Biochemistry, 1993 Nov 16, 32(45), 12096 - 104 Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA; Jones CJ et al.; The xeroderma pigmentosum group A complementing protein (XPAC) is involved in an early step of nucleotide excision repair, the main process that removes UV damage and many chemical lesions from DNA . To explore the properties and function of XPAC, recombinant protein encoded by the human XPAC cDNA was expressed with an N-terminal polyhistidine tag in Escherichia coli and purified to homogeneity . The soluble fusion protein could correct the repair defect in vitro of XP-A cell extracts . XPAC protein bound to DNA with a preference for UV-irradiated over nonirradiated DNA, as determined by a gel electrophoresis mobility shift assay with a 258 base pair DNA fragment (the association constant was approximately 3 x 10(6) M-1 for the fragment irradiated with 6 kJ/m2 UV light) . Removal of cyclobutane pyrimidine dimers from UV-irradiated DNA by enzymatic photoreactivation did not significantly reduce binding of XPAC to the irradiated fragment, indicating that binding was mostly due to (6-4) photoproducts, with a preference for a (6-4) photoproduct over an undamaged base pair up to 300-fold . Undamaged single-stranded DNA competed about 4-fold more effectively than undamaged double-stranded DNA for binding of XPAC to a UV-irradiated fragment . In addition, XPAC bound to DNA treated with the chemotherapeutic agent cis-diamminedichloroplatinum(II) . The results suggest that XPAC functions as a key component in recognition of DNA damage during repair. Biochemistry, 1993 Nov 16, 32(45), 12054 - 61 Boundary of the autoinhibitory region of smooth muscle myosin light-chain kinase; Yano K et al.; It has been proposed that myosin light-chain kinase (MLCK) activity is inhibited in the absence of Ca2+/calmodulin by a pseudosubstrate sequence {Kemp, B . E., Pearson, R . B., Guerriero, V . J., Bagchi, I., & Means, A . R . (1987) J . Biol . Chem . 262, 2542-2548} . To evaluate this hypothesis, the role of a cluster of basic residues, Arg797-Arg798-Lys799, which are essential for the pseudosubstrate sequence, in the inhibition of MLCK was studied . A full-length cDNA of chicken gizzard MLCK was obtained, and the recombinant MLCK which contains the entire amino acid sequence was expressed in Escherichia coli . The Ca2+/calmodulin-dependent activity of the recombinant MLCK was comparable to that of the naturally isolated MLCK . Two truncation mutants, MT799 and MT796, were produced, of which MT799 but not MT796 contained a cluster of basic residues . Neither MT799 nor MT796 bound calmodulin, and kinase activity was inhibited (similar to MLCK activity in the absence of Ca2+/calmodulin) . However, the kinase activity of the mutants was increased markedly by subsequent tryptic proteolysis . The tryptic digestion of the mutants initially produced a 64-kDa fragment then, subsequently, the 61-kDa fragment, and the increase in activity coincided with the appearance of the 61-kDa fragment . This was similar to the digestion profile of native MLCK, and it is known that the 61-kDa fragment is the constitutively active kinase {Ikebe, M., Stepinska, M., Kemp, B . E., Means, A . R., & Hartshorne, D . J . (1987) J . Biol . Chem . 262, 13828-13834}.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Nov 16, 32(45), 11953 - 6 The gateway to the active site of heme-copper oxidases; Lemon DD et al.; The spectroscopy and dynamics of CO binding were measured for wild-type and mutant cytochromes bo, members of the superfamily of heme-copper oxidases . The results suggest that access of ligands, including substrate O2, to the binuclear Fe-Cu active site is controlled at two levels . CO recombination to the wild-type ubiquinol oxidase exhibited saturation kinetics (kmax = 190 s-1, Km = 2.4 mM), indicative of the existence of an intermediate in the ligand-binding pathway . FTIR spectroscopy and TRIR spectroscopy were used to demonstrate conclusively that this intermediate was a CuB-CO complex . Two mutant oxidases (His333Leu, His334Leu) which lack CuB showed no evidence of saturation of CO rebinding, even up to 21 mM CO . Also, the absolute rates of CO binding to the mutant oxidases were much greater than for wild type, even at CO concentrations well below the apparent Km for wild-type enzyme . These results clearly indicate that the copper ion at the binuclear site acts as an obligatory way station, or gate, severely limiting the approach of ligands to the heme active site . Further, an analysis of the rate constants for CO binding to CuB suggests that the protein structure external to the binuclear site regulates ligand entry into this site . We propose that these control mechanisms for substrate binding are operative throughout this general class of enzymes. Biochemistry, 1993 Nov 16, 32(45), 12203 - 8 Modification of the head-group selectivity of porcine pancreatic phospholipase A2 by protein engineering; Bhat MK et al.; On the basis of the three-dimensional structures of phospholipid and porcine pancreatic phospholipase A2 (pla2), it was predicted that the removal of a negative charge in the hydrophilic region of the phospholipid binding site would influence the head-group selectivity of porcine pancreatic pla2 . To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis . The E46L mutant, expressed in Escherichia coli, was purified and characterized . The mutation did not affect the activity toward the mixed micellar substrate, but the activity of E46L toward DiC12-P, which has two negative charges on the head group, was three times higher than that of DiC12-PC, which carries no net charge in the head group . The native pla2 was inhibited by the product(s) released from DiC12-P but not the mutant enzyme . Kinetic analysis revealed that the E46L mutant and the native pla2 had comparable affinities (Km) toward monomeric and micellar phospholipids of zwitterionic type while the activity (kcat) of E46L, toward the same substrates, was approximately 50% lower compared to that of native pla2 . When micellar DiC12-P was used as a substrate, the Kmapp value for E46L was four times lower and the kcatapp/kmapp was 5-fold higher than those of native pla2 . However, the kinetic parameters of mutant and native pla2s remained unchanged for monomeric HEPG, with one negative charge in the head group . Thus, we have modified the head-group selectivity of porcine pancreatic pla2 by protein engineering. Anal Biochem, 1993 Nov 15, 215(1), 118 - 28 Membrane protein topology determination by proteolysis of maltose binding protein fusions; Miller KW et al.; A method is presented for determining the topology of Escherichia coli inner membrane proteins that is based on proteolysis of fusion proteins between maltose binding protein (MBP) and the membrane protein of interest . Fusion proteins are constructed wherein the MBP domain is fused upstream of the membrane protein domain . A secreted MBP domain is attached to the protein if its N-terminus resides in the periplasm . A cytosolic MBP domain (MBP delta 2-26) is attached to the protein when its N-terminus resides in the cytoplasm . The method has been developed using a fusion protein in which a secreted MBP domain is attached to the pBR322 tetracycline resistance protein at its first periplasmic loop . Fusion proteins are subjected to partial proteolysis in membrane vesicles under conditions where digestion does not occur in MBP . The mixture of digestion products is analyzed by Western immunoblotting using anti-MBP antiserum to detect cleavage fragments . Sites of digestion in the membrane protein are identified by comparing the mobilities of digestion products to truncated fusion standards that terminate at defined locations within the membrane protein domain . The technique has the advantage that neither overexpression of the protein nor high-quality antiserum to it are required for detection of protease fragments . Furthermore, the method can be applied to screen membrane proteins for structure alterations that have been introduced deliberately into them. Tijdschr Diergeneeskd, 1993 Nov 15, 118(22), 731 - 4 {Infectious diarrhea of calves: therapeutic and preventive possibilities based on etiological, pathophysiological and immunological data}; Vanopdenbosch E et al.; In this article the current knowledge on the development mechanisms of bacterial, viral and cryptosporidial neonatal calf diarrhoea are briefly reviewed as basis for an efficacious prophylaxis strategy . Hygienic measures will prevent the infection of the calf before colostrum intake and will diminish the infection pressure during the first weeks of life . Moreover, local intestinal immunity will be provided by early administration of immune colostrum for the prevention of E.coli diarrhoea and prolonged administration of colostrum or immune milk during the first weeks can protect against viral diarrhoea . To obtain a maximum level of E.coli antibodies in colostrum, the last vaccination has to be administered at least 8 weeks before parturition . Prolonged excretion of antiviral antibodies in the milk for several weeks, can be achieved by a 'priming' vaccination with an adjuvanted inactivated vaccine during pregnancy, followed by a booster vaccination within 24 hours after parturition . Cryptosporidium parvum diarrhoea can be prevented with halofuginone lactate. Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1552 - 7 Sensitive detection of low levels of ribonuclease H activity by an improved renaturation gel assay; Frank P et al.; Renaturation gel assays are good tools to assign enzymatic activities to protein bands . First, proteins are separated by denaturating electrophoresis on substrate-containing gels . Then, following the elimination of the denaturing agent, polypeptides are allowed to renature, thus leading to the degradation of the embedded substrate at positions at which the corresponding activity has moved . Nevertheless, this in situ technique does not only reflect a certain amount of enzyme activity, it also depends upon the ability of an enzyme to renature . Here we present a renaturation gel assay procedure with an improved sensitivity and discuss the detection of E . coli and human ribonuclease H activities as an example. Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1466 - 73 Cloning, sequencing and expression of the gene for alpha antigen from Mycobacterium intracellulare and use of PCR for the rapid identification of Mycobacterium intracellulare; Kitaura H et al.; The complete nucleotide sequence of alpha antigen secreted from Mycobacterium intracellulare (ATCC13950) was determined . The gene encoded 330 amino acids including 40 amino acids for signal peptide, followed by 290 amino acids for a mature protein with molecular mass 30,645 Da . The cloned gene was expressed in Escherichia coli by using an E . coli expression vector . Based on these results, the feasibility of rapid identification of M . intracellulare by two step polymerase chain reaction (PCR) was demonstrated. Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1376 - 82 Binding site of annexin XI on the calcyclin molecule; Watanabe M et al.; We purified rabbit calcyclin of S100 family protein and a calcyclin associated protein which has proved to be a novel annexin, annexin XI . Using a co-precipitation assay of annexin XI with phospholipid, the binding site of annexin XI on calcyclin was examined . The peptide fragment of calcyclin, CNBr-3 (residues 1-57), digested with cyanogen bromide completely inhibited the interaction of native calcyclin with annexin XI, while CNBr-1 (residues 83-90) and CNBr-2 (residues 58-82) did not affect the binding . We then constructed and expressed recombinant cDNAs for wild type and four different deletion mutants lacking N-terminal portions . The wild type (wt) and mt1 mutant lacking three amino acids from N-terminal bound to annexin XI with phosphatidylserine and Ca2+, whereas mt2, mt3 and mt4 with seven, twelve and eighteen amino acids deleted, respectively, did not bind to annexin XI . Moreover, the truncated mutant from residues 4 to 7 (mt5) decreased the binding capacity . These observations suggest that four amino acids (residues 4-7) at the N-terminal portion of calcyclin play an important role in the interaction of calcyclin with annexin XI. Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1343 - 8 Modulation of human endothelial cell tetrahydrobiopterin synthesis by activating and deactivating cytokines: new perspectives on endothelium-derived relaxing factor; Schoedon G et al.; Endothelial cells, through soluble mediators, play an important role in the regulation of the vascular tone . In the present paper we investigated whether endothelial tetrahydrobiopterin (BH4), an obligatory cofactor of nitric oxide (NO) synthase, could serve as such a regulatory mediator . By studying the human vascular endothelial hybrid cells EA hy 926, we found that 1) BH4 biosynthesis is highly regulated (70-fold) by activating and deactivating cytokines; that 2) up to 90% of the induced BH4 is released by activated endothelium, and 3) while intracellular BH4 could be related to cyclic GMP concentrations within the endothelial cells, the bulk of BH4 (up to 90 pmol/10(6) cells) appears not to serve endothelial cell requirements . Activation and deactivation of BH4 synthesis by cytokines was paralleled by other endothelial cell responses reflecting their activity . We propose that BH4 serves as an endothelial mediator augmenting the activity of cytokine-inducible NO synthase in vascular smooth muscle cells . BH4 could thereby account for endothelium-derived relaxing factor activity. Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1255 - 60 Expression of the fungal cytochrome P-450nor cDNA in Escherichia coli; Obika K et al.; We succeeded in expressing the unique cytochrome P-450 (P-450), nitric oxide reductase (P-450nor), in Escherichia coli by utilizing P-450nor cDNA and the expression vector pKK233-2 or pUC19 . The expression was confirmed by Western blot analysis and by detecting the unique nitric oxide reductase activity . The expressed protein was recovered in the soluble fraction of transformed cells, supporting that P-450nor is the first soluble P-450 of eukaryote . The results also provided conclusive evidence that the enzymatic reaction depends solely on P-450nor without support of any other electron transferring components. Biochem J, 1993 Nov 15, 296 ( Pt 1), 235 - 43 Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1-->4)-beta-D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli; Garcia-Campayo V et al.; Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli . The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing . XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa . The catalytic activity shown by both domains was optimal at 50 degrees C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum . XYLA-A1 showed a higher sensitivity to pH than XYLA-C2 . The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions . Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased . Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis . With oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues . Neither domain was able to release arabinose from oat spelt xylan. Biochem J, 1993 Nov 15, 296 ( Pt 1), 209 - 15 Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector; Sivaprasadarao A et al.; In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1 . However, this construct did not generate detectable expression of RBP in E . coli . When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm . Deletion of the 3' untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx . 20-fold) . After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin . The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e . it interacted with retinol, transthyretin and its cell-surface receptor . Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher . This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor. Biochem J, 1993 Nov 15, 296 ( Pt 1), 189 - 97 Reversible modification of rat liver glutathione S-transferase 3-3 with 1-chloro-2,4-dinitrobenzene: specific labelling of Tyr-115; Liu LF et al.; Rat liver glutathione S-transferase 3-3 (GST, EC 2.5.1.18), a triple mutant with all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were overexpressed in Escherichia coli under the control of a phoA promoter . Using this system, we obtained over 35 mg of fully active pure protein/litre of cell medium . GST 3-3 and CallS mutant were modified with 1-chloro-2,4-dinitrobenzene (CDNB), a model substrate for the enzyme, in the absence of GSH . Dinitrophenol, but not S-methylglutathione, inhibits this process . The dinitrophenyl groups are readily removed from the enzyme with GSH, but much more slowly with dithiothreitol . Results from peptide mapping and amino acid sequence analyses indicate that CDNB modifies the cysteine residues and Tyr-115 on wild-type GST 3-3, but only Tyr-115 on CallS . In addition, CDNB cannot modify the CallSY115F mutant . We propose that Tyr-115 is located at or near the H-site of GST 3-3. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10861 - 5 Cell growth and lambda phage development controlled by the same essential Escherichia coli gene, ftsH/hflB; Herman C et al.; The lambda phage choice between lysis and lysogeny is influenced by certain host functions in Escherichia coli . We found that the frequency of lambda lysogenization is markedly increased in the ftsH1 temperature-sensitive mutant . The ftsH gene, previously shown to code for an essential inner membrane protein with putative ATPase activity, is identical to hflB, a gene involved in the stability of the phage cII activator protein . The lysogenic decision controlled by FtsH/HflB is independent of that controlled by the protease HflA . Overproduction of FtsH/HflB suppresses the high frequency of lysogenization in an hflA null mutant . The FtsH/HflB protein, which stimulates cII degradation, may be a component of an HflA-independent proteolytic pathway, or it may act as a chaperone, maintaining cII in a conformation subject to proteolysis via such a pathway . Suppressor mutations of ftsH1 temperature-sensitive lethality, located in the fur gene (coding for the ferric uptake regulator), did not restore FtsH/HflB activity with respect to lambda lysogenization. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10583 - 7 A second hepatitis C virus-encoded proteinase; Grakoui A et al.; Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products . Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified . In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207 . This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3 . Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes . Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function . Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein . Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10558 - 62 Mechanistic aspects of genome-wide demethylation in the preimplantation mouse embryo; Kafri T et al.; Gene-specific methylation patterns in mammals play a role in a variety of biological processes in the embryo and adult tissues . These patterns are established during embryo development by a process that involves genome-wide demethylation in the morula and de novo methylation in the pregastrula . To elucidate the mechanism of demethylation in the early mouse embryo, we have injected mouse zygotes with gene sequences that were methylated in vitro by Hpa II methylase and analyzed the methylation status of specific sites in blastocyst DNA . Because it had been propagated in Escherichia coli, the DNA used for these injections was also methylated at adenine residues in GATC sites . This allowed us to eliminate fully methylated, unintegrated DNA by Dpn I digestion and fully unmethylated, integrated DNA that underwent several rounds of replication by Mbo I digestion . The integrated, originally injected DNA strands were in a hemimethylated state and survived this treatment . The methylation status of Hpa II sites in these molecules was analyzed by Hpa II digestion of the genomic DNA isolated from blastocysts, followed by PCR amplification using appropriate primers . The results demonstrate that demethylation is achieved by an active mechanism and that specific sites in imprinted genes escape demethylation, maintaining a methylated state throughout preimplantation development. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10444 - 8 Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface; Francisco JA et al.; We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface . This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity . Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface . Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps . The expression of antibody fragments on the surface of E . coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments. Eur J Biochem, 1993 Nov 15, 218(1), 205 - 11 Recombinant coho salmon insulin-like growth factor I . Expression in Escherichia coli, purification and characterization; Moriyama S et al.; Recombinant coho salmon insulin-like growth factor I (rsIGF-I) was produced in Escherichia coli, purified and characterized . The rsIGF-I expression vector was constructed by polymerase chain reaction and cloning into a plasmid containing a phage T7 RNA polymerase promoter . The rsIGF-I was recovered from bacterial inclusion bodies, solubilized under reducing conditions, immediately refolded, then fractionated by a two-step ion-exchange chromatography on DEAE-52 and Mono-S columns . It was further purified by HPLC on a reverse-phase Asahi-Pak C4P-50 C4 column . Purification of rsIGF-I was monitored by SDS/PAGE and immunoblot with anti-{human somatomedin C (SM C)/IGF-I} serum . The rsIGF-I appeared as a single band with molecular mass of 7 kDa, the same size as recombinant human IGF-I (rhIGF-I) and cross-reacted with anti-(human SM C/IGF-I) serum . The amino acid sequence of rsIGF-I contained an NH2-terminal methionine residue followed by the sequence predicted for mature sIGF-I . At concentrations in the range 3.9-250 ng/ml, rsIGF-I significantly stimulated sulfate uptake by the cultured branchial cartilage of coho salmon . The stimulatory effect of rsIGF-I was concentration dependent and slightly more potent than that of rhIGF-I at the highest concentration tested. Arch Biochem Biophys, 1993 Nov 15, 307(1), 46 - 51 Amino acid substitutions at position 73 in motif 2 of Escherichia coli alanyl-tRNA synthetase; Filley SJ et al.; Lysine73, located in the adenylate synthesis domain of Escherichia coli alanyl-tRNA synthetase (AlaRS), was previously indicated to be an important residue for the interaction of this enzyme with the acceptor stem of its cognate tRNA (tRNA(Ala)) . Replacement of this residue with glutamine produced a reduction in the catalytic efficiency of AlaRS in the aminoacylation assay, primarily through an increase in the apparent KM for tRNA(Ala) {Hill, K., and Schimmel, P . (1989) Biochemistry 28, 2577-2586} . Studies on the role of residue 73 in the interaction of AlaRS with its substrates have now been extended using the additional substitutions of asparagine, alanine, and glutamate . Analysis of each substituted enzyme in the ATP-PPi exchange and aminoacylation reactions reveals kinetic characteristics similar to those obtained with the glutamine substitution, except that the glutamate substitution causes a fivefold decrease in the affinity for alanine . These data verify that the positive charge on lysine 73, rather than its hydrophilic side chain, is of importance in the binding of the cognate tRNA, but do not support an ionic interaction of this residue with the RNA phosphate backbone . The collective data support the prediction that lysine73 is in motif 2 of AlaRS {Cusack, S., Hartlein, M., and Leberman, R . (1991) Nucleic Acids Res . 