Biochim Biophys Acta, 1998 Apr 23, 1384(1), 121 - 9 Functional implications of the 21-24 loop in recombinant prochymosin; Li H et al.; To investigate the role of the 21-24 (pepsin numbering) loop in prochymosin, the amino acid residues GTPP at positions 21 through 24 were replaced with GG, the equivalent loop residues from its homologous protein, penicillopepsin, or SG, GS by site-directed mutagenesis . The mutants except GTPP(21-24)GS could be expressed in Escherichia coli . Activation studies indicated that the refolded prochymosin mutants were capable of undergoing autocatalytic activation to produce pseudochymosin by cleaving its N-terminal 27 amino acid residues at pH 2 . The resulting pseudochymosin mutants were able to convert into chymosin at pH 5.5 by further autocatalytic cleavage to remove additional 15 amino acid residues . These results demonstrate that the prochymosin analogs can fold into an active state from an unfolded state and that the pseudochymosin analogs can proceed in the transformation from one active form into another active form . Spectroscopic analyses revealed that after mutation the far UV CD spectrum of prochymosin was considerably modified, showing less negative ellipticity values, and the fluorescence emission intensities of prochymosin and pseudochymosin were remarkably reduced . The stabilities of prochymosin and pseudochymosin, especially, were dramatically decreased . The stabilization energy of prochymosin was reduced by 7-8 kJ/mol . The inactivation temperature of pseudochymosin was decreased by 15-20 degrees C . The wild-type pseudochymosin was stable at pH 1.5 and 6.5, whereas the mutants were completely inactivated at the same pH values . Taken together, it is reasonable to conclude that the 21-24 loop (GTPP) plays an important role in determining the stability of prochymosin and pseudochymosin, although the mutants with mutated loop (GG or SG) still can refold into an active conformation. Infect Immun, 1998 Jun, 66(6), 2905 - 13 Signal transduction during Legionella pneumophila entry into human monocytes; Coxon PY et al.; Legionella pneumophila causes Legionnaires' disease by replication in alveolar macrophages and monocytes . The bacteria are internalized most efficiently by opsonin-dependent, CR3-mediated phagocytosis . This investigation focused on determining the role of actin polymerization and phosphorylation signals in this uptake mechanism . Uptake inhibition assays and confocal microscopic analysis indicated that entry of L . pneumophila activated tyrosine kinase (TK) and protein kinase C (PKC) and induced actin polymerization at the site of bacterial entry . Upon L . pneumophila entry, six major cellular proteins (75, 71, 59, 56, 53, and 52 kDa) were TK phosphorylated in soluble fractions of monocytes, and three of these proteins (52, 53, and 56 kDa) were consistently found in insoluble (i.e., cytoskeletal) fractions of monocytes as well . Tyrosine phosphorylation was suppressed when cells were pretreated with the kinase inhibitor genistein, tyrphostin, or staurosporine . A similar tyrosine-phosphorylated protein pattern was observed with CR3-mediated entry of avirulent L . pneumophila, Escherichia coli, or zymosan into monocytes . This study has shown that PKC and TK signals which activate actin polymerization during the process of phagocytosis are induced upon L . pneumophila entry . In addition, CR3 receptor-mediated phagocytosis into monocytes may involve tyrosine phosphorylation of similar proteins, regardless of the particle being phagocytosed . Therefore, the tyrosine-induced phosphorylation observed during opsonized L . pneumophila entry is not a virulence-associated event. Infect Immun, 1998 Jun, 66(6), 2879 - 86 Rectal and intranasal immunizations with recombinant urease induce distinct local and serum immune responses in mice and protect against Helicobacter pylori infection; Kleanthous H et al.; To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route . When mice were immunized with rUre (25 microg p.o . or rectally or 10 microg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H . pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls . Quantitative H . pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05) . Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o . immunization was greater than after i.n . administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H . pylori . However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40 . 89 ng/ml), which was detectable prior to challenge with H . pylori . Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4(+) T cells showed levels of recruitment to be dependent upon challenge with H . pylori and equivalent for all routes . These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization. Infect Immun, 1998 Jun, 66(6), 2782 - 90 Expanded CD14+ CD16+ monocyte subpopulation in patients with acute and chronic infections undergoing hemodialysis; Nockher WA et al.; Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections . The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcgamma receptor type III (CD16) . We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients . In healthy controls CD14+ CD16+ monocytes account for 8% +/- 4% of CD14+ monocytes, with an absolute number of 29 +/- 14 cells/microl . In stable HD patients the CD14+ CD16+ subpopulation was significantly elevated (14% +/- 3%, or 66 +/- 28 cells/microl), while the number of CD14(++) monocytes (monocytes strongly positive for CD14) remained constant . In HD patients suffering from chronic infections a further rise in CD14+ CD16+ monocytes was observed (128 +/- 71 cells/microl; P < 0.01) such that this subpopulation constituted 24% of all blood monocytes . In contrast, numbers of CD14++ cells did not change compared to those for stable HD patients, indicating that the CD14+ CD16+ monocyte subpopulation was selectively expanded . During acute infections the CD14+ CD16+ cell subpopulation always expanded . A whole-blood assay revealed that CD14+ CD16+ monocytes exhibited a higher phagocytosis rate for Escherichia coli bacteria than CD14++ monocytes, underlining their role during host defense . In addition, CD14+ CD16+ monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC) . Thus, CD14+ CD16+ cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients. Infect Immun, 1998 Jun, 66(6), 2655 - 9 Differential effects of myeloperoxidase-derived oxidants on Escherichia coli DNA replication; Rosen H et al.; The microbicidal myeloperoxidase (MPO)-H2O2-chloride system strongly inhibits Escherichia coli DNA synthesis . Also, cell envelopes from MPO-treated E . coli cells lose their ability to interact with hemimethylated DNA sequences of oriC, the chromosomal origin of replication, raising the prospect that suppression of DNA synthesis involves impairment of oriC-related functions (H . Rosen, et al . Proc . Natl . Acad . Sci . USA, 87:10048-10052, 1990) . To evaluate whether origin-specific DNA sequences play a role in the MPO effect on E . coli DNA synthesis, plasmid DNA replication was compared to total (chromosomal) DNA replication for six plasmids with three distinct origins of replication . Plasmid pCM700 replication, replicating from oriC, was as sensitive to MPO-mediated inhibition as was total (chromosomal) DNA replication . A regression line describing this relationship had a slope of 0.90, and the r2 was 0.89 . In contrast, the replication activities of three of four non-oriC plasmids, pUC19, pACYC184, and pSC101, demonstrated significant early resistance to inhibition by MPO-derived oxidants . The exception to this resistance pattern was plasmid pSP102, which has an origin derived from P1 phage . pSP102 replication declined similarly to that of total DNA synthesis . The regression line for pSP102 replication versus total DNA synthesis had a slope of 0.95, and the r2 was 0.92 . The biochemical requirements for P1-mediated replication are strikingly similar to those for oriC-mediated replication . It is proposed that one of these requirements, common to oriC and the P1 origin but not critical to the replication of the other non-oriC plasmids, is an important target for MPO-mediated oxidations that mediate the initial decline in E . coli chromosomal DNA synthesis. Infect Immun, 1998 Jun, 66(6), 2576 - 86 Molecular characterization of Mycoplasma arthritidis variable surface protein MAA2; Washburn LR et al.; Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability . Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M . arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2 . Triton X-114 partitioning and metabolic labeling with {3H}palmitic acid suggested lipid modification of MAA2 . Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y . The maa2 genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats . The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide . The maa2 gene was expressed in Escherichia coli from the lacZ promoter of vector pGEM-T . The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E . coli . The maa2 gene and upstream DNA sequences were cloned from M . arthritidis clonal variants differing in MAA2 expression state . Expression state correlated with the length of a poly(T) tract just upstream of a putative -10 box . Full-sized recombinant MAA2 was expressed in E . coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region. Infect Immun, 1998 Jun, 66(6), 2494 - 500 Escherichia coli cytotoxic necrotizing factor 1 effaces microvilli and decreases transmigration of polymorphonuclear leukocytes in intestinal T84 epithelial cell monolayers; Hofman P et al.; Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic Escherichia coli strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho . Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A . Nusrat, M . Giry, J . R . Turner, S . P . Colgan, C . A . Parkos, E . Lemichez, P . Boquet, and J . L . Madara, Proc . Natl . Acad . Sci . USA 92:10629-10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration . Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin . However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction . CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs . Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria. Genes Dev, 1998 May 15, 12(10), 1425 - 37 clr-1 encodes a receptor tyrosine phosphatase that negatively regulates an FGF receptor signaling pathway in Caenorhabditis elegans; Kokel M et al.; Receptor tyrosine phosphatases have been implicated in playing important roles in cell signaling events by their ability to regulate the level of protein tyrosine phosphorylation . Although the catalytic activity of their phosphatase domains has been well established, the biological roles of these molecules are, for the most part, not well understood . Here we show that the Caenorhabditis elegans protein CLR-1 (CLeaR) is a receptor tyrosine phosphatase (RTP) with a complex extracellular region and two intracellular phosphatase domains . Mutations in clr-1 result in a dramatic Clr phenotype that we have used to study the physiological requirements for the CLR-1 RTP . We show that the phosphatase activity of the membrane-proximal domain is essential for the in vivo function of CLR-1 . By contrast, we present evidence that the membrane-distal domain is not required to prevent the Clr phenotype in vivo . The Clr phenotype of clr-1 mutants is mimicked by activation of the EGL-15 fibroblast growth factor receptor (FGFR) and is suppressed by mutations that reduce or eliminate the activity of egl-15 . Our data strongly indicate that CLR-1 attenuates the action of an FGFR-mediated signaling pathway by dephosphorylation. Gene, 1998 Apr 28, 211(1), 57 - 62 Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases; Wada K et al.; Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned . The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs . MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs . The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively . Recombinant proteins of C . elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs . Digestion of gelatin was observed only with MMP-C31 . Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C . elegans MMPs are structurally closely related with those of mammalian MMPs. Genetics, 1998 May, 149(1), 7 - 16 Single-strand DNA-specific exonucleases in Escherichia coli . Roles in repair and mutation avoidance; Viswanathan M et al.; Mutations in the genes encoding single-strand DNA-specific exonucleases (ssExos) of Escherichia coli were examined for effects on mutation avoidance, UV repair, and conjugational recombination . Our results indicate complex and partially redundant roles for ssExos in these processes . Although biochemical experiments have implicated RecJ exonuclease, Exonuclease I (ExoI), and Exonuclease VII (ExoVII) in the methyl-directed mismatch repair pathway, the RecJ- ExoI- ExoVII- mutant did not exhibit a mutator phenotype in several assays for base substitution mutations . If these exonucleases do participate in mismatch excision, other exonucleases in E . coli can compensate for their loss . Frameshift mutations, however, were stimulated in the RecJ- ExoI- ExoVII- mutant . For acridine-induced frameshifts, this mutator effect was due to a synergistic effect of ExoI- and ExoVII- mutations, implicating both ExoI and ExoVII in avoidance of frameshift mutations . Although no single exonuclease mutant was especially sensitive to UV irradiation, the RecJ- ExoVII- double mutant was extremely sensitive . The addition of an ExoI- mutation augmented this sensitivity, suggesting that all three exonucleases play partially redundant roles in DNA repair . The ability to inherit genetic markers by conjugation was reduced modestly in the ExoI- RecJ- mutant, implying that the function of either ExoI or RecJ exonucleases enhances RecBCD-dependent homologous recombination. J Biol Chem, 1998 May 22, 273(21), 13307 - 12 Ada protein-RNA polymerase sigma subunit interaction and alpha subunit-promoter DNA interaction are necessary at different steps in transcription initiation at the Escherichia coli Ada and aidB promoters; Landini P et al.; The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes . This region is also a binding site for the alpha subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter . In this report, we show that deletion of the C-terminal domain of the alpha subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters . We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma70 subunit (amino acids 574-613) . Several negatively charged residues in the sigma70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription . Based on these observations, we propose that the role of the alpha subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters . However, transcription initiation is dependent on meAda-sigma70 interaction. J Biol Chem, 1998 May 22, 273(21), 13264 - 72 Assembly of iron-sulfur clusters . Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii; Zheng L et al.; An enzyme having the same L-cysteine desulfurization activity previously described for the NifS protein was purified from a strain of Azotobacter vinelandii deleted for the nifS gene . This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly . Like NifS, IscS is a pyridoxal-phosphate containing homodimer . Information gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair A . vinelandii genomic segment that includes the iscS gene . The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contained within the major nif cluster of A . vinelandii previously designated orf6 . These genes have been designated iscU and iscA, respectively . Information available from complete genome sequences of Escherichia coli and Hemophilus influenzae reveals that they also encode iscSUA gene clusters . A wide conservation of iscSUA genes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formation or repair of iron-sulfur clusters . The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A . vinelandii is supported by the presence of a cysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUA genes . O-Acetylserine synthase is the product of the cysE gene, and it catalyzes the rate-limiting step in cysteine biosynthesis . A similar cysE-like gene is also located within the nif gene cluster of A . vinelandii . The likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation . Another feature of the iscSUA gene cluster region from A . vinelandii is that E . coli genes previously designated as hscB, hscA, and fdx are located immediately downstream from, and are probably co-transcribed with, the iscSUA genes . The hscB, hscA, and fdx genes are also located adjacent to the iscSUA genes in both E . coli and H . influenzae . The E . coli hscA and hscB gene products have previously been shown to bear primary sequence identity when respectively compared with the dnaK and dnaJ gene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function . The close proximity and apparent co-expression of iscSUA and hscBA in A . vinelandii indicate that the proposed chaperone function of the hscBA gene products could be related to the maturation of iron-sulfur cluster-containing proteins . Attempts to place non-polar insertion mutations within either A . vinelandii iscS or hscA revealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele . These results reveal a very strong selective pressure against the maintenance of A . vinelandii iscS or hscA knock-out mutations and suggest that such mutations are either lethal or highly deleterious . In contrast to iscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained . These results show that the cysE-like gene located upstream from iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcription units. J Biol Chem, 1998 May 22, 273(21), 13255 - 63 Rrp6p, the yeast homologue of the human PM-Scl 100-kDa autoantigen, is essential for efficient 5.8 S rRNA 3' end formation; Briggs MW et al.; The eukaryotic 25 S, 18 S, and 5.8 S rRNAs are synthesized as a single transcript with two internal transcribed spacers (ITS1 and ITS2), which are removed by endo- and exoribonucleolytic steps to produce mature rRNA . Genetic selection for suppressors of a polyadenylation defect yielded two cold-sensitive alleles of a gene that we named RRP6 (ribosomal RNA processing) . Molecular cloning of RRP6 revealed its homology to a 100-kDa human, nucleolar PM-Scl autoantigen and to Escherichia coli RNase D, a 3'-5' exoribonuclease . Recessive mutations in rrp6 result in the accumulation of a novel 5 . 8 S rRNA processing intermediate, called 5.8 S*, which has normal 5' ends, but retains approximately 30 nucleotides of ITS2 . Pulse-chase analysis of 5.8 S rRNA processing in an rrp6- strain revealed a precursor-product relationship between 5.8 S* and 5.8 S rRNAs, suggesting that Rrp6p plays a role in the removal of the last 30 nucleotides of ITS2 from 5.8 S precursors . A portion of 5.8 S* rRNA assembles into 60 S ribosomes which form polyribosomes, suggesting that they function in protein synthesis . These findings indicate that Rrp6p plays a role in 5.8 S rRNA 3' end formation, and they identify a functional intermediate in the rRNA processing pathway. J Biol Chem, 1998 May 22, 273(21), 13230 - 5 Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7); Igarashi M et al.; A newly identified member of the fibroblast growth factor (FGF) family, designated FGF-10, is expressed during development and preferentially in adult lung . The predicted FGF-10 protein is most related to keratinocyte growth factor (KGF, or FGF-7) . The latter is unique among FGFs in that it binds and signals only through the FGF receptor (FGFR2b) isoform KGF receptor (KGFR) expressed specifically by epithelial cells . In order to examine the biological and biochemical properties of human FGF-10, we isolated the cDNA and expressed its encoded protein in bacteria . The recombinant protein (rFGF-10) was a potent mitogen for Balb/MK mouse epidermal keratinocytes with activity detectable at 0.1 nM and maximal at around 5 nM . Within this concentration range, FGF-10 did not stimulate DNA synthesis in NIH/3T3 mouse fibroblasts . rFGF-10 bound the KGFR with high affinity comparable to that of KGF, and did not bind detectably to either the FGFR1c (Flg) or FGFR2c (Bek) receptor isoforms . The mitogenic activity of FGF-10 could be distinguished from that of KGF by its different sensitivity to heparin and lack of neutralization by a KGF monoclonal antibody . These results indicate that FGF-10 and KGF have similar receptor binding properties and target cell specificities, but are differentially regulated by components of the extracellular matrix. J Biol Chem, 1998 May 22, 273(21), 13037 - 46 Identification of a nickel(II) binding site on hemoglobin which confers susceptibility to oxidative deamination and intramolecular cross-linking; Levine J et al.; Complexation of Ni(II) with native state recombinant hemoglobin is shown to produce NH2-terminal deamination and globin cross-linking in the presence of the oxidant potassium peroxymonosulfate (OxoneTM) . Both the oxidative deamination and cross-linking are exclusive to the beta chains . Recombinant hemoglobin mutants have been created to identify protein sequence requirements for these reactions . It was found that His-2 of the beta globin is required for redox active Ni(II) complexation, oxidative deamination, and cross-linking . The oxidative deamination results in the formation of a free carbonyl in place of the NH2-terminal amine of the beta chain . Most cross-linking of the beta globin occurs intramolecularly, forming beta globin dimers . Structural characterization of the beta globin dimers indicates the presence of heterogeneous cross-links within the central hemoglobin cavity between the NH2 terminus of one beta chain and the COOH-terminal region of the other. J Biol Chem, 1998 May 22, 273(21), 12887 - 92 Involvement of molecular chaperonins in nucleotide excision repair . Dnak leads to increased thermal stability of UvrA, catalytic UvrB loading, enhanced repair, and increased UV resistance; Zou Y et al.; UvrA is one of the key Escherichia coli proteins involved in removing DNA damage during the process of nucleotide excision repair . The relatively low concentrations (nanomolar) of the protein in the normal cells raise the potential questions about its stability in vivo under both normal and stress conditions . In vitro, UvrA at low concentrations is shown to be stabilized to heat inactivation by E . coli molecular chaperones DnaK or the combination of DnaK, DnaJ, and GrpE . These chaperone proteins allow sub-nanomolar concentrations of UvrA to load UvrB through >10 cycles of incision . Guanidine hydrochloride-denatured UvrA was reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity . Co-immunoprecipitation assays showed that DnaK bound denatured UvrA in the absence of ATP . UV survival studies of a DnaK-deficient strain indicated an 80-fold increased sensitivity to 100 J/m2 of ultraviolet light (254 nm) as compared with an isogenic wild-type strain . Global repair analysis indicated a reduction in the extent of pyrimidine dimer and 6-4 photoproduct removal in the DnaK-deficient cells . These results suggest that molecular chaperonins participate in nucleotide excision repair by maintaining repair proteins in their properly folded state. Biochem Cell Biol, 1997, 75(6), 771 - 5 Molecular cloning and expression of avian smooth muscle S100A11 (calgizzarin, S100C); Schonekess BO et al.; S100A11 (calgizzarin or S100C), a member of the S100 family of Ca(2+)-binding proteins, was first identified in chicken gizzard smooth muscle and subsequently detected in several mammalian species and tissues . We now report the full-length coding sequence of avian smooth muscle S100A11 . The cloned nucleotide sequence is 515 bases in length, which includes in-frame start and stop codons and encodes a protein of 101 amino acids . The chicken S100A11 sequence differs from human S100A11 at 25 positions (9 conserved) and is four residues shorter (overall identity 72.4%, similarity 81%) . The protein contains two EF hand and conserved hydrophobic residues involved in dimer formation . Cloned avian S100A11 expressed in Escherichia coli and purified by Ca(2+)-dependent hydrophobic interaction chromatography and ion-exchange chromatography was recognized by polyclonal antibodies raised against tissue-purified protein and, like tissue-purified S100A11, bound 45Ca2+ in a gel overlay assay. Anal Chem, 1998 May 1, 70(9), 1838 - 46 Electrospray mass spectrometry studies of non-heme iron-containing proteins; Lei QP et al.; The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer . The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli . ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms . Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms . Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr) . ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein . Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer . The masses of the monomer ions produced in this manner are assigned to the aposubunit, {subunit + Fe - 3H}+, and {subunit + 2Fe - 6 H}+ . Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit . Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer . Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3) . In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da . Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms. Int J Food Microbiol, 1998 Mar 3, 40(1-2), 57 - 64 An interlaboratory study to find an alternative to the MPN technique for enumerating Escherichia coli in shellfish; Ogden ID et al.; Nine laboratories in eight countries tested 16 batches of common mussels (Mytilus edulis) over a 32 week period in order to find an alternative to the Most Probable Number (MPN) technique to enumerate E . coli . The alternatives investigated included the 3M Petrifilm system, the Merck Chromocult agar method and a Malthus conductance technique . The Petrifilm was found to be unsuitable and was subsequently dropped from the trial . After 669 analyses, a correlation of 0.83 was observed for log E . coli counts between the MPN and Chromocult methods and there was no significant evidence that either method tended to give higher readings than the other . The MPN was slightly better than the Chromocult method for repeatability but the Chromocult was slightly better for reproducibility . However, the observed differences are probably too small to be of practical importance . On the basis of these data therefore, the two methods appear equally suitable for E . coli enumeration in shellfish . There were poor correlations between these methods and the Malthus technique . A small but significant number of samples tested positive on the Malthus instrument but were recorded negative on the MPN and Chromocult tests . Subsequent analysis positively identified E . coli from these Malthus assays . After statistical analysis, errors were noted in both the MPN and Chromocult methods but it was found that there would be no statistical differences if the Chromocult agar were used as an alternative to the MPN technique. FEBS Lett, 1998 Apr 24, 426(3), 309 - 13 Site-directed mutagenesis and chemical modification of the two cysteine residues of the UDP-N-acetylmuramoyl:L-alanine ligase of Escherichia coli; Nosal F et al.; Site-directed mutagenesis and chemical modification of the two cysteine residues of the MurC L-alanine-adding enzyme from Escherichia coli were undertaken to study their possible role in activity and stability . Their replacement by alanine was not critical for activity . However, C230 played a role in enzyme stability and substrate binding . N-Ethylmaleimide alkylation led to monoalkylated and dialkylated proteins . The monoalkylated protein had mostly unmodified C230 residues . The extent of alkylation of C230 paralleled the loss of activity, whereas that of C426 did not . Protection against inactivation by beta,gamma-imidoadenosine 5'-triphosphate implied the involvement of C230 in the ATP binding site. FEBS Lett, 1998 Apr 24, 426(3), 297 - 300 The isolated H4-H5 cytoplasmic loop of Na,K-ATPase overexpressed in Escherichia coli retains its ability to bind ATP; Obsil T et al.; The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from Escherichia coli cells . Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP . Isolation of a soluble H4-H5 loop with the native ATP binding site is a crucial step for detailed studies of the molecular mechanism of ATP binding and utilisation. Hum Genet, 1998 Apr, 102(4), 430 - 4 A missense mutation (His42Arg) in the T-protein gene from a large Israeli-Arab kindred with nonketotic hyperglycinemia; Kure S et al.; Nonketotic hyperglycinemia (NKH) is caused by a mutation in the genes encoding the components of the glycine cleavage multi-enzyme system . More than 80% of the patients have defects in the gene encoding P-protein, whereas the rest of the patients have defects in the gene encoding T-protein . We have found a large Israeli-Arab kindred with NKH . At least 14 children were affected, and all the patients had seizures and respiratory failure within 2 days after birth . Enzymatic analysis revealed that T-protein activity was deficient in the liver specimen from one propositus . We screened this family for a mutation in the protein-coding region and exon/intron boundaries of T-protein gene by direct sequencing analysis . A missense mutation was found in exon 2; this resulted in an amino acid substitution from histidine to arginine at position 42 (H42R) . Histidine 42 is conserved in human, bovine, chicken, pea, and Escherichia coli, suggesting that it has an important role in catalytic functions . Genotype analyses of 26 family members confirmed that the homozygous H42R mutation was completely associated with the onset of NKH . The availability of DNA testing facilitates the prenatal diagnosis of NKH and the identification of carriers, which is necessary for genetic counseling in the affected families. Biochem Biophys Res Commun, 1998 May 8, 246(1), 238 - 42 Multiple phosphorylation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B by casein kinase II and p60c-src in vitro; Jung EJ et al.; We have cloned a soluble chicken protein tyrosine phosphatase, named CPTP1, from the cDNA library of chicken intestine . The CPTP1 showed 92% sequence identity to the corresponding 321 amino acid residues of human PTP1B (HPTP1B) . CPTP1 lacked 13 amino acids of the N-terminal region compared with HPTP1B, while the C-terminal 48 amino acid sequence of this protein was distinct from those of other PTPs . In vitro phosphorylation and phosphoamino acid analysis showed that both CPTP1 and HPTP1B were phosphorylated on serine and threonine residues near their N-terminus by casein kinase II (CKII) . Furthermore, phosphorylation of CPTP1 by CKII resulted in an inhibition of its phosphatase activity in vitro . Interestingly, both CPTP1 and HPTP1B were also tyrosine-phosphorylated near their N-terminus by p60c-src . When we examined the vanadate effect, in the absence of vanadate, the tyrosine-phosphorylated CPTP1 by p60c-src was autodephosphorylated by its own phosphatase activity . These results suggest that both CPTP1 and HPTP1B might play an important role in CKII- and p60c-src-induced signal transduction cascades. Planta, 1998 May, 205(1), 121 - 31 A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis-trans-isomerase that interacts with calmodulin and a 36-kDa cytoplasmic protein; Hueros G et al.; A member of a eukaryotic gene superfamily, encoding a peptidylproline cis-trans-isomerase (rotamase) has been isolated from a maize (Zea mays L . A69Y+) endosperm cDNA library . The maize sequence (mzFKBP-66) encodes a 66-kDa polypeptide most closely related to the subclass of rotamases which bind an immunosuppressive drug, FK506, (termed FK506-binding proteins FKBPs), and possesses four tandem copies of the FKBP-like binding domain . The sequence mzFKBP-66 is expressed ubiquitously in the maize plant, and the protein encoded is present in both cytosolic and nuclear compartments within the cell . Both the native mzFKBP-66 and a recombinant protein overexpressed in Escherichia coli showed peptidylproline cis-trans-isomerase (PPIase) activity at rates comparable to those reported for mammalian immunophilins . This activity was also sensitive to inhibition by FK506 . Immunoaffinity chromatography using anti-mzFKBP66 demonstrated an association of the protein with an unknown 36-kDa polypeptide, and affinity chromatography of mzFKBP-66 on calmodulin-agarose beads indicated the presence of a calmodulin-binding site . The existence of mzFKBP-66-associated proteins suggests that plant immunophilins may act as part of multicomponent complexes, as has been shown for other representatives of this class of enzyme. Planta, 1998 May, 205(1), 12 - 22 Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata; Ishiwatari Y et al.; Rice (Oryza sativa L.) phloem sieve tubes contain RPP13-1, a thioredoxin h protein that moves around the plant via the translocation stream . Such phloem-mobile proteins are thought to be synthesized in the companion cells prior to being transferred, through plasmodesmata, to the enucleate sieve-tube members . In this study, in-situ hybridization experiments confirmed that expression of RPP13-1 is restricted to companion cells within the mature phloem . To test the hypothesis that RPP13-1 enters the sieve tube, via plasmodesmata, recombinant RPP13-1 was expressed in Escherichia coli, extracted, purified and fluorescently labeled with fluorescein isothiocyanate (FITC) for use in microinjection experiments into tobacco (Nicotiana tabacum L.) mesophyll cells . The FITC-RPP13-1 moved from the injected cell into surrounding cells, whereas the E . coli thioredoxin, an evolutionary homolog of RPP13-1, when similarly labeled and injected, failed to move in this same experimental system . In addition, co-injection of RPP13-1 and FITC-dextrans established that RPP13-1 can induce an increase in plasmodesmal size exclusion limit to a value greater than 9.4 but less than 20 kDa . Nine mutant forms of RPP13-1 were constructed and tested for their capacity to move from cell to cell; two such mutants were found to be incapable of movement . Crystal-structure prediction studies were performed on wild-type and mutant RPP13-1 to identify the location of structural motifs required for protein trafficking through plasmodesmata . These studies are discussed with respect to plasmodesmal-mediated transport of macromolecules within the companion cell-sieve tube complex. J Mol Biol, 1998 Apr 24, 278(1), 267 - 78 Asymmetry, commitment and inhibition in the GroE ATPase cycle impose alternating functions on the two GroEL rings; Kad NM et al.; The ATPase cycle of GroE chaperonins has been examined by transient kinetics to dissect partial reactions in complexes where GroEL is asymmetrically loaded with nucleotides . The occupation of one heptameric ring by ADP does not inhibit the loading of the other with ATP nor does it prevent the consequent structural rearrangement to the "open" state . However, ADP binding completely inhibits ATP hydrolysis in the asymmetric complex, i.e . ATP cannot by hydrolysed when ADP is bound to the other ring . This non-competitive inhibition of the ATPase by ADP is consistent with a ring-switching, or "two-stroke", mechanism of the type: ATP:GroEL --> ADP:GroEL --> ADP:GroEL:ATP --> GroEL:ATP --> GroEL:ADP, i.e . with respect to the GroEL rings, ATP turns over in an alternating fashion . When the