However, higher concentrations of ALCl3 inhibited the tau phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level . AlCl3 at 100 microM to 500 microM range induced non-enzymatic phosphorylation of tau with gamma-ATP, gamma-GTP, and alpha-GTP . AlCl3 activated histone phosphorylation by P34 in a similar pattern . The hyperphosphorylation of tau by Al3+ was accompanied by molecular shift and mobility retardation in SDS-PAGE . This may demonstrate the mechanism of the longterm neurological effect of Al3+ in human brain leading to the formation of the neurofibrillary tangles related to Alzheimer's disease. Eur J Biochem, 1993 Dec 1, 218(2), 657 - 67 Recombinant iron-regulatory factor functions as an iron-responsive-element-binding protein, a translational repressor and an aconitase . A functional assay for translational repression and direct demonstration of the iron switch; Gray NK et al.; The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the iron-regulatory factor . Iron-regulatory factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco) . An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein . Both hIRFvac and hIRFeco bound specifically to iron-responsive elements and were immunoprecipitated by iron-regulatory-factor antibodies . Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract . In addition, hIRFvac and hIRFeco were shown to display aconitase activity . Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in iron-responsive-element-binding of iron-regulatory factor, but caused a strong stimulation of its aconitase activity . The results establish that recombinant iron-regulatory factor is a bifunctional protein; after purification, it binds to iron-responsive elements and represses translation in vitro . Following iron treatment, iron-responsive-element binding is lost and aconitase activity is gained . No eukaryotic co-factor seems to be required for the conversion of the iron-responsive-element binding to the aconitase form of the protein. Eur J Biochem, 1993 Dec 1, 218(2), 327 - 34 Biosynthetic 15N and 13C isotope labelling of glutathione in the mixed disulfide with Escherichia coli glutaredoxin documented by sequence-specific NMR assignments; Bushweller JH et al.; A biosynthetic procedure for obtaining 13C-15N doubly labelled glutathione from readily available precursor molecules is described . Isolation of the mutant Escherichia coli {C14S}glutaredoxin from E . coli cultures grown on 15N-13C doubly labelled media in the absence of reducing agents yields the mixed disulfide labelled in both the protein and the glutathione . 15N NMR assignments for glutathione obtained from two-dimensional {15N,1H}-correlation spectroscopy (COSY), and 13C NMR assignments for the entire mixed disulfide obtained from combined use of three-dimensional ct-HA{CAN}HN experiments and HCCH-total correlation spectroscopy ({HCCH}-TOCSY) demonstrated unequivocally that the glutathione is uniformly labelled with b