19, 3489-3498}, but question the predicted alignment of this motif with other enzymes in its class. Arch Biochem Biophys, 1993 Nov 15, 307(1), 193 - 9 Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase; LoGrasso PV et al.; Yeast squalene synthetase which has been truncated by 24 amino acids at the C-terminus has been overexpressed in Escherichia coli and constitutes approximately 20% of the total soluble cell protein . For the first time, milligram quantities of this essential enzyme in the cholesterol biosynthetic pathway have been purified to near homogeneity by ammonium sulfate precipitation and Mono Q anion-exchange chromatography so that the steady-state rate constants could be measured . A combination of 10% methanol, 10% glycerol, 30 mM octyl-beta-D-glucopyranoside, 0.4% Brij-58, and 1 mM dithiothreitol in 25 mM sodium phosphate, pH 7.4, was essential for the stability and maximal enzyme activity of the near homogeneous enzyme . Kinetic analysis indicated a Km for farnesyl pyrophosphate of 2.5 microM, suggesting fairly tight binding of farnesyl pyrophosphate to truncated yeast squalene synthetase . The turnover number, kcat, for the conversion of farnesyl pyrophosphate to squalene was 0.53 s-1, and the apparent second order rate constant, kcat/Km, was 2.1 x 10(5) M-1 s-1, indicating a relatively slow conversion of farnesyl pyrophosphate to squalene and a low specificity constant for this enzyme . In addition, Km for NADPH and NADH was 0.5 and 3.6 mM, respectively . Moreover, truncated yeast squalene synthetase shows a preference for NADPH over NADH as reflected in the sevenfold higher kcat/Km value for NADPH similar to that for the native enzyme. Am J Epidemiol, 1993 Nov 15, 138(10), 849 - 69 Epidemiologic studies of Escherichia coli diarrheal infections in a low socioeconomic level peri-urban community in Santiago, Chile; Levine MM et al.; The incidence of diarrhea due to six categories of diarrheogenic Escherichia coli was determined in two pediatric cohorts in a low socioeconomic level community in Santiago, Chile, with access to chlorinated water . An age cross-sectional cohort of 340 children aged birth to 47 months was assembled . A newborn cohort was assembled by enrolling 10-12 newborns monthly for 12 months . Episodes of diarrhea were detected by twice weekly household visits . E . coli from stool cultures of cases and matched controls were hybridized with DNA probes specific for enterotoxigenic, enteroinvasive, enteropathogenic, enterohemorrhagic, enteroaggregative, and diffuse adherence E . coli . Overall, the incidence of diarrhea was low (2.1 episodes/infant/year) . Nevertheless, a putative E . coli enteropathogen was found in a large proportion of diarrheal episodes, particularly during the summer . In both cohorts, enterotoxigenic E . coli were important pathogens . Enteropathogenic E . coli were incriminated during the first year of life in the newborn cohort, where they were found significantly more often in cases (p = 0.021) than in controls; beyond this age, isolation rates were similar . In contrast, the relative risk of isolation of diffuse adherence E . coli increased with age in the age cross-sectional cohort, where, overall, the difference in rate of isolation between cases and controls was significant (p = 0.0024) . Enteroinvasive and enterohemorrhagic E . coli were isolated infrequently . Enteroaggregative E . coli were encountered equally in cases and controls . Facile transmission of E . coli enteropathogens is occurring in this community despite the availability of potable waterPIP: Researchers conducted an age cross sectional cohort analysis of 340 0-47 month old children and newborn cohort analysis of 144 newborns to determine the diarrheogenic Escherichia coli incidence in Santa Julia, a low socioeconomic community in Santiago, Chile . Children in the age cross sectional cohort had age, sex, and sector matched controls . The newborns had sex matched controls . A public health nurse or nurse auxiliary visited the household of each subject 2 times a week to detect diarrhea episodes . Between December 1986 and February 1990, the age cross sectional cohort had 1178 episodes of diarrhea and the newborn cohort had 674 episodes . The overall diarrhea incidence was only 2.1 episodes/child/year . An E . coli enteropathogen was isolated in many of these episodes, especially during the summer (e.g . enterotoxigenic E . coli {ETEC}, 2.2 cases/month in summer vs . 0.4 cases/month in winter; p = .00001) . Diffuse adherence E . coli (DAEC) and enteropathogenic E . coli (EPEC) infections also peaked in the summer . ETEC contributed greatly to diarrheal episodes in both cohorts . Among newborns, EPEC was isolated significantly more often in cases than controls during the 1st 12 months of life (6.7% vs . 2.5%; p = .021) . After 1 year, however, E . coli isolation rates were essentially the same . On the other hand, in the age cross sectional cohort, the relative risk of isolation of DAEC rose with age (e.g., 1.1 for 0.11 months, 1.4 for 36-47 months, and 2.1 for = or 48 months) . In the same cohort, DAEC infections were much more common in cases than controls (16.6% vs . 11.9%; p = .0024) . Enteroinvasive and enterohemorrhagic E . coli were the most rarely isolated E . coli types . No difference in the isolation rate of enteroaggregative E . coli existed between cases and controls . Since most households in Santa Julia have access to potable water (68%) and an indoor toilet (64%), food contamination were likely the vehicles of E . coli transmission because more than 50% of households do not have a refrigerator . J Biol Chem, 1993 Nov 15, 268(32), 24498 - 505 Kallistatin: a novel human serine proteinase inhibitor . Molecular cloning, tissue distribution, and expression in Escherichia coli; Chai KX et al.; We have recently purified a novel human serine proteinase inhibitor (serpin), designated as kallistatin, which binds to tissue kallikrein and inhibits kallikrein's kininogenase and amidolytic activities . In the present studies, we have cloned a full-length cDNA encoding kallistatin from human liver RNA by the polymerase chain reaction . The cDNA is 1284 base pairs in length and encodes 427 amino acid residues, including a 26-residue signal peptide and a 401-residue mature peptide . The translated amino acid sequence of kallistatin matches with the protein sequence and shares 44-46% sequence identity with human alpha 1-antichymotrypsin, protein C inhibitor, corticosteroid-binding globulin, alpha 1-antitrypsin, thyroxin-binding globulin, and rat kallikrein-binding protein . Kallistatin is a new member of the serpin superfamily with a unique reactive site P1-P1' of Phe-Ser . Four potential glycosylation sites are found in the translated amino acid sequence of kallistatin . In a Southern blot analysis following reverse transcription and polymerase chain reaction, kallistatin was found to be expressed in human liver, stomach, pancreas, kidney, aorta, testes, prostate, artery, atrium, ventricle, lung, renal proximal tubular cell, and a colonic carcinoma cell line T84 . A genomic Southern blot using the full-length kallistatin cDNA probe revealed simple banding patterns suggesting the gene encoding kallistatin is single-copied . The kallistatin cDNA encoding the mature peptide was expressed in Escherichia coli . The recombinant kallistatin forms an SDS-stable complex with 125I-human tissue kallikrein and has a molecular mass of 40 kDa . The cloning of human kallistatin cDNA established the identity of the novel kallikrein inhibitor and its expression in a functional form in E . coli provides means for studying its structure-function relationship through protein engineering. J Biol Chem, 1993 Nov 15, 268(32), 24491 - 7 Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides . Involvement of an integration host factor-binding sequence; Lee JK et al.; The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF . The binding of E . coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific . The recorded binding affinity was significantly lower than that of E . coli IHF to the lambda attP site . Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions . These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity . Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed . Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cis-acting regulatory sequences involved in puc operon expression. J Biol Chem, 1993 Nov 15, 268(32), 24361 - 6 Molecular cloning of fish Pit-1 cDNA and its functional binding to promoter of gene expressed in the pituitary; Yamada S et al.; Pit-1 is a pituitary-specific transcription factor responsible for activating growth hormone (GH) and prolactin genes . Here, we describe the isolation of a rainbow trout cDNA clone that contains the entire Pit-1 coding region . The deduced amino acid sequence contains 358 residues, encoding a 39-kDa protein . Comparison of the protein sequences of the rainbow trout and rat Pit-1 shows that the 160 residue POU domain at the C terminus is highly conserved (86% identical) . However, homology is much weaker in the N-terminal region (56% identical), and the rainbow trout Pit-1 contains segments of 29 and 33 amino acids that are not present in rat Pit-1 . The protein produced by expression of rainbow trout Pit-1 cDNA in Escherichia coli binds specifically to at least four sites in the rainbow trout GH gene promoter . Moreover, we demonstrate that the promoter region of salmon somatolactin gene, which belongs to the GH prolactin gene family and is also expressed specifically in the pituitary, has at least five rainbow trout Pit-1 binding sites . The consensus sequence of these binding sites closely matches the 9-base pair motif, (T/A)(T/A)TATNCAT, recognized by rat Pit-1 . Rainbow trout Pit-1 specifically activates rainbow trout GH promoter fusion gene expression, confirming the ability of Pit-1 to bind in a transcriptionally active conformation in GH gene promoter. J Biol Chem, 1993 Nov 15, 268(32), 24190 - 6 The purification and characterization of recombinant yeast dolichyl-phosphate-mannose synthase . Site-directed mutagenesis of the putative dolichol recognition sequence; Schutzbach JS et al.; Yeast dolichyl-phosphate-mannose synthase was purified from cultures of Escherichia coli carrying the gene for this enzyme in a high expression vector . The synthase contains a highly conserved hydrophobic amino acid sequence proposed to be involved in the recognition of dolichols (Albright, C . F., Orlean, P., and Robbins, P . W . (1989) Proc . Natl . Acad . Sci . U . S . A . 86, 7366-7369) and amino acid residues in this sequence were altered by site-directed mutagenesis . Conservative substitutions had no effect on the affinity of the enzyme for dolichyl-P . The substitution of asparagine for isoleucine at position 253 resulted in higher values for the apparent Km for Dol-P when assayed in detergent solutions, but this substitution had no effect on Km when the enzyme was reconstituted with phosphatidylethanolamine . Enzyme containing a deletion of the entire putative dolichol recognition sequence retained catalytic activity . The apparent Km for Dol-P was increased when this enzyme was assayed in detergent solution but was the same as wild type enzyme when reconstituted in phosphatidylethanolamine . These results suggest that the amino acid composition and sequence of the conserved domain are not critically important for the recognition and binding of Dol-P when the synthase is present in a lipid matrix. J Biol Chem, 1993 Nov 15, 268(32), 24156 - 62 Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct; Teraoka H et al.; A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N, which was derived from pUC118 by oligonucleotide-directed mutagenesis . The insert contained a 2757-base pair coding sequence for a whole human DNA ligase I and an extra ACC codon adjacent to the ATG initiation codon . This ACC codon was required for achieving high levels of expression of full-length DNA ligase I in Escherichia coli strain BL21 . The recombinant plasmid, which was designed to exploit the T7 late promoter and the ATG initiation codon for beta-galactosidase was transfected into E . coli BL21 cells that express T7 RNA polymerase . The recombinant clone produced relatively high levels of DNA ligase I with a molecular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electrophoresis . The DNA ligase was purified to near-homogeneity by the two-step column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside . The specific activity, chromatographic behavior, kinetic properties, molecular mass, and antigenicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I . Metabolically labeling experiments with 32P(i) indicate that the recombinant DNA ligase I was present as an enzyme-AMP reaction