ATP-stabilized, "open" state is challenged with hexokinase and glucose, to quench the free ATP, the open state relaxes slowly (0.44 s-1) back to the apo (or closed) conformation . This rate, however, is three times faster than the hydrolytic step, showing that bound ATP is not committed to hydrolysis . When GroES is bound to the GroEL:ATP complex and the system is quenched in the same way, approximately half of the bound ATP undergoes hydrolysis on the chaperonin complex showing that the co-protein increases the degree of commitment . Thus, non-competitive inhibition of ATP hydrolysis, combined with the ability of the co-protein to block ligand exchange between rings has the effect of imposing a reciprocating cycle of reactions with ATP hydrolysing, and GroES binding, on each of the GroEL rings in turn . Taken together, these data imply that the dominant, productive steady state reaction in vivo is: GroEL:ATP:GroES --> GroEL:ADP:GroES --> ATP:GroEL:ADP:GroES --> ATP:GroEL:ADP --> GroES:ATP:GroEL:ADP --> GroES:ATP:GroEL for a hemi-cycle, and that significant inhibi tion of hydrolysis may arise through the formation of a dead-end ADP:GroEL:ATP:GroES complex . J Mol Biol, 1998 Apr 24, 278(1), 239 - 52 The three-dimensional structure of an avian class-mu glutathione S-transferase, cGSTM1-1 at 1.94 A resolution; Sun YJ et al.; Glutathione S-transferase cGSTM1-1, an avian class-mu enzyme with high sequence identity with rGSTM3-3, was expressed heterologously in Escherichia coli . The three-dimensional structure of this protein that co-crystallized with an inhibitor, S-hexylglutathione, was determined by the molecular replacement method and refined to 1.94 A resolution . The three-dimensional structure and the folding topology of the dimeric cGSTM1-1 closely resembles those of other class-mu GSTs . The bound inhibitor, S-hexylglutathione, orients in disparate directions in the two subunits . The combined space occupied by the hexyl moiety of the inhibitors overlaps with that reported for rGSTM1-1 co-crystallized with (9 S,10 S)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene . Conformational differences at a flexible loop (residue 35 to 40) were also observed between the crystal structures of cGSTM1-1 and rGSTM1-1.cGSTM1-1 has the highest epoxidase activity among all the class-mu enzymes reported . Tyr115, has been identified as a residue that participates in the epoxidase activity of class-mu glutathione S-transferase and is conserved in cGSTM1-1 . The epoxidase and trans-4-phenyl-3-buten-2-one conjugating activity of cGSTM1-1 are decreased drastically but not abolished by replacing Tyr115 with phenylalanine . The specificity constant of the cGSTM1-1(Y115F) mutant, with 1-chloro-2,4-dinitrobenzene as substrate, is 15-fold higher than that of the wild-type enzyme . J Mol Biol, 1998 Apr 24, 278(1), 117 - 33 Structural recognition and distortion by the DNA junction-resolving enzyme RusA; Giraud-Panis MJ et al.; RusA is a relatively small DNA junction-resolving enzyme of lambdoid phage-origin . Many of the physical characteristics of this enzyme are similar to those of junction-resolving enzymes of different origins . RusA binds to DNA junctions as a dimer, with a dissociation constant of 2 to 7 nM . RusA also exists in dimeric form in free solution, with a half time for subunit exchange of 4.2 minutes . We find that RusA can cleave both fixed junctions and those that can undergo a number of steps of branch migration, and confirm that the enzyme exhibits a strong preference for cleavage 5' to a CpC sequence . We have isolated a mutant protein, RusA D70N, that is completely inactive in cleavage while binding normally to DNA junctions, suggesting a role for aspartate 70 in the cleavage reaction . Constraining the conformation of the junction by means of tethering the helical ends leads to a marked reduction in cleavage rate by RusA, suggesting that the structure must be altered for cleavage . Using comparative gel electrophoresis we find that the global structure of the DNA junction is altered on RusA binding, into a structure that is different from any that is formed by the free junction . Moreover, the structure of the complex is the same irrespective of the presence or absence of magnesium ions . Thus, like all the junction-resolving enzymes, RusA both recognises and distorts the structure of DNA junctions . J Mol Biol, 1998 Apr 24, 278(1), 105 - 16 Structural similarities between Escherichia coli RuvA protein and other DNA-binding proteins and a mutational analysis of its binding to the holliday junction; Rafferty JB et al.; Comparison of the structure of Escherichia coli RuvA with other proteins in the Protein Data Bank gives insights into the probable modes of association of RuvA with the Holliday junction during homologous recombination . All three domains of the RuvA protein possess striking structural similarities to other DNA-binding proteins . Additionally, the second domain of RuvA contains two copies of the helix-hairpin-helix (HhH) structural motif, which has been implicated in non-sequence-specific DNA binding . The two copies of the motif are related by approximate 2-fold symmetry and may form a bidentate DNA-binding module . The results described provide support for the organization of the arms of the DNA in our RuvA/Holliday junction complex model and support the involvement of the HhH motifs in DNA binding . J Mol Biol, 1998 Apr 24, 278(1), 89 - 104 Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli; Chen HW et al.; The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase . Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases . We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD . Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB*) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD*) protein . RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min-1 . The nuclease reaction catalyzed by RecB*ChD* is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD*, and RecB*ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP . RecB*ChD* also has very low ATP hydrolysis activity (approximately 10(3)-fold less than RecBCD), as do the individual mutant RecB* and hRecD* proteins (approximately 100-fold less than RecB or hRecD) . The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site . Cleavage towards the 3'-end of one oligomer (observed with RecBChD*) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB*ChD) occurs towards the 5'-end of the second bound oligomer . EMBO J, 1998 Apr 15, 17(8), 2436 - 49 Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly; Kelman Z et al.; Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called gamma complex that uses ATP to assemble the beta clamp onto DNA . We examine here the function of the psi subunit of the gamma complex clamp loader . Omission of psi from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt . We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for psi and is defective in promoting clamp loading and processive replication at elevated ionic strength . SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with psi . Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to psi . These results establish a role for the psi subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA. Pharmacology, 1998 May, 56(5), 230 - 6 Pharmacological modulation of LPS-induced MIP-1 alpha production by peripheral blood mononuclear cells; Kimata M et al.; In the present study, we investigated the effects of some anti-asthmatic drugs on the production of the CC chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), in response to lipopolysaccharide (LPS) by peripheral blood mononuclear cells (PBMC) . MIP-1 alpha production was induced by LPS in a concentration-dependent fashion and reached the maximum at 10 micrograms/ml LPS (27.5 +/- 2.3 ng MIP-1 alpha/10(6) PBMC) . At a submaximal concentration of LPS (1 microgram/ml), the release of MIP-1 alpha increased with time and reached the maximum 24 h after LPS stimulation . Actinomycin D and cycloheximide inhibited MIP-1 alpha production completely, but glucocorticoids did not completely inhibit MIP-1 alpha production, with a maximum inhibition of 70% . We examined the effect of beta-stimulants and phosphodiesterase inhibitors, which upregulate intracellular cyclic AMP levels, on MIP-1 alpha production . When PBMC were treated with beta-stimulants alone, beta-stimulants showed a slightly inhibitory effect on MIP-1 alpha production . However, the coadministration of roliplam significantly potentiated the inhibitory effect of beta-stimulants on MIP-1 alpha production . Moreover, db-cAMP suppressed MIP-1 alpha production dose-dependently . The above data indicate that the production of MIP-1 alpha is regulated by cyclic AMP and that cyclic AMP could provide a useful target for therapeutic treatment in asthmatic diseases and other diseases where MIP-1 alpha is involved in their etiology. Am J Hum Genet, 1998 May, 62(5), 1023 - 33 Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients; Reiter LT et al.; The HNPP (hereditary neuropathy with liability to pressure palsies) deletion and CMT1A (Charcot-Marie-Tooth disease type 1A) duplication are the reciprocal products of homologous recombination events between misaligned flanking CMT1A-REP repeats on chromosome 17p11 . 2-p12 . A 1.7-kb hotspot for homologous recombination was previously identified wherein the relative risk of an exchange event is 50 times higher than in the surrounding 98.7% identical sequence shared by the CMT1A-REPs . To refine the region of exchange further, we designed a PCR strategy to amplify the recombinant CMT1A-REP from HNPP patients as well as the proximal and distal CMT1A-REPs from control individuals . By comparing the sequences across recombinant CMT1A-REPs to that of the proximal and distal CMT1A-REPs, the exchange was mapped to a 557-bp region within the previously identified 1.7-kb hotspot in 21 of 23 unrelated HNPP deletion patients . Two patients had recombined sequences suggesting an exchange event closer to the mariner-like element previously identified near the hotspot . Five individuals also had interspersed patches of proximal or distal repeat specific DNA sequence indicating potential gene conversion during the exchange of genetic material . Our studies provide a direct observation of human meiotic recombination products . These results are consistent with the hypothesis that minimum efficient processing segments, which have been characterized in Escherichia coli, yeast, and cultured mammalian cells, may be required for efficient homologous meiotic recombination in humans. FEBS Lett, 1998 Apr 17, 426(2), 217 - 20 Stability and functionality of cysteine-less F(0)F1 ATP synthase from Escherichia coli; Kuo PH et al.; All 21 native cysteines in the Escherichia coli F(0)F1 ATP synthase were replaced by alanines . In isolated E . coli membranes, ATP-dependent proton pumping, turnover of ATP hydrolysis and steady-state transition state thermodynamic parameters of the cysteine-less enzyme were similar to wild-type . The cysteine-less enzyme was solubilized in n-octyl beta-D-glucopyranoside, purified by affinity chromatography, and reconstituted into pre-formed liposomes made from E . coli lipids . The properties of the reconstituted, purified enzyme were not significantly different from the membranous enzyme . These data demonstrate that cysteine-less F(0)F1 is biochemically stable and has functionality similar to wild-type. FEBS Lett, 1998 Apr 17, 426(2), 191 - 5 Regulation of capsular polysialic acid biosynthesis by N-acetyl-D-mannosamine, an intermediate of sialic acid metabolism; Revilla-Nuin B et al.; N-Acetyl-D-mannosamine (ManNAc) is a specific substrate for the synthesis of N-acetylneuraminic acid, the essential precursor of bacterial capsular polysialic acid (PA) . When Escherichia coli K92 used ManNAc as a carbon source, we