Biochemistry, 1996 Jan 30, 35(4), 1232 - 41 Thermodynamics of the membrane insertion process of the M13 procoat protein, a lipid bilayer traversing protein containing a leader sequence; Soekarjo M et al.; For the first time, the standard free energy change, delta Gzero, of a membrane-inserting protein with a leader sequence has been determined experimentally, using M13 procoat protein as an example . The partition coefficient for the distribution of the procoat protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of gamma = 6.5 x 10(5) M-1, corresponding to a delta Gzero of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC . For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: gamma = 1 x 10(5) M-1 (delta Gzero = -9.3 kcal/mol) . Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region . By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol . In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of the mutant procoat protein OM30R {which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)} was determined, yielding gamma = 0.3 x 10(5) M-1 (delta Gzero = -8.6 kcal/mol) . Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli membranes but only binds to the inner surface . The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to delta Gzero, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein . Neither the leader sequence nor the mature coat protein alone yields this free energy gain . For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase . Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane. Biochemistry, 1996 Jan 30, 35(4), 1179 - 86 Accurate kinetic modeling of alkaline phosphatase in the Escherichia coli periplasm: implications for enzyme properties and substrate diffusion; Martinez MB et al.; Alkaline phosphatase in the periplasm of Escherichia coli presents many of the complex factors that may influence enzymes in vivo . These include an environment that contains a high enzyme concentration, is densely populated with other macromolecules, and is separated from other compartments by a partial diffusion barrier . A previous study provided a partial description of this situation and developed a model that utilized kinetic behavior to estimate the permeability of the outer membrane {Martinez, M . B., et al., (1992) Biochemistry 31, 11500} . This study extends that description to provide a complete model for the enzyme at all substrate levels . Some of the parameters needed for complete modeling include the following: outer membrane permeability to the substrate and product, catalytic efficiency of the enzyme, number of enzymes per cell, and effects of the reaction product (an inhibitor) on the enzyme . The theoretical model fit the data quite well over a wide range of values for each of these parameters . The best fit of theory with experimental data required that the rate constant for product escape from the periplasm was 4-fold greater than that for substrate entry . This correlated with the relative sizes of the substrate and product . The excellent fit of theory and results suggested that alkaline phosphatase and its substrate were unaffected by the solution conditions in the periplasm . That is, the catalytic parameters (kcat and KM), determined for the enzyme in dilute solution, appeared to be unchanged by the conditions in the periplasm . The major factor that altered the kinetic behavior was the combined effect of the permeability barrier and the dense population of enzyme molecules in the periplasm . Given the large impact of these parameters on reaction properties, the excellent fit of theory and results was striking . Overall, this study demonstrated that enzyme action in the complex biological environment can be accurately modeled, if all factors that influence enzyme behavior are known. Biochemistry, 1996 Jan 30, 35(4), 1162 - 72 Mode of selectivity in cyclic AMP receptor protein-dependent promoters in Escherichia coli; Pyles EA et al.; Escherichia coli cAMP receptor protein (CRP) controls more than 20 genes . There are significant differences in the promoter regions in these genes . Thus, an elucidation of the mechanism of CRP action requires knowledge about the mode of selectivity in these promoters . An earlier study {Heyduk, T., & Lee, J . C . (1990) Proc . Natl . Acad . Sci . U.S.A . 81, 1744-8} indicates that the CRP(cAMP)1 conformer exhibits the highest affinity for the lac PI site in the lac operon . It is conceivable that the CRP conformer that binds with the highest affinity to these other sites may not be CRP(cAMP)1 . To investigate this possibility, the binding of CRP to nine CRP binding sites was studied as a function of cAMP concentration . The CRP binding sites employed in this investigation were chosen to represent the primary promoter sites from class I (lac site PI) and class II (sites PI of gal and crp) as well as secondary CRP binding sites (crp site PII and cat PII) to further understand the molecular mechanism of CRP in controlling the transcription of these bacterial genes . The affinity of CRP for three synthetic CRP binding sites was also examined to explore the contribution of the inverted repeat region and sequences surrounding the recognition motifs . The synthetic sequences are gallac which contains the lac recognition motifs in the background of gal, modified cat PII which contains an 8-base pair spacer between the recognition motifs rather than the 7-base pair sequence naturally found in cat PII, and a random sequence that has no known similarity to any CRP binding site found in nature . The apparent affinities of these sequences for CRP were quantitatively determined to be biphasic in their cAMP dependence . The CRP(cAMP)1 conformer was found to have the highest affinity for all of the DNA sequences examined . No specific affinity was observed for these sequences with free CRP and CRP(cAMP)2 . The affinity of CRP for DNA was sequence-dependent and increased in the following order: random < cat site PII, modified cat site PII, crp sites PI and PII < gal site PI < lac site PI < gallac . These results indicate that the entire CRP binding site sequence and its natural variability provide information to CRP . These promoter sites which appear to have different mechanisms at the molecular level are transcriptionally controlled by the same CRP conformer, CRP(cAMP)1 . Thus, the regulation of transcription by CRP is more subtle than choosing different conformational forms of CRP . Using "physiological" concentrations of various components, a computer simulation study was conducted to illustrate the possible consequences of the thermodynamic parameters determined in this study . It is evident that the promoters of protein systems regulating the transport and metabolism of carbohydrates are responsive to low cAMP concentrations . However, the promoter for controlling the expression of CRP is highly regulated by the fluctuation of cAMP concentration. FEBS Lett, 1996 Jan 29, 379(2), 135 - 8 Expression of human inducible nitric oxide synthase in Escherichia coli; Fossetta JD et al.; We have expressed active full-length human inducible nitric oxide synthase (iNOS) in E . coli . Expression required co-expression with calmodulin, a particularly tight-binding cofactor . The extracts also required tetrahydrobiopterin to display activity . Specific activity of the purified recombinant iNOS was similar to iNOS purified from murine macrophages . This result indicates that no special processing events unique to eucaryotic cells are necessary for iNOS activity. FEBS Lett, 1996 Jan 29, 379(2), 122 - 6 Use of immobilized synthetic peptides for the identification of contact sites between human interleukin-6 and its receptor; Weiergraber O et al.; Synthetic peptides immobilized on cellulose membranes proved to be a powerful tool for the identification of sites in the cytokine IL-6 involved in receptor binding . Similarly, a region in the extracellular part of the IL-6 receptor which is important for interaction with its ligand was identified. J Chromatogr A, 1996 Jan 26, 722(1-2), 359 - 68 Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis; Arakawa H et al.; Analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis (CE) was developed . The conformational change of single-strand DNA is caused by a mutation in a DNA fragment . The change is detected as mobility shift in CE . The effects of acrylamide gel concentration, running temperature and fragment size amplified by the polymerase chain reaction (PCR) were studied to develop the separation of SSCP . The model DNA used was the divE 42 gene carrying wild- and mutant-type (G-->A point mutation at the 141 site) . The results show that two single-strand DNA fragments that differ in one nucleotide can be separated by CE within minutes . This method was also applied to the separation of SSCP for N-ras gene including four kinds of mutations . All mutations tested in this study could be distinguished . CE is well suited for clinical analysis of SSCP because it is rapid and reproducible, allows on-line detection and is easy. Biochem Biophys Res Commun, 1996 Jan 26, 218(3), 902 - 7 Synthesis and purification of soluble ligand binding domain of the human vitamin D3 receptor; Craig TA et al.; We expressed and purified milligram quantities of the ligand binding domain of the human 1,25-dihydroxyvitamin D3 receptor using a glutathione-S-transferase (GST) fusion protein expression system . Amino acids 105-427 were expressed in E . coli as a GST fusion protein at a reduced (20 degrees C) temperature and purified on glutathione sepharose . The fusion protein adsorbed to glutathione sepharose was cleaved with thrombin to yield soluble 105-427 human 1,25-dihydroxyvitamin D3 receptor . The 105-427 human 1,25-dihydroxyvitamin D3 receptor was further purified by Mono Q ion exchange chromatography and was characterized as a single band on SDS-polyacrylamide gel electrophoresis . The 105-427 human 1,25-dihydroxyvitamin D3 receptor bound 1,25-dihydroxyvitamin D3 with high affinity (Kd approximately 10(-9)M) and with a binding capacity of 47 pmoles/nmole protein . Large scale expression of 105-427 human 1,25-dihydroxyvitamin D3 receptor will provide human 1,25-dihydroxyvitamin D3 receptor ligand binding domain suitable for structural studies. Biochem Biophys Res Commun, 1996 Jan 26, 218(3), 682 - 7 A stable phage-display system using a phagemid vector: phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline, phosphatase, and anti-HEL monoclonal antibody, HyHEL10; Maenaka K et al.; A stable expression system for displaying the pIII fusion protein on the surface of a filamentous phage was constructed . A phagemid pIII display vector, pLUCK, was constructed by inserting the gene encoding the pIII fusion protein in the opposite direction to that of the lac promoter of pTZ18U . Using this phage display system, two enzymes, hen egg-white lysozyme (HEL) and E . coli alkaline phosphatase, and the single-chain Fv fragment of anti-HEL monoclonal antibody HyHEL10, could be stably and functionally displayed . Northern and primer extension analyses showed that a small amount of the sense mRNA encoding pIII-fused HEL was transcribed from the minor phage promoter located in the region encoding the C-terminus of pIII . Repressed expression of the pIII fusion protein can lead to the display of a wide range of proteins on filamentous phages without the need for strict expression conditions. Int J Cancer, 1996 Jan 26, 65(3), 383 - 8 Immunochemical identification of novel high-molecular-weight protein isoforms of the adenomatous polyposis coli (APC) gene; Kraus C et al.; Mapping analyses of monoclonal antibodies (MAbs) directed against the amino-terminus of the adenomatous polyposis coli (APC) gene product revealed that epitopes recognized by the MAbs FE9, CF11 and AC4 constitute different peptide sequences encoded by the APC exons 1, 2 and 3, respectively . The protein pattern detected with these specificity-defined immunoreagents, however, differed depending on the particular antibody used on Western blots of cellular urea extracts . APC exon 15-positive "classic" p300apc polypeptide chains were identified by the MAb FE9, MAb CF11 and the C-terminus-specific MAb IE1, but only weak signals were obtained with the AC4 MAb, which defines an exon 3-encoded epitope . In contrast with this immunoreactivity, 2 novel high m.w . products of approx . 