intermediate, but not as a phosphoprotein, in the E . coli cells. J Biol Chem, 1993 Nov 15, 268(32), 24005 - 11 Cytosine deaminase . The roles of divalent metal ions in catalysis; Porter DJ et al.; Cytosine deaminase (CDase, EC 3.5.4.1) isolated from Escherichia coli contains a catalytically essential divalent metal ion . Fe2+ was efficiently removed from the enzyme with o-phenanthroline to yield an apoenzyme with less than 5% of the catalytic activity of native enzyme . The time courses for inactivation and for removal of Fe2+ from the enzyme by o-phenanthroline were similar . Apoenzyme reconstituted with Fe2+, Mn2+, Co2+, or Zn2+ (M2+CDase) had kcat values of 185, 88, 50, and 32 s-1, respectively . The Km values of these M2+CDases for cytosine were similar (0.22-0.39 mM) . Cytosine potently inhibited reconstitution of the apoenzyme with Fe2+ . Fe2+CDase was rapidly inactivated by 1 mM H2O2 (t1/2 < 1 s), whereas Mn2+CDase, Co2+CDase, and Zn2+CDase were not inactivated by H2O2 . CDase was also inhibited by excess divalent cations . Cu2+ and Zn2+ reversibly inhibited Fe2+CDase activity with inhibition constants of 1.8 and 5.8 microM, respectively . Cu2+ dissociated slowly from the secondary binding on CDase with a rate constant of 2 x 10(-3) s-1. J Biol Chem, 1993 Nov 15, 268(32), 23986 - 90 Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells; Wales R et al.; An Escherichia coli expression system was used to produce recombinant ricin A chain (RTA) and RTA modified either by the addition of a carboxyl-terminal endoplasmic reticulum retrieval sequence Lys-Asp-Glu-Leu (RTAKDEL) or a nonfunctional analogue Lys-Asp-Glu-Ala (RTAKDEA) . These RTA molecules can enter mammalian cells by fluid phase endocytosis . RTAKDEL was significantly more cytotoxic than either RTA or RTAKDEA to both Vero cells and HeLa cells (250- and 10-fold, respectively), despite the fact that all these RTA molecules had comparable enzymatic activities . This difference did not result from KDEL-mediated binding of RTAKDEL to the cell surface . Enhanced cytotoxicity could be correlated with an increased level of ribosome inactivation, measured as the RTA-catalyzed depurination of 28 S ribosomal RNA . These results indicate that the added KDEL sequence facilitated RTA entry into the cytosol . We propose that interaction with the intracellular KDEL receptor promotes retrograde transport of the toxin to the endoplasmic reticulum, where translocation of RTA into the cytosol occurs. J Biol Chem, 1993 Nov 15, 268(32), 23972 - 80 Phosphorylation/dephosphorylation of the receiver module at the conserved aspartate residue controls transphosphorylation activity of histidine kinase in sensor protein ArcB of Escherichia coli; Iuchi S; A membrane sensor protein, ArcB, recognizes anaerobic environments and signals the information to the cognate regulator, ArcA . This in vitro study presents the process and control of the signal-transduction phosphorylation . In the presence of ATP, the ArcB transmitter module undergoes autophosphorylation and then transfers the phosphoryl group to its own receiver module as well as to the ArcA receiver module . Results suggest that the phosphoryl group of the ArcB receiver module is released by an intrinsic phosphatase activity . D-Lactate inhibits the phosphatase activity that removes phosphoaspartate groups from the receiver module in ArcB, and the associated increase in phosphorylation of this module leads to an activation of transphosphorylation of subsequently added phosphohistidine groups on ArcB to the receiver module of ArcA . A similar effect was also observed in the presence of pyruvate, acetate, or NADH . Conversely, the non-phosphorylated ArcB receiver module completely inhibits the intermolecular transphosphorylation . Thus, the phosphorylation state of the ArcB receiver module controls signal transduction from ArcB to ArcA . Since the intrinsic phosphatase activity is inhibited by cellular metabolites that increasingly accumulate by anaerobiosis, the enzyme portion of ArcB may be involved in sensing anaerobic environments through cellular metabolites in vivo. J Biol Chem, 1993 Nov 15, 268(32), 23964 - 71 Alternative splicing in a novel tyrosine phosphatase gene (DPTP4E) of Drosophila melanogaster generates two large receptor-like proteins which differ in their carboxyl termini; Oon SH et al.; A novel Drosophila receptor-like protein tyrosine phosphatase gene, DPTP4E, was isolated and characterized . DPTP4E, located at cytological position 4E1-2, is comprised of 10 exons; its RNA products are widely expressed during embryonic development, including the developing central nervous system . DPTP4E produces three major developmentally regulated transcripts of 6.5, 7.0, and 7.5 kilobases . The two major embryonic transcripts arise as the result of the alternative splicing of exon IX; as a consequence, two proteins (200 and 183 kDa) are produced which differ in their carboxyl-terminal sequences . The deduced extracellular domain, which lies between two putative hydrophobic transmembrane segments, contains 11 fibronectin type III-like repeats and 25 putative N-glycosylation sites . A single conserved protein tyrosine phosphatase (PTPase) catalytic domain, which shows a high level of amino acid identity to the Drosophila PTPase DPTP10D and human HPTP beta, is found in the predicted intracellular domain; this PTPase domain, when expressed as a fusion protein in Escherichia coli, exhibits PTPase activity . The possible implications of these findings are discussed. J Biol Chem, 1993 Nov 15, 268(32), 23876 - 80 Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3 . Dominant role of the phosphorylation of Ser-640 (site-3a); Wang Y et al.; Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation . The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit . Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5) . Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively . The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9 . Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3 . Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity . When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted . Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude . Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3 . Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits. J Biol Chem, 1993 Nov 15, 268(32), 23837 - 42 Role of the conserved Lys-X-Gly-Gly sequence at the ADP-glucose-binding site in Escherichia coli glycogen synthase; Furukawa K et al.; Although bacterial and mammalian glycogen synthases differ in the primary structure and specificity for glucosyl donor, lysyl residues identified at their substrate-binding sites by affinity labeling are present in a conserved tetrapeptide sequence, Lys-X-Gly-Gly, where X is a residue not conserved (Tagaya, M., Nakano, K., and Fukui, T . (1985) J . Biol . Chem . 260, 6670-6676; Furukawa, K., Tagaya, M., Inouye, M., Preiss, J., and Fukui, T . (1990) J . Biol . Chem . 265, 2086-2090) . To elucidate the functional role of this conserved sequence, Lys-15, Gly-17, and Gly-18 in Escherichia coli glycogen synthase have been replaced by other amino acid residues via site-directed mutagenesis . Kinetic analyses of the Lys-15 mutant enzymes showed that the epsilon-amino group of Lys-15 is mainly involved in binding of the phosphate moiety adjacent to the glycosidic linkage in the substrate ADP-glucose, presumably through an ionic interaction . The mutant enzyme in which Ala was substituted for Gly-17 had a catalytic rate constant 3 orders of magnitude smaller than that of the wild-type enzyme with a slightly increased Michaelis constant for ADP-glucose, whereas the Gly-18-->Ala mutant showed a rate constant only 3.2-fold smaller . In addition, mutations of Gly-17 and Gly-18 resulted in marked changes in the reactivity of Lys-15 with affinity labeling reagents . These results suggest that the 2 glycyl residues in the conserved Lys-X-Gly-Gly sequence, in particular the one closer to the ADP-glucose-binding lysyl residue, participate in catalysis by assisting conformational change(s) of the active site or stabilizing the transition state. J Biol Chem, 1993 Nov 15, 268(32), 23824 - 9 Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages; Shapiro SD et al.; Human alveolar macrophages have the capacity to degrade elastin . As an approach to define proteinases responsible for this activity, we recently cloned a murine macrophage elastase cDNA and demonstrated that it is a member of the matrix metalloproteinase gene family (Shapiro, S . D., Griffin, G . L., Gilbert, D . J., Jenkins, N . A., Copeland, N . G., Welgus, H . G., Senior, R . M., and Ley, T . J . (1992) J . Biol . Chem . 267, 4664-4671) . We now report that there is a human orthologue of murine macrophage metalloelastase that we call human macrophage metalloelastase (HME) . The full-length HME cDNA spans 1.8 kilobases and contains an open reading frame of 1410 base pairs; the predicted molecular mass of the HME proenzyme is 54 kDa . HME mRNA and protein were detected in human alveolar macrophages . Similar to murine macrophage metalloelastase, HME readily undergoes NH2- and COOH-terminal processing to a mature 22-kDa form . Both recombinant HME expressed in Escherichia coli and native HME derived from human alveolar macrophage-conditioned media degraded insoluble elastin . HME is a unique human metalloproteinase that possesses elastolytic activity and is expressed in alveolar macrophages; it is therefore a candidate molecule for the causation of diseases characterized by damage to the extracellular matrix. Gene, 1993 Nov 15, 133(2), 291 - 4 Production of human plasma retinol-binding protein in Escherichia coli; Wang TT et al.; We designed a polymerase chain reaction (PCR) primer pair which allowed us to clone the cDNA coding for the human plasma retinol-binding protein (hRBP) into an Escherichia coli expression vector . Production of hRBP was confirmed by probing Western blots with antisera against plasma hRBP . Purification and characterization of the E . coli-produced plasma hRBP are also described . The availability of this expression system makes it possible to obtain large quantities of hRBP to facilitate our continuing studies of retinol and RBP metabolism. Gene, 1993 Nov 15, 133(2), 223 - 5 Translational properties of the human papillomavirus type-6 L1-coding mRNA; Tomita Y et al.; A cDNA encoding a bicistronic mRNA, E1E4L1, which was generated by double-splicing of the E1, E4 and L1 genes, of the type-6 human papillomavirus (HPV-6), was cloned . The E1E4 and L1 open reading frames (ORFs) in this cDNA were expressed in COS-1 or CV-1 cells as fusion proteins with Escherichia coli beta-galactosidase (beta Gal), and the products were analyzed by immunoprecipitation and enzyme assay . The results showed that the translational efficiency of the L1 ORF was about 9-15-fold less efficient than that of the E1E4 ORF . Substitution of the ATG of the E1E4 ORF with AAG increased translation of the L1 ORF about 30-fold . Lengthening of the intercistronic sequence to 31 bp, equivalent in length to the bicistronic HPV-1 mRNA, showed little translational effect relative to the wild-type 12-bp intercistronic sequence . (Carets {} represent splicing of RNA.) Gene, 1993 Nov 15, 133(2), 213 - 7 One-step purification and characterization of a lignin-specific O-methyltransferase from poplar; Van Doorsselaere J et al.; O-Methyltransferases (OMT; EC 2.1.1.6) play an important role in the synthesis of lignin precursors by catalyzing the O-methylation of o-diphenolic substrates such as caffeic acid (CA) and 5-hydroxyferulic acid (5OH) . Here, we report on the purification of a lignin-specific OMT (38 kDa) from poplar (Populus trichocarpa x P . deltoides) . The OMT was purified from xylem by a single affinity chromatography step on adenosine agarose . The enzyme uses both CA and 5OH as substrates . We previously have reported the cloning of a corresponding OMT cDNA {Dumas et al., Plant Physiol . 