observed a dramatic reduction (up to 90%) in in vivo PA production . Experiments in which the carbon source was changed revealed that the maximal inhibitory effect occurred when this sugar was present in the medium before the logarithmic phase of bacterial growth had started . Enzymatic analysis revealed that high concentrations of ManNAc-6-phosphate inhibit NeuAc lyase, the enzyme that synthesizes NeuAc for PA biosynthesis in E . coli . These results indicate that ManNAc-6-phosphate is able to regulate NeuAc lyase activity and modulate the PA synthesis. FEBS Lett, 1998 Apr 10, 426(1), 135 - 9 Aminoacylation of tRNA gene transcripts is strongly affected by 3'-extended and dimeric substrate RNAs; Kholod N et al.; Kinetic parameters of aminoacylation by E . coli phenylalanyl-tRNA synthetase vary for phage T5 tRNA(Phe) gene transcript from 0.950 to 2.545 microM for Km and from 550 to 400 min(-1) for kcat . To reveal the source of this variability for various RNA preparations, homogeneity of the transcripts has been examined . Presence of 3' extensions and dimer formation in transcript preparations reduced the catalytic efficiency kcat/Km several-fold . We have shown that the proportion of dimers and 3'-extended transcripts in tRNA preparations is sensitive to single-base substitutions in tRNA . While wild-type phage T5 tRNA(Phe) gene transcript contains about half of dimeric molecules, for some mutants this value increases up to 90% or drops to 0% . Phage T5 tRNA(Phe) gene with anticodon stem nucleotide substitutions used as a template in run-off transcription produces 5 times less 3'-extended molecules than the wild-type gene . In view of all these results kinetic parameters of aminoacylation reaction for many wild-type and mutant tRNA gene transcripts should be reevaluated. FEBS Lett, 1998 Apr 10, 426(1), 52 - 6 Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1; Marzocchini R et al.; The genes of the human low Mr phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E . coli as fusion proteins with glutathione S-transferase . The recombinant proteins were purified by a rapid one-step procedure allowing each enzyme to purify with high final yield and specific activity . This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies . The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver . These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose . IF1 and IF2 crystals were also grown . IF1 crystals were X-ray-grade, diffracted to better than 2.4 A and were suitable for high resolution X-ray structure determination. FEBS Lett, 1998 Apr 10, 426(1), 37 - 40 Trapping of conformations of the Escherichia coli F1 ATPase by disulfide bond formation . A state of the enzyme with all three catalytic sites of equal and low affinity for nucleotides; Aggeler R et al.; A mutant of Escherichia coli F1F0-ATPase, alphaS411C/betaY331W/betaE381C/gammaC87S, has been generated . CuCl2 treatment of this mutant led to cross-linking between alpha and beta subunits in yields of up to 90% . This cross-linking across non-catalytic site interfaces inhibited ATP hydrolysis activity . In the absence of cross-linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 microM, 6 microM and 60 microM, respectively, values that are comparable to wild-type . For MgADP, there was one tight site (0.34 microM) and two sites of lower affinity (each 27 microM), again comparable to wild-type enzyme . After cross-linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (approximately 60 microM) . Thus cross-linking fixed all three catalytic sites in the same conformation . Trypsin cleavage experiments showed that cross-linking fixed the epsilon subunit in the ATP+EDTA conformation. FEBS Lett, 1998 Apr 10, 426(1), 21 - 3 IncI1 plasmid R64 encodes the ArsR protein that alleviates type I restriction; Rastorguev SM et al.; The host-controlled EcoK restriction of unmodified phage lambda was five-fold alleviated in the wild-type Escherichia coli strain K12 carrying the R64 plasmid of the incompatibility group I1 . The relevant gene was mapped between the origin of vegetative replication (rep, oriV) and the tet(r) gene about 60 kbp downstream from the origin of transfer, oriT . We cloned this gene inside the 613 bp long EcoRI-PstI fragment and sequenced it . Only one 351 bp long open reading frame (ORF) starting at 124 bp from the beginning of the insert was found in the sequence . Computer search in the current databases revealed that the putative protein is identical to the ArsR protein specified by the IncFI plasmid R773 . ArsR is a repressor of the arsenical resistance (ars) operon, arsRDABC . There are no arsABC genes in the R64 plasmid since plasmid R64- (or pSR8)-mediated resistance of E . coli K12 cells to the arsenicals arsenate and arsenite was not detected . The gene arsR and the antirestriction genes ard (ardA and ardB) are non-homologous . However, comparison of the deduced amino acid sequence of ArsR with the ArdA and ArdB sequences revealed only one small region of similarity, a 9 amino acid motif found in different antirestriction proteins that is hypothesized to be an interaction site for antirestriction proteins with restriction endonucleases. Am J Trop Med Hyg, 1998 May, 58(5), 655 - 62 Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice; Simmons M et al.; A recombinant protein containing part of the dengue (DEN) 2 envelope protein was evaluated as a subunit immunogen for vaccination against DEN virus infection . A gene fragment encoding amino acids 298-400 (B domain) of the DEN-2 virus envelope was expressed as a fusion protein with the maltose binding protein (MBP) of Escherichia coli . This recombinant, DEN-2(B)/MBP, was purified and analyzed for its antigenicity, immunogenicity, and ability to protect mice against lethal challenge . The recombinant antigen reacted with a DEN-2 type-specific neutralizing monoclonal antibody (3H5), DEN-2 hyperimmune mouse ascitic fluid, and DEN-2 immune human sera . When administered to mice, DEN-2(B)/MBP elicited a DEN-2 virus neutralizing antibody response that conferred partial protection against challenge infection with a lethal dose of DEN-2 virus administered by intracranial inoculation . In addition, no replication of DEN-2 virus was detectable in the brains of the immunized mice as compared with control mice that were killed six days after challenge . Sera from immunized mice revealed no cross-neutralizing antibody to any of the other DEN serotypes in the plaque-reduction neutralization test . These findings warrant further studies with the DEN-2(B)/MBP antigen as a potential human vaccine candidate . An effective vaccine could prevent thousands of cases of illness and many deaths each year resulting from DEN virus infections. Crit Rev Biochem Mol Biol, 1998, 33(2), 95 - 149 Ribosomal tRNA binding sites: three-site models of translation; Burkhardt N et al.; The first models of translation described protein synthesis in terms of two operationally defined tRNA binding sites, the P-site for the donor substrate, the peptidyl-tRNA, and the A-site for the acceptor substrates, the aminoacyl-tRNAs . The discovery and analysis of the third tRNA binding site, the E-site specific for deacylated tRNAs, resulted in the allosteric three-site model, the two major features of which are (1) the reciprocal relationship of A-site and E-site occupation, and (2) simultaneous codon-anticodon interactions of both tRNAs present at the elongating ribosome . However, structural studies do not support the three operationally defined sites in a simple fashion as three topographically fixed entities, thus leading to new concepts of tRNA binding and movement: (1) the hybrid-site model describes the tRNAs' movement through the ribosome in terms of changing binding sites on the 30S and 50S subunits in an alternating fashion . The tRNAs thereby pass through hybrid binding states . (2) The alpha-epsilon model introduces the concept of a movable tRNA-binding domain comprising two binding sites, termed alpha and epsilon . The translocation movement is seen as a result of a conformational change of the ribosome rather than as a diffusion process between fixed binding sites . The alpha-epsilon model reconciles most of the experimental data currently available. Adv Exp Med Biol, 1998, 431, 221 - 6 Structure and functional relationships in human pur H; Beardsley GP et al.; 1 . The human pur H (ATIC) gene encoding a bifunctional protein, hPurH, which carries the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway, AICARFT & IMPCH, has been cloned and sequenced . The gene product, hPurH has been overexpressed in E . coli, purified to homogeneity and crystallized . 2 . The human pur H gene lies on chromosome 2, between band q34 and q35 . There is at least one intron of 278 bp near the 5' end . 3 . Truncation mutant studies demonstrate two non-overlapping functional domains in the protein arranged as indicated in Figure 5 . The existence of a linker or interaction region between the catalytic domains remains to be established . 4 . Cleland-type kinetic inhibition experiments indicate that the AICARFT reaction is of the ordered, sequential type with the reduced folate cofactor binding first . 5 . The reaction has a broad pH optimum in the alkaline range, with a maximum at about pH 8.2 . 6 . Preliminary transient phase kinetic studies show the presence of a "burst" indicating that a late step in the reaction sequence is rate limiting . 7 . A PurH crystal structure is that of a dimer, with a putative single binding site for the reduced folate cofactor formed using elements from each of the monomer subunits . Probable binding sites for AICAR and FAICAR can be identified on each monomer . 8 . Equilibrium sedimentation studies show hPurH apoprotein to be a monomer:dimer equilibrium mixture with a kD of 0.55 uM . 9 . The crystal structure has permitted identification of a number of candidate amino acid residues likely to be involved in catalysis and/or substrate binding . Among these, we have thus far completed studies on two, Lysine 265 and Histidine 266 . These appear to be critically involved in the AICARFT reaction, although whether their role(s) are in catalysis or binding remains to be determined. Int J Biochem Cell Biol, 1998 Jan, 30(1), 13 - 26 Structure, function and physiological role of glycine N-methyltransferase; Ogawa H et al.; Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the transfer of the methyl group of S-adenosylmethionine (AdoMet) to glycine to form S-adenosylhomocysteine and sarcosine . Unlike most AdoMet-dependent methyltransferases, glycine N-methyltransferase is a tetramer of identical subunits . Crystallography of recombinant rat glycine N-methyltransferase indicates that four nearly spherical subunits are arranged to form a flat, square tetramer with a large hole in the centre . The enzyme occurs abundantly in the livers of rat, rabbit and mouse . Glycine N-methyltransferases from rat, rabbit, human and pig livers are shown to have similar amino acid sequences and, with the enzymes from rat and rabbit livers, it is demonstrated that the N-terminal valine is acetylated . Glycine N-methyltransferases from livers exhibit sigmoidal rate behaviour with respect to AdoMet and hyperbolic behaviour with respect to glycine at all pH tested . However, recombinant rat glycine N-methyltransferase which lacks the N-terminal acetyl group shows no cooperativity toward AdoMet at neutral pH, suggesting that elimination of the positive charge at the N-terminus is required for cooperative behaviour . Glycine N-methyltransferase binds 5-methyltetrahydropteroylpentaglutamate tightly, resulting in inhibition of the catalytic activity . The nature of these unique functional features is discussed in the light of the three-dimensional structure of the enzyme . The tissue and subcellular localization of the enzyme and its possible