150/160 and 200 kDa were exclusively recognized by the AC4 MAb, which was shown to bind to the APC exon 3-encoded peptide sequence SRESTGYL . A molecular form of some 400 kDa was identified to represent a disulfide-bound oligomer of the p150/160apc molecules . The novel APC-related molecules did not contain exon 1- and exon 15-encoded epitopes, as confirmed with the help of the FE9 and IE1 MAbs, respectively . This observation was corroborated by the fact that these novel proteins were not truncated in a collection of familial adenomatous polyposis patients with stop mutations in exon 15 . We conclude, that APC MAb AC4-reactive p150/160 and p200 polypeptide chains represent novel genuine products of the APC gene devoid of exon 1- and exon 15-encoded protein interaction domains. J Mol Biol, 1996 Jan 26, 255(3), 425 - 34 Duplex destabilization in superhelical DNA is predicted to occur at specific transcriptional regulatory regions; Benham CJ; Analytic methods that accurately calculate the extent of duplex destabilization induced in each base-pair of a DNA molecule by superhelical stresses are used to analyze several genomic DNA sequences . Sites predicted to be susceptible to stress-induced duplex destabilization (SIDD) are found to be closely associated with specific transcriptional regulatory regions . Operators within the promoters of SOS-regulated Escherichia coli genes are destabilized by superhelical stresses, whereas closely related sequences present elsewhere on that genome are not . Analysis of genomic sequences from the budding yeast Saccharomyces cerevisiae finds a distinctive tripartite pattern, in which the 3' and 5' termini of genes are destabilized, but the sequence encoding the primary transcript is not . Three rDNA genes from higher eukaryotes exhibit a similar pattern . Implications of these results regarding possible mechanisms of activity of the regions involved are discussed . A strategy is presented for designing experiments in which the susceptibility to SIDD of a local region is altered without changing its local base sequence . The occurrence of the observed SIDD patterns provides a new approach to searching uncharacterized genomic sequences for transcriptionally active regions. J Mol Biol, 1996 Jan 26, 255(3), 373 - 86 Affinity selective isolation of ligands from peptide libraries through display on a lac repressor "headpiece dimer"; Gates CM et al.; DNA binding by the Escherichia coli lac repressor is mediated by the approximately 60 amino acid residue 'headpiece' domain . The dimer of headpiece domains that binds to the lac operator is normally formed by association of the much larger approximately 300 amino acid residue C-terminal domain . We have used in vitro selection to isolate 'headpiece dimer' molecules containing two headpiece domains connected via a short peptide linker . These proteins bind plasmid molecules with sufficient stability to allow association of a peptide epitope displayed at the C terminus of the headpiece dimer with the plasmid encoding that peptide . Libraries of peptides displayed on the C terminus of a headpiece dimer can be screened for specific receptor ligands by affinity enrichment of peptide-headpiece dimer-plasmid complexes using an immobilized receptor . After each round of enrichment, transformation of E . coli with recovered plasmids permits amplification of the selected population . After several rounds of enrichment, sequencing of individual clones reveals the structure of the selected peptides . Headpiece dimer libraries allow selection of peptide ligands of higher average affinity than similar libraries based on the intact lac repressor . Interestingly, the presence of the lac operator is not required for plasmid binding by the headpiece dimer protein. J Mol Biol, 1996 Jan 26, 255(3), 349 - 55 The N-terminal domain of the rne gene product has RNase E activity and is non-overlapping with the arginine-rich RNA-binding site; McDowall KJ et al.; The rne gene of Escherichia coli encodes a 118 kDa protein that has ribonuclease E (RNase E) activity and binds RNA . A functional rne gene product is essential for cell viability and for the processing and/or decay of a variety of RNA species, including 9 S RNA, mRNA and RNAI, the antisense RNA regulator of ColE1-type plasmid replication . By testing the ability of different segments of the Rne protein to catalyze RNA cleavage and to bind RNA, we found that the N-terminal half (residues 1 to 498) of Rne contains a catalytic function sufficient for site-specific cleavage of oligoribonucleotides and complex RNAs . The C-terminal half of the protein, which contains both an arginine-rich region (residues 597 to 684) that we show binds RNA and a segment that is essential for cell viability (residues 844 to 1061), had no detectable endoribonucleolytic activity . Our results, which map the catalytic domain of RNase E, indicate the existence of discrete functional domains within the multifaceted Rne protein. J Biol Chem, 1996 Jan 26, 271(4), 2249 - 54 Mapping and functional role of phosphorylation sites in the thyroid transcription factor-1 (TTF-1); Zannini M et al.; The phosphorylation of thyroid transcription factor-1 (TTF-1), is homeodomain-containing transcription factor that is required for thyroid-specific expression of the thyroglobulin and thyroperoxidase gene promoters, has been studied . Phosphorylation occurs on a maximum of seven serine residues that are distributed in three tryptic peptides . Mutant derivatives of TTF-1, with alanine sites, have been constructed and used to assess the functional relevance of TTF-1 phosphorylation . The DNA binding activity of TTF-1 appears to be phosphorylation-independent, as indicated also by the performance of TTF-1 purified from an overexpressing Escherichia coli strain . Transcriptional activation by TTF-1 could require phosphorylation only in specific cell types since in a co-transfection assay in heterologous cells both wild-type and mutant proteins show a similar transcriptional activity. J Biol Chem, 1996 Jan 26, 271(4), 2206 - 12 Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein co-purified with splicing factor SF2 . Hyaluronic acid-binding protein as P-32 protein, co-purified with splicing factor SF2; Deb TB et al.; The purification of a 68-kDa hyaluronic acid-binding protein (HA-binding protein), a homodimer of 34 kDa that binds specifically to hyaluronic acid, has been reported earlier by us (Gupta, S., Batchu, R.B., and Datta, K . (1991) Eur . J . Cell Biol . 56, 58-67) . Here, we report the isolation of a partial cDNA clone from a lambda gt11 cDNA expression library of human skin fibroblast by immuno-screening with HA-binding protein antiserum . The internal polypeptide sequence (83 residues) of the purified hyaluronic acid-binding protein is identical to the predicted protein sequence derived from hyaluronic acid-binding protein cDNA, suggesting the authenticity of the clone . Interestingly, this hyaluronic acid-binding protein cDNA sequence has complete homology with the cDNA sequence of a protein P-32, co-purified with the human pre-mRNA splicing factor SF2 (Krainer, A.R., Mayeda, A., Kozak, D., and Binns, G . (1991) Cell 66, 383-394) . Furthermore, the data on the N-terminal sequence of hyaluronic acid-binding protein and the predicted polypeptide of P-32 revealed the identical coding sequence of 209 amino acids for both the proteins . As the identity and functional characterization of P-32 have not yet been reported, P-32 cDNA was expressed in Escherichia coli, and the recombinant P-32 protein was purified by hyaluronic acid-Sepharose affinity chromatography . The recombinant P-32 protein showed immunocross-reactivity with the polyclonal antibodies raised against HA-binding protein . The predicted amino acid sequence of the protein fulfilled the minimal criteria for binding to hyaluronic acid, i.e . two basic amino acids flanking a seven-amino acid stretch, as reported for other hyaluronic acid affinity of the recombinant P-32 protein was confirmed by biotinylated hyaluronic acid binding assay . The binding of recombinant P-32 protein to biotinylated hyaluronic acid binding assay . The binding of recombinant P-32 protein to biotinylated hyaluronic acid can be competed only with excess unlabeled hyaluronic acid, confirming its specificity toward hyaluronic acid . All these results suggest that both P-32, co-purified with the human pre-mRNA splicing factor SF2, and 34-kDa hyaluronic acid-binding protein reported by us are the same protein and that it is a new member of the hyaluronic acid-binding protein family, the "hyaladherins." J Biol Chem, 1996 Jan 26, 271(4), 2057 - 62 Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex and reconstitution of recombinant protein with three-subunit core complex; Chen YR et al.; Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/RSIV . Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 degrees C in LB medium . Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin . When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant subunit IV of R . sphaeroides cytochrome b-c1 complex . Although the isolated recombinant subunit IV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa . The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution . When the three-subunit core cytochrome b-c1 complex, purified from RS delta IV-adapted chromatophores containing a fraction of the wild type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased . Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used . The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex . A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex. J Biol Chem, 1996 Jan 26, 271(4), 1998 - 2004 Promoter selectivity of Escherichia coli RNA polymerase E sigma 70 and E sigma 38 holoenzymes . Effect of DNA supercoiling; Kusano S et al.; The functional specificity was compared between two sigma factors, sigma 70 (the major sigma at exponentially growing phase) and sigma 38 (the essential sigma at stationary growth phase), of Escherichia coli RNA polymerase . The core enzyme binding affinity of sigma 38 was less than half the level of sigma 70 as measured by gel filtration column chromatography or by titrating the concentration of sigma required for the maximum transcription in the presence of a fixed amount of core enzyme . In addition, the holoenzyme concentration required for the maximum transcription of a fixed amount of templates was higher for E sigma 38 than E sigma 70 . The transcription by E sigma 38 was, however, enhanced with the use of templates with low superhelical density, in good agreement with the decrease in DNA superhelicity in the stationary growth phase . We thus propose that the selective transcription of stationary-specific genes by E sigma 38 holoenzyme requires either a specific reaction condition(s) or a specific factor(s) such as template DNA with low superhelical density. J Biol Chem, 1996 Jan 26, 271(4), 1966 - 71 Strand displacement synthesis of the long terminal repeats by HIV reverse transcriptase; Fuentes GM et al.; According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo . We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer . The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation . Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not . The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis . Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA . This observation suggests that recombination is favored during strand displacement synthesis in vivo. J Biol Chem, 1996 Jan 26, 271(4), 1853 - 6 CTG triplet repeats from human hereditary diseases are dominant genetic expansion products in Escherichia coli; Ohshima K et al.; The relative ability of the 10 triplet repeat sequences to be expanded in Escherichia coli was determined . Surprisingly, CTG tracts are expanded at least 8 times more frequently than any of the other nine triplets . Low levels of expansion were found also for CGG, GTG, and GTC . Thus, the structure of the CTG repeats and/or their utilization by the DNA synthetic systems in vivo must be quite different from the other triplets . These data further validate this genetically defined system for elucidating molecular mechanisms of expansion and may explain why most triplet repeat hereditary neuromuscular and neurodegenerative disease genes contain CTG repeats. J Biol Chem, 1996 Jan 26, 271(4), 1833 - 6 Regulation of fatty acid elongation and initiation by acyl-acyl carrier protein in Escherichia coli; Heath RJ et al.; Long chain acyl-acyl carrier protein (acyl-ACP) has been implicated as a physiological inhibitor of fatty acid biosynthesis since acyl-ACP degradation by thioesterase overexpression leads to constitutive, unregulated fatty acid production . The biochemical targets for acyl-ACP inhibition were unknown, and this work identified two biosynthetic enzymes that were sensitive to acyl-ACP feedback inhibition . Palmitoyl-ACP inhibited the incorporation of {14C}malonyl-CoA into long chain fatty acids in cell-free extracts of Escherichia coli . A short chain acyl-ACP species with the electrophoretic properties of beta-hydroxybutyryl-ACP accumulated concomitant with the overall decrease in the amount of {14C}malonyl-CoA incorporation, indicating that the first elongation cycle was targeted by acyl-ACP . All of the proteins required to catalyze the first round of fatty acid synthesis from acetyl-CoA plus malonyl-CoA in vitro were isolated, and the first fatty acid elongation cycle was reconstituted with these purified components . Analysis of the individual enzymes and the pattern of intermediate accumulation in the reconstituted system identified initiation of fatty acid synthesis by beta-ketoacyl-ACP synthase III (fabH) and enoyl-ACP reductase (fabI) in the elongation cycle as two steps attenuated by long chain acyl-ACP. Nature, 1996 Jan 25, 379(6563), 311 - 9 The 2.0 A crystal structure of a heterotrimeric G protein; Lambright DG et al.; The structure of a heterotrimeric G protein reveals the mechanism of the nucleotide-dependent engagement of the alpha and beta gamma subunits that regulates their interaction with receptor and effector molecules . The interaction involves two distinct interfaces and dramatically alters the conformation of the alpha but not of the beta gamma subunits . The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation with respect to the membrane surface and an activated heptahelical receptor. Biochemistry, 1996 Jan 23, 35(3), 990 - 8 Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli; Sun J et al.; Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation {Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H . R . (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H . R . (1984) Biochemistry 23, 3688} . In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance . Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants . Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism . Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity . Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation. Biochemistry, 1996 Jan 23, 35(3), 948 - 54 Role of glutamate-104 in generating a transition state analogue inhibitor at the active site of cytidine deaminase; Carlow DC et al.; The 19F-NMR resonance of 5-{19F}fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rate 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine . Comparison of pKa values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M . Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and kcat has been reduced by a factor of 10(8) . 5-{19F}Fluoropyrimidin-2-one ribonucleoside is strongly fluorescent, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (Kd = 4.4 x 10(-8) M) than does the native enzyme (Kd = 1.1 x 10(-7) M) . 19F-NMR indicates, however, that the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state . These results are consistent with a major role for Glu-104, not only in stabilizing the ES++ complex in the transition state, but also in destabilizing the ES complex in the ground state. Biochemistry, 1996 Jan 23, 35(3), 922 - 9 Preparation and characterization of a disulfide-linked bioconjugate of annexin V with the B-chain of urokinase: an improved fibrinolytic agent targeted to phospholipid-containing thrombi; Tanaka K et al.; A conjugate of annexin V and the B-chain of urokinase was prepared and its fibrinolytic properties were studied . First, a mutant of annexin V was constructed with an N-terminal extension of six amino acids (Met-Ala-Cys-Asp-His-Ser) and with Cys316 mutated to Ser; this molecule was expressed in Escherichia coli . The urokinase B-chain was prepared by limited reduction of the interchain disulfide bond between the A- and B-chains of urokinase . These two molecules were then then connected by a disulfide bond and purified to yield a 1:1 stoichiometric conjugate . The conjugate had the same catalytic activity as urokinase against a synthetic substrate, Glt-Gly-Arg-MCA, and a similar plasminogen activating activity . The conjugate showed the same binding affinity for phosphatidylserine-containing membranes as annexin V . The in vitro fibrinolytic activity of the conjugates on clots prepared from platelet-rich plasma was comparable to that of urokinase . However, the conjugate showed 3-4-fold stronger in vivo thrombolytic activity than urokinase in a rat pulmonary embolism model, while having essentially the same plasma clearance rate as urokinase or B-chain . These results show that annexin V is a useful agent for targeting plasminogen activators to phospholipid-containing thrombi. Biochemistry, 1996 Jan 23, 35(3), 743 - 8 Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis; Mollah AK et al.; The crystal structure of an engineered monomer of the lambda Cro repressor shows unexpected expansion of the hydrophobic core of the protein and disorder of the five C-terminal residues {Albright et al . (1996) Biochemistry 35, 735-742} . This structural information has guided the construction of a second generation of monomeric Cro proteins by combinatorial mutagenesis of selected core and C-terminal residues . Clones were identified in a library of randomized cro genes by a genetic screen for protein accumulation in Escherichia coli . Sequencing of candidate genes followed by purification and analysis of their product proteins has identified alternative arrangements of hydrophobic core residues which result in substantial increases in thermal stability . In contrast, residue replacements at the C-terminus have minor effects on stability but may increase protein expression levels. FEBS Lett, 1996 Jan 22, 379(1), 94 - 6 Cloning, purification, and crystallization of Escherichia coli cystathionine beta-lyase; Laber B et al.; The metC gene coding for cystathionine beta-lyase of Escherichia coli has been cloned and used to construct an overproducing E . coli strain . An efficient purification scheme has been developed and the purified enzyme has been crystallized by the hanging drop vapour diffusion method using either ammonium sulfate or polyethyleneglycol 400 as precipitating agent . The crystals belong to the orthorombic space group C222 . Their unit cell parameters are a = 60.9 A, b = 154.7 A and c = 152.7 A . Consideration of the possible values of VM accounts for the presence of one dimer per asymmetric unit . The crystals are suitable for X-ray analysis and a complete native date set to 1.83 A resolution has been collected using synchrotron radiation. FEBS Lett, 1996 Jan 22, 379(1), 47 - 50 15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1-24); Uegaki K et al.; For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required . A direct over-expression system by E . coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells . We here developed an over-expression system by means of thioredoxin gene fusion system . The fused protein composed of thioredoxin and the objective peptide was expressed in E . coli and then the peptide part was released by enterokinase . This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis. FEBS Lett, 1996 Jan 22, 379(1), 107 - 11 Molecular cloning of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A; Tanabe O et al.; Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or delta) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library . The cDNA had an open reading frame encoding an M(r) 66,138 protein of 570 amino acids . Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit . The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72 . Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, and SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence . The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues. J Theor Biol, 1996 Jan 21, 178(2), 183 - 204 Evolution of transcriptional regulation system through promiscuous coupling of regulatory proteins with operons; suggestion from protein sequence similarities in Escherichia coli; Otsuka J et al.; As an advanced molecular study of the problems of the evolution of organisms, the transcriptional regulation system is studied by investigating the amino acid sequence similarities between the proteins in the regulation system of Escherichia coli in which the data of sequenced proteins as well as of regulator-regulon relationships are accumulated . The similarities between the proteins are calculated by the FASTA algorithm and their homology is also evaluated in terms of statistical significance with the use of the RDF2 program . This investigation reveals that the similarity between the regulatory protein and the regulated protein is hardly found, but many similarities are found between regulatory proteins and between regulated proteins . These similarity relations are compared with the regulator-regulon relationships ascertained experimentally . From this comparison, it is found that similar regulatory proteins rarely regulate the transcription of similar protein genes . As most of the highly similar proteins are considered to have diverged from a common ancestral protein, this finding strongly suggests the possibility that descendant regulatory proteins have been promiscuously coupled with descendant operons, independently of their ancestral regulator-regulon relationship, and that some of the couplings have been fixed by selection to form the present system of transcriptional regulation . The compatibility of such promiscuous coupling with regulatory organization is illustrated in the carbohydrate transport systems and the succeeding metabolic pathways, whose organization is comprehensive in sending nutritious substances to the central path of glycolysis under different environmental conditions . The benefit of flexibility in regulator-regulon relationships in evolutionary processes is also discussed in connection with the punctuational divergence of species in macroevolution and the cell differentiation in multicellular organisms. Hum Gene Ther, 1996 Jan 20, 7(2), 173 - 82 Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration; Geschwind MD et al.; The neurotrophins are a family of growth factors that play an important role in the development and maintenance of the nervous system . Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that appears to participate in the maturation and function of mammalian auditory neurons . Forms of deafness due to varied injurious stimuli that are amenable to treatment with implantable prosthetic devices require the survival of these BDNF-responsive auditory neurons for effective outcome . To evaluate the feasibility of developing a gene therapy for deafness that may be used in conjunction with a prosthetic device, we constructed replication-defective herpes simplex virus (HSV) amplicon vectors that carry the human BDNF cDNA . Using these vectors, HSVbdnf and HSVbdnflac (expresses BDNF and Escherichia coli beta-galactosidase), we evaluated the expression and biological activity in established cell lines and explant cultures prepared from spiral ganglia of the murine ear . Gene transfer with HSVbdnf resulted in the efficient expression of human BDNF mRNA in murine fibroblasts . Using two BDNF-responsive cell lines, PC12trkB and MG87trkB, we demonstrate efficient secretion of biologically active BDNF . Finally, transduction of explanted spiral ganglia with HSVbdnflac elicited robust neuritic process outgrowth comparable to exogenously added BDNF . Overall, these data demonstrate that HSV vectors can efficiently transfer and express the BDNF gene in many cell types, including auditory neurons . Moreover, they suggest that similar vectors may be used to express the neurotrophin in auditory neurons in vivo and perhaps as adjunctive gene therapy for deafness. J Biol Chem, 1996 Jan 19, 271(3), 1726 - 31 Expression and biological activity of mouse fibroblast growth factor-9; Santos-Ocampo S et al.; Receptor specificity is an essential mechanism governing the activity of fibroblast growth factors (FGF) . To begin to understand the developmental role of FGF-9/glial activating factor, we have cloned and sequenced the murine FGF-9 cDNA and expressed the protein in mammalian cells and in Escherichia coli . We demonstrate that the FGF-9 protein is highly conserved between mouse and human . Receptor specificity was determined by direct binding to soluble and cell surface forms of FGF receptor (FGFR) splice variants and by the mitogenic activity on cells, which express unique FGF receptor splice variants . Our data demonstrate that FGF-9 efficiently activates the "c" splice forms of FGFR2 and FGFR3, receptors expressed in potential target cells for FGF-9 . Significantly, FGF-9 also binds to and activates the "b" splice form of FGFR3, thus becoming the first FGF ligand besides FGF-1 to activate this highly specific member of the FGF receptor family. J Biol Chem, 1996 Jan 19, 271(3), 1435 - 40 Monovalent cation activation and kinetic mechanism of inosine 5'-monophosphate dehydrogenase; Xiang B et al.; Human type II inosine 5'-monophosphate dehydrogenase has been purified to homogeneity from an Escherichia coli strain that express large quantities of the enzyme from the cloned gene . Steady state kinetic studies have been used to characterize the activation by monovalent cations, including Li+, Na+, K+, Rb+, Cs+, Tl+, NH4+, and N(CH3)4+ . The enzyme has less than 1% of the maximal activity in the absence of an added monovalent cation, such as K+, Na+, Rb+, Tl+, or NH4+ . The enzyme is activated by K+ and Tl+ at lower concentrations than those of other monovalent cations . Li+ and N(CH3)4+ do not activate the enzyme, nor do they inhibit the K(+)-activated enzyme, implying that ionic radius is important in binding selectivity . The Km values for both substrates and Vmax differ with different monovalent cations . Initial velocity and product inhibition kinetic data are consistent with an ordered steady state mechanism in which the enzyme binds K+ first, TMP second, and then NAD; the product NADH is released before xanthosine 5'-monophosphate . Substrate and product binding experiments support this mechanism and show the presence of one substrate binding site per subunit . Several rate constants were obtained from a computer simulation of the complete steady state rate equation. J Biol Chem, 1996 Jan 19, 271(3), 1416 - 23 Novel regulation of keratin gene expression by thyroid hormone and retinoid receptors; Tomic-Canic M et al.; Expression of keratin proteins, markers of epidermal differentiation and pathology, is uniquely regulated by the nuclear receptors for retinoic acid (RAR) and thyroid hormone (T3R) and their ligands: it is constitutively activated by unliganded T3R, but it is suppressed by ligand-occupied T3R or RAR . This regulation was studied using gel mobility shift assays with purified receptors and transient transfection assays with vectors expressing various receptor mutants . Regulation of keratin gene expression by RAR and T3R occurs through direct binding of these receptors to receptor response elements of the keratin gene promoters . The DNA binding "C" domain of these receptors is essential for both ligand-dependent and -independent regulation . However, the NH2-terminal "A/B" domain of T3R is not required for either mode of regulation of keratin gene expression . Furthermore, v-ErbA, an oncogenic derivative of cT3R, also activates keratin gene expression . In contrast to the previously described mechanism of gene regulation by T3R, heterodimerization with the retinoid X receptor is not essential for activation of keratin gene expression by unliganded T3R . These findings indicate that the mechanism of regulation of keratin genes by RAR and T3R differs significantly from the mechanisms described for other genes modulated by these receptors. J Biol Chem, 1996 Jan 19, 271(3), 1400 - 4 Evidence that transmembrane segment 2 of the lactose permease is part of a conformationally sensitive interface between the two halves of the protein; Jessen-Marshall AE et al.; A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), is found in a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane . This motif is located within the cytoplasmic side of transmembrane domain 2 (TM-2) and extends through the hydrophilic loop that connects transmembrane domains 2 and 3 . The motif is repeated again in the second half of the protein . In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A . E., Paul, N . J., and Brooker, R . J . (1995) J . Biol . Chem . 270, 16251-16257), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-directed mutations were found to greatly diminish the rate of transport . In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental strains to isolate second site suppressor mutations that restore transport function . A total of 10 different second site mutations were identified among a screen of 19 independent mutants . One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine . Since the conserved aspartate and position 45 are at opposite ends of TM-2, these results suggest that the role of the conserved aspartate residue in loop 2/3 is to influence the topology of TM-2 . Surprisingly, the majority of suppressor mutations were found in the second half of the permease . All of these are expected to alter helix topology in either of two ways . Some of the mutations involved residues within transmembrane segments 7 and 11 that produced substantial changes in side chain volume: TM-7 (Cys-234-->Trp or Phe, Gln-241-->Leu, and Phe-247-->Val) and TM-11 (Ser-366-->Phe) . Alternatively, other mutations were highly disruptive substitutions at the ends of transmembrane segments or within hydrophilic loops (Gly-257-->Asp, Val-367-->Glu, Ala-369-->Pro, and a 5-codon insertion into loop 11/12) . It is hypothesized that the effects of these suppressor mutations are to alter the helical topologies in the second half of the protein to facilitate a better interaction with the first half . Overall, these results are consistent with a transport model in which TM-2 acts as an important interface between the two halves of the lactose permease . According to our tertiary model, this interaction occurs between TM-2 and TM-11. J Biol Chem, 1996 Jan 19, 271(3), 1349 - 56 Transcriptional regulation of the yeast DnaJ homologue SIS1; Zhong T et al.; The Saccharomyces cerevisiae SIS1 gene encodes an essential heat shock protein with similarity to the Escherichia coli DnaJ protein . In sis 1-85 and sis1-86 mutants, the sis1 RNA is induced to high levels at room temperature and without heat shock . The presence of wild type SIS1 in the sis1-85 mutant represses the overexpression of SIS1-85 protein . Furthermore, overexpression of wild type SIS1 reduces the beta-galactosidase activity expressed from a SIS1:lacZ fusion . These results suggest that SIS1 negatively regulates its own expression . The autoregulation of SIS1 transcription is mediated through a 39-base pair cis-element containing the SIS1 heat shock element plus additional flanking sequences on one side . Although SIS1 transcription is constitutive, it is transiently induced upon heat shock . In addition, SIS1 transcription is regulated by SSA (a class of HSP70 proteins) function . The elevated transcription of SIS1 in ssa1 ssa2 mutants is mediated solely through the SIS1 heat shock element . Therefore, the SIS1 autoregulatory element is different from the SSA-responsive element, suggesting that the mechanism involved in autoregulation of SIS1 is distinct from regulation of SIS1 by SSA proteins. J Biol Chem, 1996 Jan 19, 271(3), 1314 - 21 Folding-related dimerization of human cystatin C; Ekiel I et al.; With the aim to improve our understanding of the structural basis for protein self-association and aggregation, in particular in relationship to protein refolding and amyloid formation, folding-related processes for human cystatin C have been studied . Using NMR spectroscopy together with chromatographic and electrophoretic methods, a self-association process resulting in dimer formation for protein samples treated with denaturing agents as well as for samples subjected to low pH or high temperature conditions could be studied with amino acid resolution . In all three cases, the dimerization involves properly folded molecules and proceeds via the reactive site of the inhibitor, which leads to complete loss of its biological activity . This dimerization process has potential relevance for amyloid formation by the brain hemorrhage-causing Leu58-Gln variant of cystatin C . The results also indicate that cystatin C dimerization and inactivation may occur in acidified compartments in vivo, which could be relevant for the physiological regulation of cysteine proteinase activity. J Biol Chem, 1996 Jan 19, 271(3), 1285 - 94 Effects of assembly and mutations outside the active site on the functional pH dependence of Escherichia coli aspartate transcarbamylase; Yuan X et al.; Electrostatics are central to the function and regulation of Escherichia coli aspartate transcarbamylase, and modeling has suggested that long range electrostatic effects are likely to be important (Glackin, M . P., McCarthy, M . P., Mallikarachchi, D., Matthew, J . B., and Allewell, N . M . (1989) Proteins Struct . Funct . Genet . 5, 66-77; Oberoi, H., Trikha, J., Yuan, X., and Allewell, N . M . (1995) Proteins Struct . Funct . Genet., in press) . To investigate this possibility from an experimental standpoint, we have examined the effects both of assembly and of removing ionizable and polar side chains outside the active site (Glu-50, Tyr-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase . The holoenzyme (c6r6) assembles from three regulatory dimers (r2) and two catalytically active trimers (c3) . pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of the catalytic subunit (c3) and holoenzyme (c6r6) with carbamyl phosphate are different . In contrast, the Michaelis complex appears similar for both c3 and c6r6, except for pK shifts of approximately 1 pH unit . Results also indicate that the catalytic mechanism of the holoenzyme does not involve reverse protonation, as has recently been proposed for the catalytic trimer (Turnbull, J . L., Waldrop, G . L., and Schachman, H . K . (1992) Biochemistry 31, 6562-6569) . The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the cluc4 interface) and which is disrupted in the T --> R transition . The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type . Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wild-type catalytic subunit . Like Y165F, E50A has low activity and affinity over the entire pH range . Linkage analysis indicates that there is long range energetic coupling among the active site, the ear subunit interfaces, and residue Y165 . The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14-16 A from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site. Nature, 1996 Jan 18, 379(6562), 277 - 80 Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2; Eck MJ et al.; The domain organization of many signalling proteins facilitates a segregation of binding, catalytic and regulatory functions . The mammalian SH2 domain protein tyrosine phosphatases (PTPs) contain tandem SH2 domains and a single carboxy-terminal catalytic domain . SH-PTP1 (PTP1C, HCP) and SH-PTP2 (Syp, PTP2C, PTP1D) function downstream from tyrosine kinase-linked insulin, growth factor, cytokine and antigen receptors . As well as directing subcellular localization by binding to receptors and their substrates, the two SH2 domains of these PTPs function together to regulate catalysis . Here we report the structure of the tandem SH2 domains of SH-PTP2 in complex with monophosphopeptides . A fixed relative orientation of the two domains, stabilized by a disulphide bond and a small hydrophobic patch within the interface, separates the peptide binding sites by approximately 40 A . The defined orientation of the SH2 domains in the structure, and data showing that peptide orientation and spacing between binding sites is critical for enzymatic activation, suggest that spatial constraints are important in this multidomain protein-protein interaction. Nature, 1996 Jan 18, 379(6562), 225 - 32 Structure and mechanism of DNA topoisomerase II; Berger JM et al.; The crystal structure of a large fragment of yeast type II DNA topoisomerase reveals a heart-shaped dimeric protein with a large central hole . It provides a molecular model of the enzyme as an ATP-modulated clamp with two sets of jaws at opposite ends, connected by multiple joints . An enzyme with bound DNA can admit a second DNA duplex through one set of jaws, transport it through the cleaved first duplex, and expel it through the other set of jaws. Biochem Biophys Res Commun, 1996 Jan 17, 218(2), 461 - 5 Inhibition of the H+/peptide cotransporter in the human intestinal cell line Caco-2 by cyclic AMP; Muller U et al.; Treatment of Caco-2 cells with cholera toxin inhibits the activity of the H+/peptide cotransporter . The effect of cholera toxin is mimicked by E . coli heat-labile enterotoxin, forskolin and isobutylmethylxanthine and is associated with an increase in cAMP levels in the cells . The inhibition is due to a decrease in the maximal velocity of the transport system . Inhibitors of protein kinase A and protein kinase C block the effect of cholera toxin . Interestingly, the H+/peptide cotransporter in Caco-2 cells does not possess any putative site for phosphorylation by protein kinase A but does possess sites for phosphorylation by protein kinase C . It appears that the cAMP-dependent inhibition of the H+/peptide cotransporter in Caco-2 cells is mediated through activation of protein kinase C. Biochemistry, 1996 Jan 16, 35(2), 659 - 65 3'- and 5'-strand cleavage reactions catalyzed by the Fpg protein from Escherichia coli occur via successive beta- and delta-elimination mechanisms, respectively; Bhagwat M et al.; The Fpg protein from Escherichia coli is a multifunctional protein that excises damaged purine bases from DNA to generate aldehydic abasic sites and then catalyzes the successive cleavage of the phosphodiester bonds first on the 3'-side and then on the 5'-side of the abasic site to generate 5'- and 3'-phosphate ends, respectively, thereby excising the deoxyribose residue . The mechanisms of the 3'- and 5'-strand cleavage reactions have been studied by nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS) . The 3'-strand cleavage reaction is a beta-elimination reaction in which the 2'-hydrogen is abstracted and the 3'-phosphate is eliminated . The 5'-strand cleavage reaction is a delta-elimination reaction in which the 4'-hydrogen is abstracted and the 5'-phosphate is eliminated . Two types of experiments were performed to establish the occurrence of the sequential elimination reactions . First, when the reaction was performed in H2(18)O, 31P NMR demonstrated that neither phosphate group contained 18O . Second, the five-carbon product derived from the deoxyribose residue was stabilized by reduction with NaBH4 and characterized by GC-MS . The mass spectrum of the reduced product was identical to that of authentic 4-oxo-2-pentenal, the tautomerized product of successive beta- and delta-elimination reactions. Biochemistry, 1996 Jan 16, 35(2), 608 - 15 Evidence that specificity of microhelix charging by a class I tRNA synthetase occurs in the transition state of catalysis; Gale AJ et al.; Determinants for the identities of tRNAs are located in the acceptor stem and, commonly, in the anticodon as well . Although the anticodon is an important determinant for the identity of methionine tRNA, RNA microhelices whose sequences are based on the acceptor stem alone can be aminoacylated by the class I methionyl-tRNA synthetase . We show here that specific nucleotide substitutions in a microhelix significantly reduced its rate of aminoacylation . In contrast, affinity coelectrophoresis analysis showed that microhelix binding to the enzyme was not significantly affected by the same substitutions . These and additional experiments and considerations imply that specific determinants for microhelix aminoacylation are needed for orientation of the acceptor stem in the transition state of catalysis rather than for enhanced binding interactions . The effect of linking together acceptor stem interactions with those in the anticodon, as occurs in the whole tRNA molecule, was also evaluated . This analysis showed that linkage results in some of the favorable acceptor stem and anticodon interactions being used to offset the free energy cost of straining the structure of the enzyme-tRNA complex. Biochemistry, 1996 Jan 16, 35(2), 601 - 7 Aminoacyl-tRNA synthetases optimize both cognate tRNA recognition and discrimination against noncognate tRNAs; Sherman JM et al.; Specific protein--nucleic acid interactions are usually the product of sequence-dependent hydrogen bonding . However, in the crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln, leucine 136 (Leu136) stabilizes the disruption of the weak first (U1-A72) base pair in tRNAGln by stacking between A72 and G2 . We have demonstrated, by a combined in vivo and in vitro mutational analysis, that Leu136 is important for tRNA specificity despite making no hydrogen bonds with tRNAGln . Both more (L136F) and less (L136V, L136M, L136A, and L136T) mischarging mutants of GlnRS have been identified . GlnRS(L136F) is more mischarging and less specific than wild-type GlnRS in vivo, due not to an increased affinity for the noncognate tRNAs but to a decreased affinity for tRNAGln . Also, unlike other mischarging mutants of GlnRS that have been characterized, it does not exhibit generally relaxed tRNA specificity in vivo and mischarges only a subset of the tRNAs tested . A possible sequence preference for a Py1-Pu72/Pu2-Py71 combination is suggested . The L136A/M/T/V mutants are the first GlnRS variants, including wild-type, expressed on pBR322 which no longer mischarge tyrT(UAG) in vivo . We have shown that, while the L136A mutant is less mischarging than wild-type both in vivo and in vitro, it is not more specific as it also exhibits reduced affinity for its cognate glutamine tRNA . On the basis of these results, we suggest that the aminoacyl-tRNA synthetases have evolved to balance cognate tRNA recognition and discrimination against noncognate tRNAs. Biochemistry, 1996 Jan 16, 35(2), 545 - 53 Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains; Newton DL et al.; The gene for human angiogenin (Ang), a member of the ribonuclease superfamily, was fused to a gene encoding a single-chain antibody (sFv) against the human transferrin receptor . Three Ang single-chain immunofusion proteins (AngsFvs) were constructed with variations in the type of linker connecting the VL and VH chain {EGKSSGSGSESKEF, L1 or (GGGGS)3, L2} as well as with or without a spacer (FB) connecting the Ang and sFv (AngFBsFvL1 or L2; AngsFv(L2)} . Although the nature of the linker did not affect the enzymatic activity of the FB-containing fusion proteins, the fusion protein containing the L2 linker was 2.3-fold more effective than the L1 linker in competing with the labeled monoclonal IgG1 antibody for binding to the transferrin receptor . The fusion protein containing the L2 linker without the FB spacer exhibited a 13-fold decrease in binding to the transferrin receptor as well as a decrease in its capacity to degrade tRNA and to inhibit translation in the rabbit reticulocyte lysate compared to its counterpart containing the FB spacer . Binding of placental ribonuclease inhibitor (PRI) to Ang also was affected by the nature of the linker and by the presence or absence of a spacer . PRI bound to Ang and AngFBsFv(L2) and inhibited their ribonuclease activity . A 3-fold greater concentration of PRI, however, did not affect the activity of AngFBsFv(L1) or AngsFv(L2), suggesting that the conformation of these fusion proteins was altered . Binding of monoclonal and polyclonal anti-Ang antibodies to AngsFvs was also used to investigate conformational alterations of the fusion proteins . AngFBsFv(L2) was the least altered while AngFBsFv(L1) exhibited the greatest change in structure . Yet maximal concentrations of all AngsFvs elicited angiogenesis in the chick chorioallantoic membrane assay, demonstrating that Ang in all three fusion proteins remained functionally active . Consistent with all the activities, the fusion protein containing the FB spacer and L2 linker was the most cytotoxic to three different human tumor cell lines . The fusion protein lacking the FB spacer exhibited the least cytotoxicity . These data demonstrate that the linker connecting the VH-VL chains can affect the binding and cellular cytotoxicity of Ang immunofusions and that placement of a spacer between the antibody binding domains and Ang is necessary for optimal activity . Thus, a new class of targeted therapeutic agents containing Ang as the toxic moiety can be designed that potentially will be less immunogenic and less toxic than immunotoxins available currently. Biochemistry, 1996 Jan 16, 35(2), 433 - 43 Phosphotransfer and CheY-binding domains of the histidine autokinase CheA are joined by a flexible linker; Zhou H et al.; Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli . This fragment (CheA1-233) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region . Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA1-233 remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA124-257 . Relaxation properties of the backbone 15N nuclei were measured to study the rotational correlations of the two domains and properties of the linker region . Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model . The experimental T1 and T2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible . Dynamics data of CheA1-233 were also compared with those of CheA1-134 . Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions . These observations suggest a model for the graded regulation of CheA autophosphorylation activity . In this model, the various activity states of the receptor are generated by controlling the access of the mean position of the kinase domain to the phosphotransfer domain . This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values. J Neurosci Res, 1996 Jan 15, 43(2), 235 - 45 Possible involvement of tyrosine kinase activation in lipopolysaccharide-induced expression of Ca(2+)-insensitive but calmodulin-coupling nitric oxide synthase in rat glial cells; Kitamura Y et al.; To clarify the properties of an inducible type of nitric oxide synthase (i-NOS) in the brain, we examined whether lipopolysaccharide (LPS) induces NOS in glial cells cultured from neonatal rats . NOS activities (NO2- accumulation and L-{14C}citrulline formation) were detected by treatment with LPS at 10 micrograms/ml for 6-72 hr . L-{14C}citrulline formation by LPS-induced i-NOS was inhibited by NG-monomethyl-L-arginine (a NOS inhibitor) and diphenyleneiodonium (a flavo-protein inhibitor) . The activity was not markedly changed in the presence or absence of Ca2+ . The induction of i-NOS by LPS was abolished by cycloheximide, actinomycin D, or dexamethasone . In addition, the induction was inhibited by herbimycin A (a tyrosine kinase inhibitor), but was not by staurosporine, W-7, or FK-506 . After LPS stimulation, 130 kDa proteins were reacted with anti-rat liver i-NOS antibody 5-72 hr . i-NOS induced from glial cells coupled tightly with endogenous calmodulin (CaM) even in the absence of Ca2+ . These results suggest that LPS induces expression of 130-kDa i-NOS through an activation of tyrosine kinase, after which i-NOS couples with CaM, and that NO is formed for 6-72 hr in glial cells. J Neurosci Res, 1996 Jan 15, 43(2), 146 - 60 Proximal regions of the olfactory marker protein gene promoter direct olfactory neuron-specific expression in transgenic mice; Walters E et al.; Olfactory marker protein (OMP) expression is highly restricted to mature olfactory neurons (ON) . Less than 0.3 kb of upstream 5' flanking sequence of the OMP gene directs lacZ expression preferentially to ON in several independently derived lines of transgenic mice . A larger transgene with 0.8 kb of upstream flanking sequence also gave lacZ expression in ON and in a few ectopic sites in the central nervous system (CNS) . In addition to the main olfactory epithelium, endogenous OMP is also expressed in chemosensory neurons of the vomeronasal and septal organs, and lacZ expression was detected in neurons of these sites as well . This confirmed the presence of regulatory sequences in the proximal portion of the OMP gene . Endogenous OMP expression in ON was normal in all transgenic lines . Strikingly, in several transgenic lines lacZ expression was restricted to subsets of ON . In one such line, ON axons were intensely stained for lacZ and projected to a subset of olfactory bulb glomeruli . Although identifiable subsets of ON and their termination fields have been described previously, this is the first demonstration of this phenomenon in transgenic mice . These lines of transgenic mice thus provide in vivo models for characterization of genetic elements regulating developmental and functional organization of the olfactory neuroepithelium. Structure, 1996 Jan 15, 4(1), 89 - 96 The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations; Bujacz G et al.; BACKGROUND: Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity . So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E . coli and human immunodeficiency virus 1 (HIV-1) RNase H . Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes . RESULTS: The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution . A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination . The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN . CONCLUSIONS: Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN . This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site . Only a single divalent cation was observed even at extremely high concentrations of the metals . We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein . The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism. Structure, 1996 Jan 15, 4(1), 67 - 77 The complex of the anti-cancer therapeutic, BW1843U89, with thymidylate synthase at 2.0 A resolution: implications for a new mode of inhibition; Stout TJ et al.; BACKGROUND: Thymidylate synthase (TS) is critical to DNA synthesis as it catalyzes the rate limiting step in the only biosynthetic pathway for deoxythymidine monophosphate (dTMP) production . TS is therefore an important target for anti-proliferative and anti-cancer drug design . The TS enzymatic mechanism involves the reductive methylation of the substrate, deoxyuridine monophosphate (dUMP), by transfer of a methylene group from the co-factor, methylenetetrahydrofolate (CH2H4folate), resulting in the production of deoxythymidine monophosphate (dTMP) and dihydrofolate (H2folate) . Previous drug design efforts based on co-factor analogues have produced good inhibitors of TS, but poor bioavailability and toxicity have limited their usefulness . BW1843U89, a folate analogue, is a recently developed compound which is an exceptionally strong inhibitor (Ki = 0.09 nM), has good bioavailability and in clinical trials thus far has not demonstrated significant toxicity . RESULTS: We report the crystal structure of E . coli TS in ternary complex with dUMP and BW1843U89 at 2.0 A resolution . Although the benzoquinazoline ring system of the inhibitor binds to TS in much the same manner as previously determined for H2folate and CB3717, the larger size of the ligand is accommodated by the enzyme through a local distortion of the active site, that is not strictly conserved in both monomers in the asymmetric unit . Several conserved waters that had been previously implicated in mechanistic roles have been displaced . CONCLUSIONS: BW1843U89 forms a ternary complex with dUMP and completes with CH2H4 folate at the active site . Inhibition of TS by BW1843U89 shows four unique aspects in its mechanism of action . BW1843U89 prevents the Michael addition of dUMP to Cys146, in contrast to the mechanisms implicated from crystallography of other quinazoline based inhibitors; displaces a catalytic water from the active site; reorders a peptide loop (Leu72-Trp83) in the active site; and is unique amongst the antifolates in inactivating TS at a stoichiometric ratio of one molecule per TS dimer . Thus, it exploits the principles of negative cooperativity that are increasingly being recognized in the catalytic mechanism of the enzyme per se . The structure suggests that this 'half-the-sites' effect is catalytic and not related to ligand binding . Therefore BW1843U89 is both a competitive inhibitor (at the binding site) and a non-competitive inhibitor at the other site. Rev Prat, 1996 Jan 15, 46(2), 189 - 95 {Travelers' diarrhea}; Chagnon A; Up to fifty per cent of travellers going from temperate countries to tropical or subtropical countries present a diarrhoea . Enterotoxigenic Escherichia coli remain the most frequent bacterial cause, being identified in 40 to 70% of cases . Laboratory investigations are reserved to grave or protracted cases or those which resist to empirical therapy . Careful selection of food and beverage can limit its incidence . In adults at high risk of complications (sick or aged people) or who cannot suffer an interruption of their activity, one can prefer to a chemoprophylaxis with possible occurrence of adverse effects the early administration of a fluoroquinolone, which besides is a first-choice treatment of serious cases. Eur J Biochem, 1996 Jan 15, 235(1-2), 438 - 43 Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides; Willows RD et al.; The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase . The Rhodobacter sphaeroides genes, bchI and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3) . The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid . The production of large amounts of the 140-kDa BchH protein in E . coli was accompanied by an accumulation of protoporphyrin IX . The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1 . All three recombinant proteins were soluble; BchH was monomeric, Bchl was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa . The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified . Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP . One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio . The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+-ATP, and a metal-insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH . The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion. Eur J Biochem, 1996 Jan 15, 235(1-2), 372 - 81 Human biliverdin IXalpha reductase is a zinc-metalloprotein . Characterization of purified and Escherichia coli expressed enzymes; Maines MD et al.; Biliverdin IXalpha reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin . BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity . A cDNA clone encoding human BVR was isolated from a gamma library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA . This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed . The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein . The cDNA hybridized with a single message of approximately 1.2 kb in human kidney poly(A)-rich RNA, and appeared, by Southern blot analysis, to be the product of a single-copy gene . Sequence analysis indicated that the human reductase shows approximately 83% identity, at both the nucleotide and amino acid levels, with rat BVR . In some regions including the carboxyl terminus, protein sequence identity drops to 45% . Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat) . The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence . However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions . Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants . Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio . That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli . Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent, activity . Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity. Eur J Biochem, 1996 Jan 15, 235(1-2), 256 - 61 Comparison of self-sustained sequence-replication reaction systems; Gebinoga M et al.; The 3SR (self-sustained sequence-replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro . This method requires three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H . We have modified the original protocol by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E . coli ribonuclease H . Comparison of the incorporation kinetics between the conventional three-enzyme 3SR and our two-enzyme 3SR shows differences in the kinetic behaviour . Furthermore, by the new two-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three-enzyme 3SR . The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments. Eur J Biochem, 1996 Jan 15, 235(1-2), 248 - 55 Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI; Nakari-Setala T et al.; The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose-containing medium, was shown to encode a novel fungal hydrophobin . The encoded 97-amino-acid protein is cysteine-rich and has a typical signal sequence for secretion . Signal-sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids . Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T . reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose-containing cultures . The identity of HFBI was verified by N-terminal and peptide sequencing of proteins purified both from the cell wall and culture medium . In the cell wall most of the HFBI formed SDS-insoluble complexes that could be extracted with trifluoroacetic acid . Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus. Eur J Biochem, 1996 Jan 15, 235(1-2), 215 - 24 Molecular characterisation of plant endoplasmic reticulum . Identification of protein disulfide-isomerase as the major reticuloplasmin; Coughlan SJ et al.; Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two-step centrifugation procedure . These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components . A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity-purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide-isomerase . Northern and Southern blot analysis showed that the mRNA from a single-copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein . Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide-isomerase catalytic activity, thus confirming identity of this protein. Eur J Biochem, 1996 Jan 15, 235(1-2), 207 - 14 Influence of the signal sequence and chaperone SecB on the interaction between precursor protein prePhoE and phospholipids; Van Raalte AL et al.; To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of Escherichia coli outer-membrane protein PhoE . It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole2GroPGro) and dioleoylglycerophosphoethanolamine (Ole2GroPEtn), this insertion was mainly driven by hydrophobic forces . Compared with previous results obtained with PhoE signal peptide, the full-length precursor protein does not show the specific interaction with acidic lipids . PrePhoE inserted into a Ole2GroPGro monolayer occupies an area of 28 +/- 3 {corrected} nm2/molecule, which is approximately 10-fold larger than the area occupied by the PhoE signal peptide . The purified mature PhoE protein has a lower capacity to insert into Ole2GroPGro and Ole2GroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinase K after interacting with a Ole2GroPGro monolayer . The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers . It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB . Complexation with SecB increases the insertion of (pre)PhoE into acidic lipid monolayers . The high lipid affinity of prePhoE was also demonstrated by vesicle-binding experiments which showed that SecB dissociates from the SecB-prePhoE complex upon binding of the precursor to the bilayer . The implications of these findings for preprotein translocation are discussed and in addition some extrapolations to the insertion of PhoE into the outer membrane are made. Eur J Biochem, 1996 Jan 15, 235(1-2), 173 - 9 Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase and kinetic properties of the recombinant enzymes expressed in Escherichia coli; Duggleby RG et al.; UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP-Glc plus MgPPi . Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library . Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts . These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library {Peng, H.-L . & Chang, H.-Y . (1993) FEBS Lett . 329, 153-158} and it is proposed that these liver and muscle forms are derived from different genes . The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised . Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis-Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi . The liver form shows Michaelis-Menten kinetics towards MgUTP . For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration. Eur J Biochem, 1996 Jan 15, 235(1-2), 167 - 72 Redox properties of wild-type, Cys69Ala, and Cys69Ser Azotobacter vinelandii flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy,; Steensma E et al.; This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478) . Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue . Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin . In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode . However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response . EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry . The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK . Instead, the presence of at least two redox-linked protonation sites is suggested: pKred.1 = 5.39 +/- 0.08, pKox = 7.29 +/- 0.14, and pKred.2 = 7.84 +/- 0.14 with Em.7 = -459 +/- 4 mV, and a constant redox potential at high pH of -485 +/- 4 mV . The dependence of the semiquinone/hydroquinone redox potential on temperature is -0.5 +/- 0.1 mV . K(-1), yielding delta H degrees = 28.6 +/- 1.5 kJ . mol(1) and delta S degrees = -50.0 +/- 6.2 J . mol(-1) . K(-1) . No significant differences in redox properties of wild-type, C69A, and C69S flavodoxin were observed . The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A . vinelandii flavodoxin II. Eur J Biochem, 1996 Jan 15, 235(1-2), 152 - 8 Isolation and characterisation of dhel II, a DNA helicase from Drosophila melanogaster embryos stimulated by Escherichia coli-type single-stranded-DNA-binding proteins; Thommes P et al.; We have purified a DNA helicase from Drosophila embryos by following unwinding activity during the purification of the cellular single-stranded DNA-binding protein dRP-A . This DNA helicase unwinds DNA 5' to 3', has a salt-tolerant activity, and has a preference for purine triphosphates as cofactors for the unwinding reaction . The purified enzyme consists of a single polypeptide of 120 kDa, which cosediments with the helicase activity . Sedimentation analysis suggests that this polypeptide exists as a monomer under high and low salt conditions . Dhel II is able to unwind long stretches of DNA, but with decreased efficiency . Addition of Escherichia coli-like single-stranded DNA-binding proteins stimulates the unwinding activity at least 10-fold on substrates greater than 200 nucleotides . In particular, the mitochondrial single-stranded DNA-binding protein isolated from Drosophila embryos is able to stimulate unwinding by dhel II . These properties show that the helicase described is different from another Drosophila helicase dhel I; it has thus has been classified as dhel II. Nucleic Acids Res, 1996 Jan 15, 24(2), 348 - 53 DNA detection by strand displacement amplification and fluorescence polarization with signal enhancement using a DNA binding protein; Walker GT et al.; Strand displacement amplification (9SDA) is an isothermal in vitro method of amplifying a DNA sequence prior to its detection . We have combined SDA with fluorescence polarization detection . A 5'-fluorescein-labelled oligodeoxynucleotide detector probe hybridizes to the amplification product that rises in concentration during SDA and the single- to double strand conversion is monitored through an increase in fluorescence polarization . Detection sensitivity can be enhanced by using a detector probe containing an EcoRI recognition sequence at its 5'-end that is not homologous to the target sequence . During SDA the probe is converted to a fully double-stranded form that specifically binds a genetically modified form of the endonuclease EcoRI which lacks cleavage activity but retains binding specificity . We have applied this SDA detection system to a target sequence specific for Mycobacterium tuberculosis. EMBO J, 1996 Jan 15, 15(2), 408 - 17 A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates; Szabo A et al.; The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo . Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide . DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution . In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein . Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine-phenylalanine region following it are required for interactions with DnaK . However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase . As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins . Interestingly, binding experiments and cross-linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state. EMBO J, 1996 Jan 15, 15(2), 399 - 407 Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria; Komiya T et al.; Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria . Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner . Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70 . Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane . The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP . The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added . The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins. EMBO J, 1996 Jan 15, 15(2), 392 - 98 Preferential binding of an unfolded protein to DsbA; Frech C et al.; The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate . During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA . To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1 . We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol . This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions . The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds . Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 275 - 80 4-Phospho-hydroxy-L-threonine is an obligatory intermediate in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12; Zhao G et al.; We show that thrB-encoded homoserine kinase is required for growth of Escherichia coli K-12 pdxB mutants on minimal glucose medium supplemented with 4-hydroxy-L-threonine (synonym, 3-hydroxyhomoserine) or D-glycolaldehyde . This result is consistent with a model in which 4-phospho-hydroxy-L-threonine (synonym, 3-hydroxyhomoserine phosphate), rather than 4-hydroxy-L-threonine, is an obligatory intermediate in pyridoxal 5'-phosphate biosynthesis . Ring closure using 4-phospho-hydroxy-L-threonine as a substrate would lead to formation of pyridoxine 5'-phosphate, and not pyridoxine, as the first B6-vitamer synthesized de novo . These considerations suggest that E . coli pyridoxal/pyridoxamine/pyridoxine kinase is not required for the main de novo pathway of pyridoxal 5'-phosphate biosynthesis, and instead plays a role only in the B6-vitamer salvage pathway. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 245 - 9 Comparison of cytosolic levels of calcium and G actin in diffuse and localised adherent Escherichia coli-infected HeLa cells; Karmaker S et al.; In the present study we compared the intracellular level of free calcium ({Ca2+}i) and monomeric (G)/total (G+F) actin ratio in HeLa cells infected with diffuse (DAEC) and localised adherent Escherichia coli (LAEC) . The level of {Ca2+}i was increased in both DAEC- and LAEC-infected HeLa cells . However, studies with EGTA- and dantrolene-treated cells and also suspension of cells in Ca(2+)-free buffer suggested that the rise of {Ca2+}i in DAEC-infected cells was due to the influx of Ca2+ from extracellular medium, whereas Ca2+ mobilisation from the intracellular stores was responsible for the enhancement of {Ca2+}i in LAEC-infected cells . It was also evident that the infection of HeLa cells with DAEC and LAEC caused alteration of G/G+F actin ratio as compared to that of control cells . The ratio was much lower in LAEC-infected cells than that of DAEC-infected ones . Moreover, cytochalasin B inhibited both DAEC and LAEC invasion to HeLa cells, suggesting further the role of microfilaments in the invasion process. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 237 - 43 A stable Escherichia coli-mycobacteria shuttle vector 'pSO246' in Mycobacterium bovis BCG; Matsumoto S et al.; The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum . The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated . The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG) . The plasmid vector pSO246 was stable in BCG for at least 50 generations. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 213 - 21 Identification of sequences important for recognition of vnf genes by the VnfA transcriptional activator in Azotobacter vinelandii; Woodley P et al.; To analyze regulation of the vanadium-dependent nitrogenase of Azotobacter vinelandii, plasmids carrying vnfE-, vnfH-, or vnfD-lacZ fusions were transferred to Escherichia coli . These genes were expressed only if VnfA was present . Deletions of the vnfE upstream region were constructed and comparison of a region necessary for expression with sequences upstream of other vnf genes indicated a substantially conserved motif, GTAC-N6-GTAC, hypothesized to be the binding site for VnfA . This motif was duplicated with 17 or 18 bases lying between each in the vnfH and vnfD promoters . Deletion analysis of the vnfH promoter indicated that both motifs were necessary for full expression . In footprinting experiments, VnfA significantly protected from methylation the guanine residues within or immediately adjacent to the proposed VnfA recognition motifs . The active form of VnfA is probably interacting dimers, a tetramer, or a higher order oligomer since two regions of dyad symmetry are required for its interaction with the DNA. Biochem J, 1996 Jan 15, 313 ( Pt 2), 589 - 96 Biosynthesis of dermatan sulphate . Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism; Hannesson HH et al.; The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ({GlcA(beta 1-->3)GalNAc(beta 1-->4)}n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues . Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis . Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units . A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate) . Vmax was about 4 times higher with dermatan than with the K4 substrate . A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-{5-3H}glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme . Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA . A corresponding incubation of dermatan yielded approx . 22% GlcA, which contained virtually all the 3H label . These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base . Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed. Biochem J, 1996 Jan 15, 313 ( Pt 2), 479 - 86 Administration of Escherichia coli endotoxin to rat increases liver mass and hepatocyte volume in vivo; Qian D et al.; We have established, in vivo, an increase in liver mass and hepatocyte volume after a single intraperitoneal administration, to fasted rats, of Escherichia coli lipopolysaccharide (0127:B8) at 3 mg/kg . The phenomenon was time- and dose-dependent and could be prevented by treatment with polyclonal antiserum against tumour necrosis factor-alpha (TNF-alpha) before the endotoxin injection . Endotoxin caused an increase of 26% in the hepatic mass compared with fasted controls at 24 h . An increase of 27% in the hepatic water content underlay the altered hepatic mass which could not be accounted for by a change in the volume of hepatic blood and/or interstitial fluid (measured in vivo), suggesting an expansion in the hepatocellular volume . This is supported by an increase of 25% in the K+ content of the endotoxic livers . Morphometric study confirmed a 15% increase in hepatocyte volume after endotoxin administration . The data are discussed in the light of possible metabolic effects of increased hepatocyte volume. Biochem J, 1996 Jan 15, 313 ( Pt 2), 415 - 21 Evidence for a covalent intermediate in the S-adenosyl-L-methionine-dependent transmethylation reaction catalysed by sirohaem synthase; Woodcock SC et al.; CysG, also known as uroporphyrinogen III methylase and sirohaem synthase (CysG; EC 2.1.1.107), is a multifunctional enzyme that is able to transform uroporphyrinogen III into sirohaem via two S-adenosyl-L-methionine (AdoMet)-dependent transmethylations, an NAD(+)-dependent dehydrogenation and a ferrochelation . The apparent tight binding of AdoMet to this multifunctional enzyme is investigated . The use of a rapid AdoMet binding assay demonstrates that CysG becomes labelled with both {methyl-3H}AdoMet and {carboxyl-14C}AdoMet . Further experiments show that the CysG-AdoMet complex is subsequently able to methylate uroporphyrinogen III . CysG remains associated with the labelled constituents of the AdoMet even after denaturation with urea and SDS/PAGE, suggesting that the AdoMet has become covalently linked to the protein . A rapid examination of some of the other transmethylases involved in corrin biosynthesis reveals that they bind the AdoMet in a similar fashion . A multistep transmethylation mechanism is proposed to explain the observed results. Biochem J, 1996 Jan 15, 313 ( Pt 2), 409 - 14 Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease; Doskeland AP et al.; The phosphorylation of human phenylalanine hydroxylase by cyclic AMP-dependent protein kinase was studied using recombinant enzyme expressed as a fusion protein in the pMAL system of Escherichia coli . Using the target sequence of the restriction protease enterokinase (Asp4-Lys) as the linker peptide, 100% full-length human phenylalanine hydroxylase was obtained on protease cleavage . The fusion protein and human phenylalanine hydroxylase were both phosphorylated at Ser-16 with a stoichiometry of 1 mol of Pi/mol of subunit . The rate of phosphorylation of human phenylalanine hydroxylase was inhibited about 40% by the cofactor tetrahydrobiopterin, and this inhibition was completely prevented by the simultaneous presence of L-phenylalanine (i.e . at turnover conditions) . Phosphorylated enzyme revealed a 1.6-fold higher specific activity than the non-phosphorylated enzyme form, and it also required a lower concentration of L-Phe for substrate activation . Pre-incubation with L-Phe increased the specific activity of phenylalanine hydroxylase 2- to 4-fold, L-Phe acting with positive cooperativity . Thus, the basic catalytic and regulatory properties of recombinant human phenylalanine hydroxylase, as well as those observed for the enzyme as a fusion protein, are similar to those previously reported for the rat liver enzyme . When the target sequence of the restriction protease factor Xa (Ile-Glu-Gly-Arg) was used as the linker between maltose-binding protein and human phenylalanine hydroxylase, cleavage of the fusion protein gave a mixture of full-length hydroxylase and a truncated form of the enzyme lacking the 13 N-terminal residues . Interestingly, phosphorylation of the fusion protein, before exposure to factor Xa, almost completely protected against secondary cleavage by this restriction protease at Arg-13 of phenylalanine hydroxylase. Mol Gen Genet, 1996 Jan 15, 250(1), 81 - 8 Escherichia coli cis- and trans-acting mutations that increase glyA gene expression; Lorenz E et al.; We used an Escherichia coli strain blocked in serine biosynthesis and carrying a partial glyA deletion to isolate strains with altered regulation of the glyA gene . The glyA deletion results in 25% of the normal serine hydroxymethyltransferase activity . Three classes of mutants with increased glyA expression were isolated on glycine supplemented plates . One class of mutations increased glyA expression 10-fold by directly altering the -35 consensus sequence of the glyA promoter . The two other classes increased glyA expression about 2- and 6-fold, respectively . The latter two classes of mutations also affected regulation of the metE gene of the folate branch of the methionine pathway, but not metA in the nonfolate branch of the methionine pathway, or the gcv operon, encoding the glycine cleavage enzyme system . The mutations were mapped to about minute 85.5 on the E . coli chromosome. J Clin Invest, 1996 Jan 15, 97(2), 359 - 65 A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide; Somerville JE Jr et al.; A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells . A mutation was identified in the msbB gene of E . coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A . Unlike all previously reported lipid A mutants, the msbB mutant was not conditionally lethal for growth . Viable cells or purified LPS from an msbB mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF alpha production by adherent monocytes . The cloned msbB gene was able to functionally complement the msbB mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells . Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain . These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism . In addition, the msbB mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells. Arch Biochem Biophys, 1996 Jan 15, 325(2), 270 - 8 The sequencing expression, purification, and steady-state kinetic analysis of quinolinate phosphoribosyl transferase from Escherichia coli; Bhatia R et al.; The nadC gene from Escherichia coli was isolated and sequenced . The gene was then cloned into an expression vector and, following transformation, the resulting bacteria were able to produce quinolinate phosphoribosyl transferase as about 2% of the soluble protein . The enzyme was purified in five steps leading to a homogeneous preparation . The enzyme reaction shows an ordered binding mechanism where the magnesium ion complex of 5-phosphoribosyl-1-pyrophosphate binds first followed by quinolinic acid . The products are pyrophosphate CO2, and nicotinate mononucleotide . Product inhibition studies show that nicotinate mononucleotide is a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate while pyrophosphate is noncompetitive with respect to both 5-phosphoribosyl-1-pyrophosphate and quinolinic acid . Phthalic acid and fructose-1,6-bisphosphate were used as dead-end inhibitors . Phthalate was competitive with respect to quinolinic acid but uncompetitive with respect to 5-phosphoribosyl-1-pyrophosphate . Fructose-1,6-bisphosphate was a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate and noncompetitive with respect to quinolinic acid . The Km values for the substrates are 15.6 microM for 5-phosphoribosyl-1-pyrophosphate and 6.4 microM for quinolinic acid. FEBS Lett, 1996 Jan 15, 378(3), 286 - 90 Effects of N-terminal deletions on 1-aminocyclopropane-1-carboxylate synthase activity; Li N et al.; A series of nested N-terminal deletions were made on the full-length (wt) and C-terminal deleted (Cdel) 1-aminocyclopropane-1-carboxylate synthase cDNAs . These wt and mutant ACC synthases were over-expressed in a heterologous E . coli expression system . It was found that removal of an amino acid region (residues 2-12) from the non-conserved N-termini of wt and Cdel ACC synthases led to a slight increase in both in vivo ACC production and in vitro ACC synthase activity . Further deletion of 11 amino acids through Glu-23 from the N-termini of both wt and Cdel ACC synthases resulted in a substantial reduction in both in vivo ACC production and in vitro enzyme activity . Deletion of an amino acid region, residues 3 through 27, from the N-terminus of ACC synthase abolished enzyme activity completely . Kinetic analysis of a highly purified double-deletion mutant (NCdel-1) of ACC synthase demonstrated that the Km of this mutant is 42 microM, which is much smaller than that of the corresponding Cdel (280 microM) and closer to that of wt (22 microM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function. J Immunol, 1996 Jan 15, 156(2), 778 - 85 Modulation of endotoxin-induced histamine synthesis by cytokines in mouse bone marrow-derived macrophages; Takamatsu S et al.; The aim of this study was to examine the roles of various cytokines in histamine synthesis by macrophages in bone marrow . Pure (> 99% nonspecific esterase-, CD14-, and Mac-1-positive) macrophage populations were obtained after long term culture of bone marrow cells (bone marrow-derived macrophages) . Culture of the cells in the presence of Escherichia coli LPS caused a slight, but dose-dependent, increase in histidine decarboxylase-associated histamine synthesis with a concomitant increase in the expression of CD14, a LPS receptor, as well as the Mac-1 Ag on their surface . Granulocyte/macrophage CSF (GM-CSF) or IL-3 strongly enhanced LPS-induced histamine formation and expression of CD14 on bone marrow-derived macrophages, without affecting the expression of Mac-1 Ag . GM-CSF and IL-3 also caused a marked accumulation of both histidine decarboxylase and IL-6 mRNAs in the cells . Macrophage CSF and IL-1-alpha also potentiated LPS-dependent histamine formation when it was stimulated with GM-CSF or IL-3 . In contrast to these cytokines, IFN-gamma suppressed LPS-induced histamine production regardless of whether it was stimulated by GM-CSF or IL-3, and inhibited CD14 expression . Neither IL-6 nor granulocyte CSF had any appreciable effect on LPS-induced histamine production even in combination with GM-CSF or IL-3 . These results suggest that GM-CSF and IL-3 enhance LPS-induced histamine production in bone marrow-derived macrophages and that macrophage CSF and IL-1 alpha augment the actions of GM-CSF and IL-3 . Possible implication of CD14 molecule in the reactions is discussed. Mol Cell Biochem, 1996 Jan 12, 154(1), 9 - 16 Biochemical and immunological identification and enrichment of poly(A) polymerase from human thymus; Kyriakopoulou C et al.; Human thymus poly(A) polymerase (EC 2.7.7.1