98 (1992) 796-797} . Expression of this OMT cDNA in Escherichia coli further confirmed the identity of the clone . Genomic hybridization demonstrates the presence of one or two OMT genes per haploid poplar genome . RNA gel blot hybridization shows high levels of steady-state OMT mRNA in the xylem of young poplar trees, as compared to the levels in leaves. FEBS Lett, 1993 Nov 15, 334(2), 165 - 9 The antirepressor of phage P1 . Isolation and interaction with the C1 repressor of P1 and P7; Riedel HD et al.; Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in-frame start codon . Another open reading frame, icd, 5' upstream of and overlapping ant1 is required for ant1 expression . Using appropriate ant gene-carrying plasmids we have overproduced and purified Ant1/2 in the form of a protein complex and Ant2 as a single protein . Sequence analysis confirmed the N-terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N-terminal methionine is missing in the Ant2 protein . Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co-precipitate with the Ant1/2 complex but not with Ant2 protein alone . The results suggest that the antirepressor may exert its C1-inactivating function by a direct protein-protein interaction. Gene, 1993 Nov 15, 133(2), 237 - 42 Isolation of the chicken NF-kappa B p65 subunit-encoding cDNA and characterization of its products; Ikeda T et al.; NF-kappa B is a heterodimeric transcription factor consisting of subunits of 50 kDa (p50) and 65 kDa (p65) . cDNA clones encoding the chicken NF-kappa B p65 subunit were isolated . Sequence analysis showed that chicken p65 is approximately 55% identical to the mouse and human p65 proteins, and contains the Rel homology domain (RHD) in its N-terminal 286 amino acids (aa) and the putative transactivation domain in its C-terminal region . The RHD is particularly highly conserved between the chicken and mammalian p65 proteins . Northern blot hybridization analysis detected the expression of a 2.6-kb transcript of p65 in various organs, with the highest level in spleen . A fusion protein containing the RHD of chicken p65 was found to bind to a consensus kappa B-site in an electrophoretic mobility shift assay (EMSA) . This binding was specifically inhibited by the presence of fusion proteins containing the C-terminal ankyrin repeats domain (ARD) of chicken p105, the precursor protein for the p50 subunit . Immunoprecipitation analysis showed that p65 formed a complex(es) with multiple cellular proteins, including p50, p105 and c-Rel in chicken spleen cells. Arch Biochem Biophys, 1993 Nov 15, 307(1), 165 - 74 Threonine synthase of Escherichia coli: inhibition by classical and slow-binding analogues of homoserine phosphate; Farrington GK et al.; L-threo-3-Hydroxyhomoserine phosphate, derived from the antimetabolites L-threo-3-hydroxyaspartate and L-threo-3-hydroxyhomoserine {Shames, S . L., Ash, D . E., Wedler, F . C., and Villafranca, J . J . (1984) J . Biol . Chem . 258, 15331-15339}, is a classical competitive inhibitor of threonine synthase (Ki = 6 microM) with structural elements of both substrate and product . L-2-Amino-5-phosphonovaleric acid also inhibits the enzyme competitively with a Ki (31 microM), comparable to Km for L-homoserine phosphate . In contrast, a structural analogue of Hse-P, L-2-amino-3-{(phosphonomethyl)thio}propionic acid exhibits a Ki = 0.11 microM (ca . 100-fold less than Km for L-Hse-P), along with "slow, tight" inhibition kinetics . Nuclear magnetic resonance was used with these inhibitors to probe for pyridoxal phosphate-catalyzed hydrogen-deuterium exchange reactions characteristic of substrates . With L-threo-3-hydroxy-homoserine phosphate, H-D exchange occurs only at the C-alpha position, but for homoserine in the presence of phosphate and for L-2-amino-5-phosphonovaleric acid and L-amino-3{(phosphonomethyl)thio}propionic acid (APMTP), H-D exchange occurs at C-alpha and stereospecifically at C-beta . For L-homoserine plus phosphate and L-2-amino-5-phosphonovaleric acid, the rate of H-D exchange at C-alpha is 8-45 times faster than at C-beta . For L-2-amino-3-{(phosphonomethyl)thio}propionic acid, the C-alpha to C-beta exchange rate ratio is near unity, due to a 700-fold decrease in the C-alpha rate for the analogue . Taken with information from molecular modeling, these data can be interpreted in terms of the current working hypothesis for the catalytic mechanism . Specifically, the slow, tight inhibition by APMTP results from its being carried further into the catalytic cycle than other analogues prior to forming an intermediate that is blocked from further catalysis. J Biol Chem, 1993 Nov 15, 268(32), 24149 - 55 Evidence for a catalytic role of glutamic acid 129 in the NAD-glycohydrolase activity of the pertussis toxin S1 subunit; Antoine R et al.; The S1 subunit of pertussis toxin is an ADP-ribosyl-transferase capable of transferring the ADP-ribose moiety of NAD+ to nucleotide-binding signal-transducing proteins of the Gi/G(o) family . In the absence of G proteins, the enzyme also catalyzes the hydrolysis of NAD+ . Glu-129 was previously shown to be critical for both enzymatic activities . In this study, site-directed mutagenesis was used to make the conservative substitution of aspartate for Glu-129 . The recombinant wild type and mutant proteins were purified to near homogeneity and used for enzymatic analyses . Kinetic experiments showed that the kcat of the mutant protein was about 200 times less than that of the wild type enzyme, whereas the Km for NAD+ of the two proteins were very similar, suggesting that Glu-129 is a catalytic residue for the NAD-glycohydrolase reaction of S1 . This hypothesis was confirmed by a less than 2-fold change in Kd as measured by fluorescence quenching studies, indicating that the binding of NAD+ is not affected in the mutant protein in any important way . In another experiment, the replacement of Glu-129 by cysteine resulted in a disulfide bridge between Cys-129 and Cys-41 in rS1d-E129C, suggesting that the folding of the polypeptide chain is such that the catalytic Glu-129 residue is close to the amino-terminal NAD-binding site of S1 . These findings imply that Glu-129 plays a key role in catalysis of the NAD-glycohydrolase reaction, possibly by electrostatically stabilizing a cationic transition state intermediate, or by serving as a general base to deprotonate the ADP-ribosyl acceptor substrates. J Biol Chem, 1993 Nov 15, 268(32), 24074 - 7 Specificity of the Escherichia coli chaperone DnaK (70-kDa heat shock protein) for hydrophobic amino acids; Richarme G et al.; Molecular chaperones form a class of proteins that bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides . This property is the basis of their implication in many cellular processes such as protein folding, protein targeting to membranes, or protein renaturation after stress . It has been suggested that the recognition of non-native proteins by chaperones is mediated by their binding to exposed hydrophobic areas, to the polypeptide backbone, or to specific secondary structures . We show in the present study that DnaK, the 70-kDa chaperone of Escherichia coli specifically recognizes hydrophobic amino acids . The peptide-dependent ATPase activity of DnaK is specifically stimulated by Ile, Phe, Leu, and Val in a manner which is consistent with an interaction of these amino acids with the polypeptide binding site of DnaK . Two classes of amino acid binding site can be distinguished, one being specific for the aliphatic amino acids and the other for the aromatic amino acids . Since the hydrophobic amino acids are buried inside the hydrophobic core of native proteins and are exposed in non-native forms, their interaction with DnaK could be the basis of the specific interaction of the chaperone with non-native proteins in protein folding, protein targeting to membranes, or protein renaturation. J Biol Chem, 1993 Nov 15, 268(32), 24330 - 8 The cystic fibrosis transmembrane conductance regulator . Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein; Ko YH et al.; The first nucleotide binding fold (NBF1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and its disease-causing mutant form (delta F508,NBF1) were overexpressed in high yield in Escherichia coli in fusion with the maltose-binding protein (MBP) . The rationale for producing the chimerae was to aid in domain purification, solubilization, and crystallization and to examine the effect of protein-protein interactions on the properties of the mutant NBF1 . Both the purified wild type and delta F508 mutant fusion proteins fold into functional nucleotide binding domains as determined by using the fluorescent nucleotide analog TNP-ATP (2'-(3')-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate) . Moreover, the prominent secondary structural features of the two proteins as assessed by ultraviolet circular dichroism spectropolarimetry are very similar, as is the higher order structure evident in three separate protease digestion patterns . Finally, the stability of the nucleotide binding function of the two proteins is similar as assessed by sensitivity to urea . Gel filtration chromatography and electron and confocal microscopy reveal that both fusion proteins, but not MBP alone, form organized fibers, suggesting that NBF1 self-associates, thus raising the possibility that CFTR may be oligomeric in the plasma membrane . Significantly, in the presence of high salt, these fusion proteins also have a propensity to form microcrystals . Finally, the two separate domains (NBF1 and MBP) constituting the fusion proteins appear to interact quite strongly as both proteins remain associated even after cleavage of their fusion junction . The possible relevance of these novel findings to those approaches that might be taken to elucidate the three-dimensional structural differences between the wild type and delta F508 mutant forms of CFTR, as well as to ameliorate the severity of cystic fibrosis, is discussed. Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1214 - 20 Characterization of cDNA for a dehydration-inducible gene that encodes a CLP A, B-like protein in Arabidopsis thaliana L; Kiyosue T et al.; Sequence was obtained from a cDNA clone, designated ERD1, isolated from a cDNA library of 1-hour-dehydrated plants of Arabidopsis thaliana L . The clone (3150 bp) contains an open reading frame of 946 amino acid residues with greater than 34% sequence identity to the regulatory subunit of the Clp ATP-dependent protease in Escherichia coli and contains a putative chloroplast-targeting signal at the N-terminus . Southern blot analysis suggested the presence of additional ERD1-related genes in A . thaliana . The expression of ERD1 gene was strongly induced by dehydration-stress but not by heat-, cold-, or heavy-metal-stress . In addition ERD1 gene-expression was not strongly affected by treatment with plant growth regulators, such as auxin, cytokinin, abscisic acid, and gibberellic acid, or by starvation-stress for 10 hours. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10653 - 7 Permeability properties of a large gated channel within the ferric enterobactin receptor, FepA; Liu J et al.; FepA is an Escherichia coli outer membrane receptor protein for the siderophore ferric enterobactin . Prior studies conducted in vivo suggested that FepA and other TonB-dependent outer membrane proteins transport ligands by a gated-channel mechanism . To corroborate and extend these findings we have determined the permeability properties of the FepA channel in vitro, by measuring the diffusion rates of hydrophilic nonelectrolytes through the FepA channel in liposome swelling experiments . Like porins, the FepA deletion mutant delta RV showed a size-dependent permeability to oligosaccharides, indicating that it forms a nonspecific, hydrophilic pore . Unlike OmpF and other E . coli porins, however, delta RV proteoliposomes transported stachyose (666 Da) and ferrichrome (740 Da) . These data, and other uptake results with a series of maltodextrins of increasing size, confirm the existence of a channel domain within FepA that is considerably larger than OmpF-type pores . These results represent a reconstitution of the channel function of a TonB-dependent receptor protein and establish that FepA contains the largest channel that has been characterized in the E . coli outer membrane. J Med Chem, 1993 Nov 12, 36(23), 3542 - 5 Aminoacyl analogs of chloramphenicol: examination of the kinetics of inhibition of peptide bond formation; Drainas D et al.; Two aminoacyl analogs and one peptidyl analog of chloramphenicol (1, Cl2CHCO-CA) were prepared . These are 2 (L-Phe-CA), 3 (Gly-CA), and 4 (L-Phe-Gly-CA) . The kinetics of inhibition of peptide bond formation by these analogs were examined in a cell-free system which was derived from E . coli and used previously for the study of 1 (Drainas; et al . Eur . J . Biochem . 