role in methionine metabolism are reviewed. Can J Anaesth, 1998 Apr, 45(4), 352 - 9 Tumour necrosis factor-alpha, but not septic plasma depresses cardiac myofilament contraction; Gu M et al.; PURPOSE: In sepsis, myocardial depression may be caused by mediators released as part of the inflammatory reaction, lumour necrosis factor alpha (TNF alpha) is one mediator that may contribute to this depression . In the present study, we contrasted the effects of TNF alpha and septic plasma fraction (SP) obtained from an E . coli model on contractile tension in intact and skinned canine ventricular trabecular (VT) preparations . The objectives were to determine whether SP or TNF alpha could impair contractile tension at the level of the myofilaments, and to determine the extent to which TNF alpha may account for myocardial depression found in E . coli sepsis . METHODS: Measurements of isometric tensions were made after TNF alpha and SP (10,000 to 30,000 MW fraction) were added to respective intact or skinned canine VT preparations . In the skinned preparation, trabeculae were chemically skinned with Triton X-100 . RESULTS: Septic plasma caused a decrease in contraction in the intact preparation compared with preseptic plasma (50 +/- 7 vs 33 +/- 7%, P < 0.05), but had no effect in the skinned preparation . On the other hand, TNF alpha (30 ng.ml-1) caused an approximately 50% reduction in tension (29 +/- 2 mg vs 16 +/- 5 mg) in the skinned preparation (P < 0.05), but had no effect in the intact preparation . CONCLUSION: These results suggest that TNF alpha and SP act through different mechanisms . While SP requires an intact membrane, TNF alpha impairs function by a direct effect on the myofilaments. Zhonghua Yi Xue Za Zhi, 1997 Apr, 77(4), 274 - 7 {The inhibitory effect of recombinant human C3 fragment on murine endotoxic shock}; Liang H et al.; OBJECTIVE: To study the potentially practical use of C3 inactive fragment in anti-inflammation . METHODS: A vector expressing RGD polypeptide derived from the alpha chain segment of human C3 was constructed by using PCR and genetic engineering methods and a recombinant protein (namely C 33) was expressed with high efficiency in E . coli . RESULTS: The analysis of SDS-PAGE showed the molecular weight of C 33 was about 15 KD . Its purity was above 95% after purification . The amino acid composition was inconsistent with the theoretical values . U937 cells stimulated by low dosage PMA adhered with coated C 33, and the adhesion was blocked by anti-CD 11b monoclonal antibody . After injection of purified C 33 into mice which were consequently challenged by dead E . coli, the mortality of the endotoxic shock was significantly reduced . CONCLUSION: C 33 can specifically bind to CD 11b/CD 18 . C 33 as a ligand for CD 11b/CD 18 might be potentially used as an anti-inflammatory agent. Zhonghua Jie He He Hu Xi Za Zhi, 1996 Aug, 19(4), 202 - 5 {Effect of inhaled nitric oxide on endotoxin induced acute lung injury in rabbit}; Liao J et al.; OBJECTIVE: In order to observe the effects of inhaling nitric oxide (NO) on acute lung injury (ALI) . METHODS: 24 rabbits divided into 4 groups . Six rabbits injured with intravenous E . Coli endotoxin, then followed by treatment of inhaling 80 ppm NO in inspired gas . Before and after the infusion of endotoxin, the mean pulmonary arterial pressure (mPAP), mean systemic arterial pressure (mPSA) and the PaO2 were examined . The venous methemoglobin (MHb) was measured by using spectrophometer colorimitry . The extravasculur lung water was evaluated with rate of dried to wet lung weight at the end of study . RESULTS: The rabbits injured with endotoxin inhaling 80 ppm NO could rapidly reduce the mPAP, increase the PaO2 and without inducing significant change of mPSA, MHb and extravasculur lung water . CONCLUSIONS: Inhalation of 80 ppm NO can selectively cause pulmonary artery dilatation, reduce mPAP, improve pulmonary gas exchange, without producing system vasodilation and toxic effects to the rabbits. Biochim Biophys Acta, 1998 Apr 10, 1380(2), 209 - 22 Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs; Jeyaseelan K et al.; Cardiotoxins are the most abundant toxin components of cobra venom . Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported . In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N . n . sputatrix by cDNA cloning . This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra . The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli . The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing . These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells . Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N . n . sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins . The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N . n . atra and N . n . naja, indicating that it may be universally present in all Naja naja subspecies . Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4) . We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts . Biochemistry, 1998 Apr 28, 37(17), 6199 - 204 Inhibition of type I and type II phospholipase A2 by phosphatidyl-ethanolamine linked to polymeric carriers; Dan P et al.; We have previously shown that cell surface proteoglycans protect the cell membrane from the action of extracellular phospholipase A2 (PLA2) enzymes {Dan, P., Nitzan, D . W., Dagan, A., Ginsburg, I., and Yedgar, S . (1996) FEBS Lett . 383, 75-78} . Cell-impermeable PLA2 inhibitors (ExPLIs) were prepared by linking phosphatidylethanolamine (PE) to polymeric carriers, specifically, carboxymethylcellulose, heparin, or hyaluronic acid . The structure of these inhibitors enables the incorporation of their PE moiety into the membrane while the polymer remains at the membrane surface . In the present study, we show that the ExPLIs are effective inhibitors of the hydrolysis of different phospholipids in biological (Escherichia coli) and model (phospholipid vesicle) membranes, by diverse types of PLA2 enzymes, specifically human recombinant synovial fluid and C . atrox (type II), as well as Naja mocambique and porcine pancreatic (type I) PLA2 . It is proposed that the external polymers of the ExPLIs, which are anchored to the membrane by the PE, mimic the naturally occurring cell surface proteoglycans and similarly protect membranes from the action of exogenous PLA2. Biochemistry, 1998 Apr 28, 37(17), 6114 - 23 The flavoprotein component of the Escherichia coli sulfite reductase: expression, purification, and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors; Zeghouf M et al.; The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain . SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E . coli and purified as a monomer . The N-terminal part of the native protein contains thus all the determinants required for the polymerization . SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SiR-FP . Thus, SiR-FP60 can be considered as a reliable simplified model of the sulfite reductase flavoprotein component . The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH . Generation of FMNH* is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP . The neutral FAD semiquinone can be observed only within SiR-FP43, the isolated FAD-binding domain . NADPH was used as a titrant or in excess to demonstrate that electron transfer is possible only because the FMN cofactor is coupled to FAD as an electron acceptor in the protein . The electron distribution within the various reduced forms of SiR-FP60 has been compared with that of the reduced forms of cytochrome P450 reductase, bacterial cytochrome P450, and nitric-oxide synthase . Despite the conservation of the bi-flavin-domain structure between these proteins over evolutionary time, each of them provides significantly different flavin reactivities. Biochemistry, 1998 Apr 28, 37(17), 6106 - 13 NADPH-flavodoxin reductase and flavodoxin from Escherichia coli: characteristics as a soluble microsomal P450 reductase; Jenkins CM et al.; In addition to their endogenous roles as an activation system for various Escherichia coli metabolic pathways, the soluble flavoproteins flavodoxin (Fld) and NADPH-flavodoxin (ferredoxin) reductase (Fpr) can serve as an electron-transfer system for microsomal cytochrome P450s . Furthermore, since Fld and Fpr are structurally similar to the functional domains (FMN binding and NADPH/FAD binding domains, respectively) of NADPH-cytochrome P450 reductases (P450 reductases), these bacterial proteins represent a potentially useful model system for eukaryotic P450 reductases . Here we delineate similarities and differences between the E . coli Fpr-Fld system and rat P450 reductase as electron donors to bovine 17alpha-hydroxylase/17,20-lyase P450 (P450c17) . Importantly, recombinant Fpr, in combination with recombinant Fld, supports both the hydroxylase and lyase activities of P450c17 to the same proportional extent (hydroxylase-to-lyase ratio) as does P450 reductase . Maximum P450c17 turnover {5-6 mol of 17alpha-OH-progesterone (mol of P450c17)-1 min-1} was achieved using a large molar excess (50-100-fold over P450c17) of a 1:1 ratio of Fpr-Fld, although this rate was an order of magnitude less than the maximal P450 reductase-supported activity . Using these conditions, we have examined the effects of increasing ionic strength and the presence of cytochrome b5 (b5) on these two systems . Critical Fld-P450c17 electrostatic interactions are disrupted at moderate ionic strength (>100 mM NaCl) as evidenced by significant inhibition (>50%) of Fpr-Fld-supported P450c17 activity while much higher ionic strength (300 mM NaCl) is required to disrupt P450 reductase-P450c17 interactions to the same extent . Interestingly, cytochrome b5 was found to dramatically inhibit both P450 reductase- and Fpr-Fld-supported P450c17 progesterone 17alpha-hydroxylase activity while in contrast 17alpha-OH-pregnenolone lyase activity was stimulated by b5 . Investigation of the fate of reducing equivalents from NADPH added to Fpr under aerobic conditions revealed that the majority of the protein-bound FAD of Fpr is converted to the hydroquinone form . In constrast, the FMN of Fld is reduced by Fpr to a stable blue, neutral semiquinone which serves as the predominant electron donor to P450c17 in reconstitution assays . Thus, while the Fpr-Fld system and P450 reductase are fundamentally different with respect to their electrostatic interactions with P450c17, their ability to support maximal P450c17 turnover, and the FMN redox states (one-electron-reduced for Fld and two-electron-reduced for P450 reductase) capable of transferring electrons to microsomal cytochrome P450s, these differences do not appear to influence the relative catalytic efficiency of the P450c17 hydroxylase and lyase reactions. Biochemistry, 1998 Apr 28, 37(17), 6041 - 9 Role of base G-2 of pre-tRNAfMet in cleavage site selection by Escherichia coli RNase P in vitro; Lazard M et al.; In this study, a protocol for the purification of fully active Escherichia coli RNase P holoenzyme from a strain overproducing both the C5 protein and the M1 RNA components is described . A total of 0 . 8 mg of homogeneous enzyme, with a 1:1 protein/RNA subunit stoichiometry, was recovered from a 1 L bacterial culture . In addition, a convenient and reliable method based on capillary gel electrophoresis was developed to measure initial rates of pre-tRNA maturation by RNase P . Using these tools, the kinetic parameters of cleavage by RNase P of various mutants of pre-tRNAfMet showing maturation defects in vivo {Meinnel and Blanquet (1995) J . Biol . Chem . 270, 15906-15914} were investigated in vitro and the locations of cleavage sites were determined from the length of the various products of the reaction . The nucleotide at position -2 of pre-tRNAfMet is shown to be important only in the selection of the cleavage site, whereas it has no role in the efficiency of the reaction . It is concluded that base G-2 acts as an antideterminant by preventing an alternative cleavage by RNase P . In addition, the presence of G-2 alone is enough to fully compensate for the lack of a G at position +1 of pre-tRNAfMet. Biochemistry, 1998 Apr 28, 37(17), 5953 - 60 Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone; Hennig L et al.; In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C . 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A . We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E . coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases . The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E . coli parvulin and its Cys69Ala variant with juglone . For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4 . 5 x 10(-)3 s-1 . The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone . Redox reactions did not contribute to the inactivation process . Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation . When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation . Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity. Biochemistry, 1998 Apr 28, 37(17), 5939 - 46 Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus; Ogasahara K et al.; The thermostability of methionine aminopeptidase from a hyperthermophile P . furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2 . To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP . (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak . Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3 . The heat denaturation of PfMAP was not reversible . (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC . DeltaCp was estimated to be 0.82 J K-1 g-1 . (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves . The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures . It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses . This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it . (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP. Biochemistry, 1998 Apr 28, 37(17), 5849 - 57 Preparation, characterization, and complete heteronuclear NMR resonance assignments of the glutaredoxin (C14S)-ribonucleotide reductase B1 737-761 (C754S) mixed disulfide; Berardi MJ et al.; The first committed step in de novo DNA biosynthesis involves the conversion of ribonucleotides to the corresponding deoxyribonucleotides catalyzed by the enzyme ribonucleotide reductase . Reduction of disulfides in ribonucleotide reductase is essential and is catalyzed by the protein disulfide reductants glutaredoxin or thioredoxin . The interaction region between Escherichia coli glutaredoxin-1 and E . coli ribonucleotide reductase has been localized to the C-terminal end of the B1 subunit of ribonucleotide reductase . We have demonstrated that a 25-residue peptide corresponding to this C-terminal sequence is a very good substrate for glutaredoxin via a fluorescence assay and that this peptide binds in a specific manner via isothermal titration calorimetric measurements . By selectively mutating the two cysteines in the peptide, we have identified the electrophilic cysteine as C759 (B1 numbering) and prepared a mixed disulfide between E . coli glutaredoxin-1 (C14 --> S) and the C759 monothiol form of the peptide . The peptide and the protein have been labeled with 13C and 15N, and complete heteronuclear NMR resonance assignments have been completed for both the peptide and the protein in the complex . By using half-filtered NOESY spectra, intermolecular NOEs between the protein and the peptide have been identified and the binding site on glutaredoxin has been mapped . The electrostatic charge distribution of the protein in this region is very positive, thus providing an excellent match for the highly negatively charged peptide . In addition, the electrostatic potential of the peptide provides a rationale for the observed cysteine selectivity in the reaction between glutaredoxin and the B1 peptide. Biochemistry, 1998 Apr 28, 37(17), 5840 - 8 Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography; Tong W et al.; Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of ribonucleotides to deoxyribonucleotides . It is composed of two homodimeric subunits, R1 and R2 . R2 contains the diferric-tyrosyl radical cofactor essential for the nucleotide reduction process . The in vitro mechanism of assembly of this cluster starting with apo R2 or with a diferrous form of R2 has been examined by time-resolved physical biochemical methods . An intermediate, Fe3+/Fe4+ cluster (intermediate X), has been identified that is thought to be directly involved in the oxidation of Y122 to the tyrosyl radical (*Y122) . An R2 mutant in which phenylalanine has replaced Y122 has been used to accumulate intermediate X at sufficient levels that it can be studied using a variety of spectroscopic methods . The details of the reconstitution of the apo and diferrous forms of Y122F R2 have been examined by stopped-flow UV/vis spectroscopy and by rapid freeze quench electron paramagnetic resonance, and Mossbauer spectroscopies . In addition the structure of this mutant, crystallized at pH 7.6 in the absence of mercury, at 2.46 A resolution has been determined . These studies suggest that Y122F R2 is an appropriate model for the examination of intermediate X in the assembly process . Studies with two mutants, Y356F and double mutant Y356F and Y122F R2, are interpreted in terms of the possible role of Y356 in the putative electron transfer reaction between the R1 and R2 subunits of this RNR. Biochemistry, 1998 Apr 28, 37(17), 5831 - 9 Fidelity of mutant HIV-1 reverse transcriptases: interaction with the single-stranded template influences the accuracy of DNA synthesis; Kim B et al.; We have used random sequence mutagenesis and complementation in a bacterial selection system to establish a large library of immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with amino acid substitutions in the beta3-beta4 region of the fingers subdomain {Kim, B., Hathaway, T . R., and Loeb, L . A . (1996) J . Biol . Chem . 271, 4872-4878} . We show here that one of these mutants, D76V, exhibits increased accuracy of copying both DNA and RNA templates in a primer extension assay with biased dNTP pools . More detailed analysis of DNA-dependent polymerization showed that the D76V mutation conferred an up to 14-fold increase in fidelity of nucleotide insertion and a 9-fold reduced mutation rate in an M13mp2 lacZalpha forward mutation assay . Substitution at D76 with positively charged (D76R) and nonpolar (D76V and D76I) residues increased replicational accuracy, while substitutions with negatively charged (D76E) and polar residues (D76S and D76C) had little effect on fidelity . We propose that D76 affects replicational accuracy by mediating interaction between the fingers subdomain and the single-stranded template . Our work shows that the Escherichia coli complementation system can yield HIV RT mutants with increased fidelity that have not been isolated from the natural host and that are valuable in understanding the molecular bases of replicational accuracy. J Biol Chem, 1998 Apr 17, 273(16), 10026 - 35 Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine; Purmal AA et al.; A major stable oxidation product of DNA cytosine is uracil glycol (Ug) . Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties . Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo-) . During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined . Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg . Ug opposite G and A was also recognized and removed in vitro by the E . coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase . The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate. J Biol Chem, 1998 Apr 17, 273(16), 9927 - 34 The proton motive force, acting on acidic residues, promotes translocation of amino-terminal domains of membrane proteins when the hydrophobicity of the translocation signal is low; Delgado-Partin VM et al.; We have shown previously that the first transmembrane segment of leader peptidase can function to translocate the polar amino-terminal Pf3 domain across the membrane into the periplasm independently of the proton motive force (pmf) (Lee, J . I., Kuhn, A., and Dalbey, R . E . (1992) J . Biol . Chem . 267, 938-943) . We now show that when the first transmembrane segment lacks a strong hydrophobic character, the pmf is required for translocation . In addition, we find that the amino-terminal acidic residue proximal to the transmembrane domain plays a critical role in pmf-dependent amino-terminal translocation . Moreover, the pmf is required to hold the amino-terminal domain in the periplasm to prevent it from slipping such that the amino terminus is no longer exposed to the periplasm . In all cases, translocation occurs under conditions in which the function of the Sec machinery is impaired . These studies show that the low hydrophobicity of the first apolar domain (the translocation signal) can be compensated for by a negative charge in the amino-terminal region, upon which the pmf acts. J Biol Chem, 1998 Apr 17, 273(16), 9872 - 7 Reduction in abortive transcription from the lambdaPR promoter by mutations in region 3 of the sigma70 subunit of Escherichia coli RNA polymerase; Sen R et al.; Transcription initiation by Escherichia coli RNA polymerase at most promoters is associated with a reiterative synthesis and release of short abortive RNA products . We have investigated the mechanism of abortive RNA synthesis by using holoenzymes containing mutant sigma70 subunits with changes in region 3 (S506F and P504L), which reduce the ratio of abortive to full-length products . Binary complexes formed by these mutant enzymes at a modified lambdaPR promoter contained a smaller fraction of open complexes than for normal polymerase, suggesting an involvement of region 3 in melting duplex DNA or in maintenance of the open complex . The half-lives of the majority of binary complexes formed by the mutant enzymes were less than 1 min, in contrast to 30 min for the wild-type complexes . The time courses of transcription and pulse-labeling assays showed that moribund complexes, which generate only abortive products (Kubori, T., and Shimamoto, N . (1996) J . Mol . Biol . 256, 449-457), were formed by the mutant enzymes . However, they accumulated to a lesser extent than for the wild-type enzyme, due both to faster dissociation and conversion into inactive complexes . This is the main cause of the low degree of abortive transcription displayed by the mutant enzymes on this promoter. J Biol Chem, 1998 Apr 17, 273(16), 9695 - 702 Characterization and functional analysis of the cis-autoproteolysis active center of glycosylasparaginase; Guan C et al.; Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing . This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing . Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors . This binding site is sequestered in the wild-type precursor . Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids . The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation . Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180 . The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis. J Biol Chem, 1998 Apr 17, 273(16), 9602 - 7 Re-evaluating the role of His-143 in the mechanism of type I dehydroquinase from Escherichia coli using two-dimensional 1H,13C NMR; Leech AP et al.; Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate . pH/log Vmax profiles of the enzyme indicate the presence of a single ionizing group with a pKa of 6.2 . Chemical modification experiments with diethyl pyrocarbonate have identified the conserved residue His-143 as essential for catalysis in this enzyme and the pKa for this modification is also 6.2, implying that this is the single ionizing residue in dehydroquinase that may be acting as a general base in the catalytic mechanism . Subsequent mutagenesis of this residue (Leech, A . P., James, R., Coggins, J . R., and Kleanthous, C . (1995) J . Biol . Chem . 270, 25827-25836) further suggested that His-143 may be involved in Schiff base formation/breakdown as well as being the proton abstracting general base . The importance of this residue was confirmed by recent x-ray crystallographic data showing His-143 to be at the center of a hydrogen-bonded triad, flanked by the essential Schiff base forming residue Lys-170 and Glu-86 . In the present study, we have used mutagenesis and 1H and 13C NMR to assign the resonance of His-143 and probe its ionization state to define more precisely its role in the mechanism of type I dehydroquinase . Following isotopic enrichment of wild-type and H143A dehydroquinase enzymes with {2-13C}histidine, the resonance for His-143 was assigned by comparing their 1H,13C heteronuclear single quantum correlation NMR spectra . pH titrations revealed that whether in the liganded or unliganded state, His-143 does not ionize over the pH range 6-9.5 and so cannot possess a pKa of 6.2 . The NMR data are consistent with this residue remaining unprotonated at pH values optimal for the activity of this enzyme (pH > 7) . The role of His-143 is re-evaluated in light of these and the recent structural data, and an alternative candidate for the pKa of 6.2 is discussed. J Biol Chem, 1998 Apr 17, 273(16), 9501 - 9 Proteasome activation by REG molecules lacking homolog-specific inserts; Zhang Z et al.; The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28 . The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts . These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai . The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome . Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts . By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein . REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai . At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert . These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation . By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes . On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome . We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes. Protein Eng, 1997 Dec, 10(12), 1461 - 3 Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein; Brenner C et al.; Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors . Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis . Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate-bound state . Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog . Single crystals obtained by vapor diffusion against ammonium sulfate diffracted X-rays to beyond 2.75 A resolution . High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form. Protein Eng, 1997 Dec, 10(12), 1453 - 9 A form of anti-Tac(Fv) which is both single-chain and disulfide stabilized: comparison with its single-chain and disulfide-stabilized homologs; Rajagopal V et al.; Disulfide-stabilized Fvs (dsFvs) are recombinant proteins composed of a heavy-chain variable domain (VH) of an antibody connected via a disulfide bond to the light-chain variable domain (VL) . In single-chain Fvs (scFvs), a peptide connector links VH and VL . The dsFv form of the anti-Tac monoclonal antibody which reacts with the alpha subunit of the IL2 receptor was recently reported to be more stable and to aggregate less during renaturation than anti-Tac(scFv) . In addition, it could be produced in a better yield owing to less aggregation . However, the yields are still too low to permit the production of material for clinical trials in which the dsFv will be used to image or treat IL2 receptor (CD25)-containing tumors . To increase the efficiency by which VH and VL associate and form a disulfide bond during renaturation, we have prepared an Fv form of anti-Tac which is both single chain and disulfide stabilized (scdsFv) . The recombinant protein is expressed in Escherichia coli, where it accumulates in inclusion bodies . Using inclusion body protein as the reference point, the yield of purified anti-Tac(scdsFv) was 13% compared with 2% for anti-Tac(dsFv) . Anti-Tac(scdsFv) has equivalent binding affinity, immunoreactivity after radiolabeling and stability . The results show that a linker between VH and VL facilitates heterodimer formation and leads to disulfide bond formation in a higher percentage of the molecules renatured . Thus anti-Tac(scdsFv) is the preferred form of anti-Tac(Fv) to be used for clinical studies . We anticipate that scdsFvs will be the optimum recombinant form of Fv to produce from bacteria. Protein Eng, 1997 Dec, 10(12), 1425 - 32 Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin; de Lamotte-Guery F et al.; Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix . The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein . We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli . The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations . Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3 . The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible . These predictions were totally in agreement with the experimental results . The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31 . The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol . However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation . Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased . The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT . Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein. J Clin Microbiol, 1998 Apr, 36(4), 857 - 61 Enzyme-linked immunosorbent assay using recombinant OspC and the internal 14-kDa flagellin fragment for serodiagnosis of early Lyme disease; Rauer S et al.; The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo of Borrelia burgdorferi sensu stricto were expressed as recombinant proteins in Escherichia coli and were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC-14-kDa antigen ELISA) . No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA . The recombinant OspC-14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract from Borrelia afzelii PKo as the antigen . According to the manufacturer's information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens . The specificity was adjusted to 95% on the basis of data for 154 healthy controls . On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%) . However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%) . OspC displays sequence heterogeneity of up to 40% according to the genomospecies . However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains of B . burgdorferi sensu stricto (strain GeHo) and B . afzelii (strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen . This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study . In conclusion, we suggest that the OspC-14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease. Plant Physiol, 1998 Apr, 116(4), 1339 - 50 Cloning and characterization of AtRGP1 . A reversibly autoglycosylated arabidopsis protein implicated in cell wall biosynthesis; Delgado IJ et al.; A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides . We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene . Polyclonal antibodies detect homologs in both dicot and monocot species . The patterns of expression and intracellular localization of the protein were examined . AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells . Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes . We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates . Possible sites for UDP-sugar binding and glycosylation are discussed . Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown. Plant Physiol, 1998 Apr, 116(4), 1271 - 8 The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis; Yamaguchi S et al.; The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf . Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS) . Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype . A genomic clone coding for KS, AtKS, was isolated from A . thaliana using CmKS cDNA as a heterologous probe . The corresponding A . thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein . The fusion protein showed enzymatic activity that converted {3H}copalyl diphosphate to {3H}ent-kaurene . The recombinant AtKS protein derived from the ga2-1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro . Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2-1 mutant . Taken together, our results show that the GA2 locus encodes KS. J Mol Biol, 1998 Apr 3, 277(3), 723 - 32 Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues; Scholz C et al.; The trigger factor is associated with bacterial ribosomes and catalyzes proline-limited protein folding reactions . Its folding activity is very high and conserved in evolution, as shown for the homologous enzymes from Escherichia coli and Mycoplasma genitalium . The folding protein substrate (a variant of ribonuclease T1) binds with high affinity to the trigger factors, and permanently unfolded proteins are strong, competitive inhibitors . We used this inhibition to characterize the substrate binding sites of the trigger factors . Unfolded alpha-lactalbumin binds very tightly and inhibits the trigger factor from M . genitalium with a KI value of 50 nM . The binding of inhibitory proteins is independent of proline residues, as shown for unfolded tendamistat, which binds to the trigger factor with equal affinity in the presence and in the absence of its three proline residues . The good inhibition by a non-folding variant of ribonuclease T1 that lacks Pro39 showed that this proline, at which the catalysis of folding occurs, is dispensable for substrate binding . The trigger factors cannot catalyze prolyl isomerization when proteins are partially folded already . They preferentially recognize unstructured protein chains, which bind with high affinity to a site distinct from the catalytic prolyl isomerase center in the FKBP domain . J Mol Biol, 1998 Apr 3, 277(3), 707 - 22 Structural stability and internal motions of Escherichia coli ribonuclease HI: 15N relaxation and hydrogen-deuterium exchange analyses; Yamasaki K et al.; The relationship between the structural stability and the internal motions of proteins was investigated through measurements of 15N relaxation and hydrogen-deuterium exchange rates of ribonuclease HI from Escherichia coli and its thermostable quintuple mutant (Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His), which has a higher melting temperature by 20.2 degreesC . For most of the residues, the generalized order parameters (S2) obtained from 15N relaxation analyses as well as the localized hydrogen-bond-breaking motions (local breathing) observed as fast H-D exchange rates were largely unaffected by the mutations, indicating no global mutational effect on the internal motions . Several local mutational effects were observed for residues close to the mutation sites as follows . The S2 value significantly increased for Lys96 and Val98, which indicated that motions on the pico- to nanosecond time-scale became restricted within a protruding region including the Lys95-->Gly mutation site . In contrast, slight decreases in S2, and drastic increases in the chemical exchange motion on the micro- to millisecond time-scale (Deltaex), were observed for residues located in the joining region between the protrusion and the major domain of the protein . These changes may be caused by the elimination of the bulky Lys95 side-chain at the center of the protrusion . Deltaex observed for residues in alpha-helix I of the wild-type protein was reduced for the mutant, probably because a cavity in the hydrophobic core is filled by the Val74-->Leu mutation . The local breathing at position 134 was restricted by the Asp134-->His mutation, probably because the reduction of the negative charge repulsion contributes t