1987, 164, 53-58) . In the absence of inhibitor, the reaction follows first-order kinetics for the entire course of the reaction . In the presence of the analog the reaction gives biphasic log-time plots . The kinetic information pertaining to the initial slope of the plot is analyzed (initial-slope analysis) . This information differentiates the analogs from the parent compound 1 . The parent compound 1 gives complex inhibition kinetics; increasing the concentration of 1 changes the inhibition from competitive to mixed noncompetitive (Drainas; et al . Eur . J . Biochem . 1987, 164, 53-58) . In contrast, the analogs give competitive kinetics even at high concentrations of the inhibitor . The following Ki values have been determined: 18.0 microM for 2, 5.5 microM for 3, 1.5 microM for 4 . If we were to assume that compounds 2, 3 and 4 behave as classical competitive inhibitors, we could say that 4 is 12 times more potent than 3 and 4 times more potent than 2 . On this assumption we could also compare 1 with 4 and see that 4 is 2 times weaker than 1 . It is suggested that as compared with 1, the two aminoacyl analogs and the dipeptidyl analog have increased structural similarity to the 3'-terminus of aminoacyl-tRNA or of peptidyl-tRNA and that this similarity results in a more pronounced competitive inhibition. Nucleic Acids Res, 1993 Nov 11, 21(22), 5110 - 6 Reconstitution of mammalian excision repair activity with mutant cell-free extracts and XPAC and ERCC1 proteins expressed in Escherichia coli; Park CH et al.; Nucleotide excision repair in humans involves the coordinated actions of 8-10 proteins . To understand the roles of each of these proteins in excision it is necessary to develop an in vitro excision repair system reconstituted entirely from purified proteins . Towards this goal we have expressed in E . coli two of the 8 genes known to be essential for the excision reaction . XPAC and ERCC1 were expressed as fusion proteins with the Escherichia coli maltose binding protein (MBP) and purified to > 80% homogeneity by affinity chromatography . The purified proteins either as fusions or after cleavage from the MBP were able to complement the CFE of cells with mutations in the corresponding genes in an excision assay with thymine dimer containing substrate. Nucleic Acids Res, 1993 Nov 11, 21(22), 5025 - 33 The structure of the initiation complex at the replication origin, oriC, of Escherichia coli; Woelker B et al.; Two distinct regions in the replication origin, oriC, of Escherichia coli are separately distorted upon initiation complex formation by the initiator protein DnaA . The AT-rich region in the left part of oriC and the start site region in the right part of oriC . Chemical modification of single-stranded DNA was observed at both regions whereas endonuclease recognition of DNA mini-bulges specifically occurred in the start site region . We show that the helical phasing of binding sites for DnaA protein in oriC is important for origin function . An insertion or deletion of one helical turn between the two rightmost binding sites does not alter the efficiency of replication initiation, whereas all modifications of distance by less or more than one helical turn result in inactivation of oriC . DnaA binding and helical distortions in the AT-rich region as well as in the start site region are not affected in the distance mutants irrespective of their functionality in vivo . We propose a specific compact nucleoprotein structure for the initiation complex. Nature, 1993 Nov 11, 366(6451), 178 - 82 Tandem binding in crystals of a trp repressor/operator half-site complex; Lawson CL et al.; The crystal structure of trp repressor tandemly bound in a 2:1 complex to a 16-base-pair palindromic DNA containing a central trp operator half-site has been determined and refined to 2.4 A resolution . Despite dramatically different DNA sequence contexts and crystallization conditions, the protein/DNA interface is essentially identical to that seen in the original trp repressor/operator complex structure . Water-mediated sequence recognition by trp repressor is likely to be related to the unusual end-on approach of the recognition helix (E), which allows sharing of the major groove by tandem dimers . The tandem complex model accounts for the mutational sensitivity of all trp operator base pairs . The structure also provides the first detailed view of the tandem interaction, revealing a key role for the amino-terminal arms. Nucleic Acids Res, 1993 Nov 11, 21(22), 5050 - 8 Single exchanges of amino acids in the basic region change the specificity of N-Myc; Feldmann T et al.; We exchanged specific amino acids in the basic region of the murine N-Myc protein and tested the mutant proteins for their DNA binding specificity . The amino acids we exchanged were chosen in analogy to residues of the homologous basic regions of bHLH and bZIP proteins . Mutant N-Myc peptides were expressed in Escherichia coli and specific DNA binding was monitored by gel shift experiments . For this we used palindromic target sequences with systematic base pair exchanges . Several mutants with altered DNA binding specificity were identified . Amino acid exchanges of residues -14 or -10 of the basic region lead to specificity changes (we define leucine 402 of N-Myc as +1; comparable to GCN4 see (1)) . The palindromic N-Myc recognition sequence 5'CACGTG is no longer recognized by the mutant proteins, but DNA fragments with symmetrical exchanges of the target sequence are . Exchanges at position -15 broaden the binding specificity . These data were used to build a computer based model of the putative interactions of the N-Myc basic DNA binding region with its target sequence. Nucleic Acids Res, 1993 Nov 11, 21(22), 5074 - 8 Competition between frameshifting, termination and suppression at the frameshift site in the Escherichia coli release factor-2 mRNA; Adamski FM et al.; Competition between frameshifting, termination, and suppression at the frameshifting site in the release factor-2 (RF-2) mRNA was determined in vitro using a coupled transcription-translation system by adding a UGA suppressor tRNA . The expression system was programmed with a plasmid containing a trpE-prfB fusion gene so that each of the products of the competing events could be measured . With increasing concentrations of suppressor tRNA the readthrough product increased at the expense of both the termination and the frameshifting product indicating all three processes are in direct competition . The readthrough at the internal UGA termination codon was greater than that at the natural UGA termination codon at the end of the coding sequence . The results suggest that this enhanced suppression may reflect slower decoding of the internal stop codon by the release factor giving suppression a competitive advantage . The internal UGAC stop signal at the frameshift site has been proposed to be a relatively poor signal, but in addition the release factor may be less able to recognise the signal with the mRNA in such a constrained state . Consequently, the frameshifting event itself will be more competitive with termination in vivo because of this longer pause as the release factor is decoding the stop signal. Biochim Biophys Acta, 1993 Nov 10, 1203(1), 27 - 35 Physico-chemical characterization of a recombinant cytoplasmic form of lysine: N6-hydroxylase; Thariath AM et al.; A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity . IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH . The enzyme is stringently specific with L-lysine and (S)-2-aminoethyl-L-cysteine serving as substrates . Protonophores, FCCP and CCCP, as well as cinnamylidene, have been found to serve as potent inhibitors of lysine: N6-hydroxylation by virtue of their ability to interfere in the reduction of the flavin cofactor. Biochim Biophys Acta, 1993 Nov 10, 1203(1), 155 - 61 Expression, purification and binding to the receptor of human insulin-like growth factor II; Miyagishima T et al.; Human insulin-like growth factor II (IGF-II) was expressed as a fused protein with 14 additive amino acids in Escherichia coli with a high yield by an expression system using T7 RNA polymerase . Purification of the expressed protein was simply performed using only differential ultrafiltrations, giving a homogeneous preparation upon polyacrylamide gel electrophoresis and high-performance liquid chromatography . The expressed peptide was reacted with a monoclonal antibody raised against native IGF-II on a blotted membrane . Furthermore, the peptide was bound to IGF-II receptor in solubilized rat fetus membrane, though the affinity was slightly inferior to that of native IGF-II . In addition, fusion IGF-II immobilized on a gel matrix was useful for one-step purification of the IGF-II receptor with a high yield from solubilized rat fetus membranes. Biochemistry, 1993 Nov 9, 32(44), 11923 - 8 Functional expression in Escherichia coli of the mitotic regulator proteins p24ran and p45rcc1 and fluorescence measurements of their interaction; Klebe C et al.; The gene products for the mitotic regulator genes RCC1 and Ran, p45rcc1 and p24ran, were expressed in Escherichia coli, purified in large amounts, and characterized for their biochemical properties . p24ran binds guanine nucleotide as a 1:1 complex, which is only slowly released from the protein . p45rcc1 catalyzes the exchange of nucleotide bound to the guanine nucleotide binding protein p24ran in the same way as the protein purified from HeLa cells . Likewise, the nucleotide dissociation from HeLa cell-derived p24ran protein is equally efficient with recombinant and nonrecombinant proteins . The recombinant proteins form a strong complex which contains no bound nucleotide . The kinetics of nucleotide exchange on p24ran in the presence or absence of p45rcc1 can be conveniently monitored either by the direct tryptophan fluorescence of p24ran or by fluorescence energy transfer measurements involving fluorescent nucleotides. Biochemistry, 1993 Nov 9, 32(44), 11794 - 801 Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds as DNA-protein cross-linking agents: potential suicide DNA lesions; Van Houten B et al.; Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds having the generalized formula M(a)NH2(CH)4NH2M(b) are shown to form specific DNA lesions which can efficiently cross-link proteins to DNA . In this study, the homodinuclear case is represented by M(a) = M(b) = {cis-Pt(Cl2)-(NH3)} and the heterodinuclear case is represented by M(a) = {cis-RuCl2(DMSO)3} and M(b) = {cis-PtCl2(NH3)} . Native and denaturing polyacrylamide gel electrophoresis was used to show the formation of ternary coordination complexes between the metal-treated 49-bp DNA fragment and the Escherichia coli UvrA and UvrB DNA repair proteins . Treatment with proteinase K results in loss of the DNA-protein cross-links . DNA-protein cross-links formed between UvrA and DNA previously modified with the dinuclear metal compounds are reversible with the reducing agent beta-mercaptoethanol . The DNA lesion responsible for efficient DNA-protein cross-linking is most probably a DNA-DNA interstrand cross-link in which each metal atom is coordinated with one strand of the DNA helix . The formation of DNA repair protein associated DNA cross-links, potential "suicide adducts", suggests a novel action mechanism for these anticancer compounds . In addition, these dinuclear metal compounds should be very useful agents for the investigation of a wide range of protein-DNA interactions. Biochemistry, 1993 Nov 9, 32(44), 11769 - 75 Solution oligomerization of the rev protein of HIV-1: implications for function; Cole JL et al.; rev is an RNA-binding protein of human immunodeficiency virus-1 and is required for the expression of incompletely spliced viral transcripts . Oligomerization of rev is thought to be associated with RNA binding and rev function . Here, we have characterized the oligomerization of rev using equilibrium analytical centrifugation . rev is predominantly monomeric at low concentrations, but reversibly polymerizes to produce large aggregates at higher concentrations . The data fit well to an unlimited isodesmic self-association model in which the association constants for the addition of a monomer to each aggregate are equal {K = 1.08 x 10(6) M-1 at 4 degrees C} . The association constant is essentially independent of monovalent salt concentration from 0.15 to 2 M at pH 6-9 . Thermodynamic parameters derived from the temperature dependence of the association constant over the limited range of 0-30 degrees C reveal that the primary contribution to the free energy of oligomerization is a large negative enthalpy . Binding of rev to the rev-responsive element of RNA was characterized under the same conditions as the centrifugation experiments using a nitrocellulose filter assay . rev binds to the RRE at a protein concentration where rev is predominantly monomeric, suggesting that solution multimerization of rev is not required for rev function. FEBS Lett, 1993 Nov 8, 334(1), 55 - 9 Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli; Liang NS et al.; A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli . Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin . The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies . It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple . Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography . The correct N-terminus was confirmed by protein sequencing . Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity. FEBS Lett, 1993 Nov 8, 334(1), 1 - 2 A comment on the absence of calcium regulation of human thioredoxin reductase; Oblong JE et al.; It has been previously suggested that human thioredoxin reductase activity is regulated by calcium . However, the activity of a purified form of human placental thioredoxin reductase was found to not be affected by mM concentrations of calcium, well above intra- and extracellular physiological levels . Furthermore, the suggestion that an E-F hand is present in Escherichia coli thioredoxin reductase is strongly contested . These current results suggest that human thioredoxin reductase is not regulated by calcium. Biochim Biophys Acta, 1993 Nov 7, 1179(2), 134 - 40 Primed pentose cycle activity supports production and elimination of superoxide anion in Kupffer cells from rats treated with endotoxin in vivo; Spolarics Z et al.; Glucose use and pentose cycle activity were determined in freshly isolated rat Kupffer cells 3 h after an i.v . injection of Escherichia coli endotoxin (0.1 mg/kg body weight), by using {1-14C}, {6-14C} and {2-3H}glucose . Endotoxin treatment in vivo caused a 5-fold increase in the basal glucose uptake in Kupffer cells . Pentose cycle activity was elevated from 8.7 to 13.6 nmol/h per 10(7) cells after endotoxin . In vitro treatment of the cells from saline- and endotoxin-treated animals with phorbol ester (10(-6) M) increased pentose cycle activity 2-fold and 8-fold, respectively . Phorbol ester caused a 50% increase in glucose uptake in both groups . t-Butyl hydroperoxide (0.5 mM) caused a similar increase in pentose cycle activity as phorbol ester . Glucose oxidation in the Krebs cycle was also doubled after endotoxin . KC from endotoxin-treated animals produced O2- spontaneously, and were primed to produce additional large amounts of O2- upon phorbol ester treatment . Addition of t-butyl hydroperoxide inhibited O2- production by Kupffer cells . Depletion of glutathione by N-ethylmaleimide (0.1 mM), or inhibition of NADPH oxidase by diphenyliodonium (0.1 mM) inhibited both the pentose cycle activity and the O2- production . Increasing the concentration of exogenous glucose in the cell medium elevated the glycolytic rate, while pentose cycle flux was not affected either under basal conditions or following subsequent challenges by phorbol ester or t-butyl hydroperoxide . Our data suggest that the endotoxin-induced elevated glucose use in Kupffer cells is accompanied by a primed state of the pentose cycle . This condition supports superoxide and macromolecule synthesis and could also represent a potentiated protective mechanism against oxidative cellular injury during bacterial infections. J Chromatogr A, 1993 Nov 5, 653(2), 241 - 6 Characterization of further association of the trimeric membrane protein porin by low-angle laser-light scattering photometry coupled with high-performance gel chromatography; Watanabe Y et al.; Porin (OmpF), a trimeric membrane protein, in an extract of the outer membrane of Escherichia coli gave a twin-peaked elution pattern on Sephacryl S-300HR gel chromatography in the presence of sodium dodecyl sulphate . The species eluting earlier and later were found to be the hexamer and trimer, respectively, from molecular mass determination by low-angle laser-light scattering photometry coupled with TSK-G3000SWXL gel chromatography . As the hexamer was dissociated into the trimer under the conditions of sodium dodecyl sulphate polyacrylamide gel electrophoresis, its presence had been overlooked . The addition of lipopolysaccharide, another component of the outer membrane, and subsequent dialysis induced association of the trimer, the product containing an appreciable amount of the hexamer. J Chromatogr A, 1993 Nov 5, 653(2), 207 - 18 Anion-exchange chromatographic behavior of recombinant rat cytochrome b5 . Thermodynamic driving forces and temperature dependence of the stoichiometric displacement parameter Z; Roush DJ et al.; The HPLC anion-exchange isocratic retention behavior of the recombinant soluble core of wild type rat cytochrome b5 on Mono Q HR 5/5 was investigated as a function of temperature and sodium chloride concentration at fixed eluent flow-rates . Retention was measured over a range of eluent flow-rates at a specified temperature to determine if true adsorption equilibrium could be approximated by the HPLC method . Apparent Van 't Hoff enthalpies of adsorption obtained from the HPLC retention data were positive, indicating an entropically driven spontaneous adsorption process, and were found to decline with increasing ionic strength . The retention results were interpreted in terms of the stoichiometric displacement model to obtain the apparent number of binding sites in the contact region, Z, as a function of temperature and of protein concentration . Z was found to depend significantly on temperature, even under conditions of nearly complete protein recovery, but did not depend on protein concentration at the low loadings studied. Science, 1993 Nov 5, 262(5135), 867 - 73 Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription; Erie DA et al.; Pre-steady state kinetics of misincorporation were used to investigate the addition of single nucleotides to nascent RNA by Escherichia coli RNA polymerase during transcription elongation . The results were fit with a branched kinetic mechanism that permits conformational switching, at each template position, between an activated and an unactivated enzyme complex, both of which can bind nucleotide triphosphates (NTPs) from solution . The complex exists most often in the long-lived activated state, and only becomes unactivated when transcription is slowed . This model permits multiple levels of nucleotide discrimination in transcription, since the complex can be "kinetically trapped" in the unactivated state in the absence of the correct NTP or if the 3' terminal residue is incorrectly matched . The transcription cleavage factor GreA (or an activity enhanced by GreA) increased the fidelity of transcription by preferential cleavage of transcripts containing misincorporated residues in the unactivated state of the elongation complex . This cleavage mechanism by GreA may prevent the formation of "dead-end" transcription complexes in vivo. J Mol Biol, 1993 Nov 5, 234(1), 87 - 98 A regulatory cascade in the induction of rhaBAD; Egan SM et al.; The RhaS and RhaR regulatory proteins are encoded in the Escherichia coli L-rhamnose gene cluster . We used complementation analysis and DNA mobility shift assays to show that RhaR is not the direct activator of the L-rhamnose catabolic operon, rhaBAD . An in-frame deletion of rhaS (rhaS-rhaR+) eliminated expression from the rhaBAD promoter, pBAD, while overexpression of rhaS greatly speeded the normally slow induction of transcription from pBAD . Expression from pBAD in a coupled transcription-translation assay was only detected when rhaS+ DNA was added to allow synthesis of RhaS protein . RhaS thus appears to be the direct L-rhamnose-specific activator of rhaBAD expression . Deletion mapping located the binding site for the L-rhamnose-specific regulator to a region overlapping position -70 relative to the rhaBAD transcription start site . Deletion mapping and DNA mobility shift assays located a CRP binding site just upstream from the binding site for the L-rhamnose-specific regulator . Quantitative primer extension analysis showed that induction of both the rhaBAD and rhaSR messages was unusually slow, requiring 40 to 50 minutes to reach a steady-state level . Induction of rhaBAD apparently involves a regulatory cascade in which RhaR first induces rhaSR expression, then RhaS accumulates and induces rhaBAD expression. J Mol Biol, 1993 Nov 5, 234(1), 8 - 13 Assembly of multimeric proteins . Effect of mutations in the alpha-subunit on membrane assembly and activity of pyridine nucleotide transhydrogenase; Ahmad S et al.; The pyridine nucleotide transhydrogenase of Escherichia coli is an inner membrane protein of two different subunits (alpha and beta) . It functions as a proton pump . The highly hydrophilic carboxy-terminal tail of ten amino acid residues in the alpha-subunit determines the correct folding and proper assembly of the beta-subunit leading to a functional enzyme . Premature termination of the alpha-subunit six amino acid residues from the carboxy-terminal end abolishes the activity completely . Although the two subunits are still assembled into the membrane, the conformation of the beta-subunit is perturbed . Systematic truncation and site-directed substitutions revealed that at least one positive charge in the carboxy-terminal region is required for efficient assembly of the two subunits to give a functional enzyme, while a phenylalanine residue, essential for activity, has no apparent effect on the extent of assembly of the two subunits. J Mol Biol, 1993 Nov 5, 234(1), 72 - 86 Negative co-dominant inhibition of recA protein function . Biochemical properties of the recA1, recA13 and recA56 proteins and the effect of recA56 protein on the activities of the wild-type recA protein function in vitro; Lauder SD et al.; We have investigated the biochemical properties of several Escherichia coli mutant recA proteins that display a null phenotype . These are the recA1, recA13 and recA56 proteins, each of which carries a single missense mutation . These proteins all share a common defect which is the inability to adopt the high affinity DNA binding state normally elicited by the nucleotide cofactor ATP . Consequently, other than the ability to bind ssDNA, they possess none of the in vitro enzymatic activities of recA protein . However, each protein has characteristics that are unique, leading to the conclusion that the observed mutant phenotypes arise through fundamentally different mechanisms . Despite the magnitude of these defects, the recA56 protein is able to differentially inhibit various activities of wild-type recA protein . Incorporation of recA56 protein into a presynaptic filament with the wild-type recA protein does not affect the ability of the wild-type protein to hydrolyze ATP, as judged by the turnover number (kcat), provided that the ssDNA concentration is not limiting; however, the affinity of wild-type recA protein for ATP is lowered by the presence of recA56 protein . Similarly, the ability to cleave lexA protein is only modestly inhibited . However, both the ability to compete with SSB protein for ssDNA binding sites and the DNA strand exchange activity of wild-type recA protein are severely inhibited by the presence of recA56 protein . These results suggest that individual monomeric components of the recA protein-DNA filament are translated through protein-protein contacts to become macroscopic properties of the filament. J Mol Biol, 1993 Nov 5, 234(1), 14 - 27 Translation initiation complex formation with 30 S ribosomal particles mutated at conserved positions in the 3'-minor domain of 16 S RNA; Ringquist S et al.; Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting . These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex . Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants . Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA(fMet) and tRNA(Phe) for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA(fMet) . The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m2(6)A, in the synthetic RNA . Synthetic particles containing the mutation U1512C, which converts the universal U.G pair to C.G enhanced both tRNA(fMet) binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect . Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3'-minor domain, also enhanced tRNA(fMet) selectivity, in this case by reducing complex formation with elongator tRNA . Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels . The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence . This result had not been detected by standard tRNA-30 S binding assays . Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA(fMet). J Immunol Methods, 1993 Nov 5, 166(1), 1 - 10 Detection and quantification of secreted soluble Fc gamma RIIA in human sera by an enzyme-linked immunosorbent assay; Astier A et al.; Fc gamma RIIA can be produced in a soluble form that contains both the extracellular and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-coding exon . We have developed an enzyme-linked immunosorbent assay (ELISA) that permits the specific detection and quantification in human sera of this secreted soluble Fc gamma RIIA . It uses the monoclonal antibody (MAb) IV.3 as capture antibody and rabbit polyclonal IgG directed against the intracellular region of Fc gamma RIIA as detector antibodies . The enzymatic reaction was amplified using an NADH/NAD+ amplification system . As little as 0.8-1.5 ng/ml (20-38 pM) of purified recombinant secreted Fc gamma RIIA could be detected . The serum levels of secreted sFc gamma RIIA ranged from 0 to 30 ng/ml in sera from 51 healthy donors . The mean value was 11.9 ng/ml +/- 6.55 (297 pM +/- 163) and the median value was 10.6 ng/ml (265 pM) (range: 0-764 pM). J Biol Chem, 1993 Nov 5, 268(31), 23685 - 96 Characterization of the structural requirements for assembly and nucleotide binding of an ATP-binding cassette transporter . The maltose transport system of Escherichia coli; Panagiotidis CH et al.; The periplasmic maltose-binding protein-dependent, maltose transport system of Escherichia coli is a well studied member of the ATP-binding cassette family of transport ATPases . In addition to the water-soluble maltose-binding protein, the system comprises three membrane proteins, MalF, MalG, and MalK, which form a heterotetrameric complex (FGK2) in the cytoplasmic membrane . The purified complex exhibits transport-associated ATPase activity . To characterize the requirements for nucleotide binding and hydrolysis by the FGK2 complex, we used plasmids to express different combinations of the individual subunits as well as mutant forms of the MalK subunit . Prior to measuring nucleotide binding, we examined membrane preparations for the presence of each subunit from strains that contained all possible permutations of the three structural genes, malF, malG, and malK . We found that when all three genes were present or when malF and malK were present together, the corresponding antigens were detected easily on Western immunoblots and were soluble in the non-ionic detergent, Triton X-100 . In contrast, all other permutations resulted in decreased amounts of antigen or antigen that was Triton X-100-insoluble . We relied on photocross-linking with 8-azido-{32P}ATP and ATP hydrolysis as indicators of the ability of the transport complex to interact with purine nucleotides . 8-Azido-{32P}ATP was photocross-linked to the MalK subunit . Photolabeling of MalK was inhibited by ATP, ADP, and GTP and not by other nucleotides . Photolabeling of MalK required the presence of MalF but not MalG . Mutations in malK that affect amino acid residues thought to be directly involved in nucleotide binding did indeed abolish labeling and resulted in loss of transport activity without affecting protein stability . In general, ATP hydrolysis correlated with the photocross-linking . A notable exception is the MalK941 mutant protein which retained the ability to be labeled by 8-azido-{32P}ATP but was unable to catalyze detectable levels of ATP hydrolysis . Some, but not all, of the malK mutations were dominant to wild type . To study the mechanism of dominance we devised a means of measuring the ability of different wild-type and mutant MalK proteins to interact with the MalF and MalG subunits . This assay relies on the fact that, when a bifunctional MalK-LacZ hybrid protein is associated with the MalF and MalG subunits, it is membrane-bound . Excess MalK competed with the MalK-LacZ hybrid protein for sites in the membrane and resulted in the hybrid fractionating as a soluble protein.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1993 Nov 5, 268(31), 23580 - 4 Calcium-dependent activation of protein kinase C . The role of the C2 domain in divalent cation selectivity; Luo JH et al.; Activation of certain isoforms of protein kinase C (cPKCs) requires Ca2+ and is associated with a conserved C2 domain that is not present in Ca(2+)-independent isoforms (nPKCs) . The site(s) of Ca2+ binding and the role of the C2 domain have not been previously identified . We have analyzed phosphatidylserine-dependent Ca2+ binding to fusion proteins expressed in Escherichia coli that carry various modifications in the regulatory region of cPKC beta 1 or nPKC epsilon . Ca2+ is bound mainly to the C1 domain of PKC beta 1, but the C2 domain confers specificity for Ca2+ binding when compared with Mg2+ and Mn2+ . We propose that in cPKCs there is selective binding of Ca2+ to a pocket formed by the C1 and C2 domains . This induces a change in conformation that activates the enzyme . In nPKCs, the cation binding pocket is less specific for Ca2+ because it lacks the C2 domain . Therefore, divalent cations like Mg2+ can bind to it, thereby abrogating the requirement of Ca2+ for enzyme activation. J Biol Chem, 1993 Nov 5, 268(31), 23524 - 30 Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis; Sakumi K et al.; 8-Oxoguanine (8-oxo-7, 8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by active oxygen species normally formed during cellular metabolic processes . 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies, and transversion mutation ensues . Human cells contain enzyme activity, which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, and this enzyme is responsible for preventing misincorporation of 8-oxoguanine into DNA . We purified this particular human enzyme to physical homogeneity and determined a partial amino acid sequence . We then cloned the cDNA for human 8-oxo-dGTPase and examined its nucleotide sequence . The human protein comprises 156 amino acid residues and has some sequence homology with the Escherichia coli MutT protein, which has a distinct 8-oxo-dGTPase activity . When the human cDNA was expressed in E . coli mutT- mutant cells, there was a significant amount of 8-oxo-dGTPase activity . In such cells, the frequency of spontaneous mutation was greatly reduced . We propose that the human 8-oxo-dGTPase protects genetic information from the untoward effects of endogenous oxygen radicals. J Biol Chem, 1993 Nov 5, 268(31), 23519 - 23 Reconstitution of recombinant 40-kDa subunit of the clathrin-coated vesicle H(+)-ATPase; Peng SB et al.; We have proposed a model of the ATP hydrolytic sector of the clathrin-coated vesicle H(+)-ATPase wherein significant catalysis requires four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D . K . (1988) J . Biol . Chem . 263, 9859-9867) . We have cloned and expressed the 40-kDa component in Escherichia coli and have purified the recombinant protein to homogeneity . This subunit lacks ATP hydrolytic capacity, but when reconstituted to a 40 kDa-depleted hydrolytic sector, there is a greater than 20-fold increase in calcium-activated, N-ethylmaleimide-sensitive ATP hydrolysis, indicating that this subunit is required for vacuolar-type proton pump function. J Biol Chem, 1993 Nov 5, 268(31), 23477 - 82 Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli; Borukhov S et al.; The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R . L . (1988) Nucleic Acids Res . 16, 9789-9809) . We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes . By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex . The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding . A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation. J Biol Chem, 1993 Nov 5, 268(31), 23427 - 34 The replication fate of R- and S-styrene oxide adducts on adenine N6 is dependent on both the chirality of the lesion and the local sequence context; Latham GJ et al.; In order to deduce the biological fate of adducts formed by the reaction of styrene oxide, a suspected carcinogen, with DNA, four oligodeoxynucleotides were synthesized which contained either R- or S-styrene oxide lesions on the N6 position of neighboring adenines within the human N-ras codon 61 . When these adducted oligodeoxynucleotides were ligated into the single-stranded vector M13mp7L2 and the modified DNA used to transform repair-deficient Escherichia coli, the resultant plaque-forming abilities were found to vary as much as 300-fold, depending on the stereochemical configuration of the styrene oxide lesion and the sequence context in the vicinity of the damage . The frequency of mutations caused by the various styrene oxide adducts were similarly dependent on both their chirality and local sequence context . Oligodeoxynucleotide templates bearing these same four adducts were also constructed in order to evaluate their replication in vitro by the Klenow fragment . Three of the four styryl-modified templates yielded significant levels of fully extended primer upon polymerization . In contrast, the template containing R-styrene oxide at the second position of N-ras 61 was a very poor substrate for replication, a result which correlates well with the observed lethality of this lesion in vivo. J Biol Chem, 1993 Nov 5, 268(31), 23239 - 49 Dependence of trp repressor-operator affinity, stoichiometry, and apparent cooperativity on DNA sequence and size; Liu YC et al.; A series of chemically synthesized trp and mutant operator DNAs was employed to examine trp repressor binding . Although only a single repressor-operator complex was observed for most DNAs as reported previously, varying DNA sequence revealed two retarded complexes with an additional band of faster mobility . The relative intensity of the two retarded bands with varying repressor concentrations suggests that cooperative interactions between dimers may occur in the formation of the predominant repressor-operator complex . Direct stoichiometry measurements demonstrated that a 2:1 stoichiometry (two dimers per operator) is found in the primary repressor-operator complex band and that a 1:1 stoichiometry is observed, when present, for the minor repressor-operator complex band of faster mobility . Similar retardation patterns with a single complex of 2:1 stoichiometry were observed for 40-base pair (bp) trp operators corresponding to TrpEDCBA, aroH, and Trp-PL (a derivative of TrpEDCBA with increased symmetry) operator sequences as well as to hybrid operators containing a half-binding site from TrpEDCBA in conjunction with lac operator sequences, although the apparent affinity for the half-site DNAs was diminished by 10-fold . In contrast, the prominence of the 1:1 dimer-operator complex for Trp-PR, a different derivative of TrpEDCBA with increased symmetry, suggests that sequence context may diminish cooperativity between dimers . The stoichiometry observed was also dependent on the length of TrpEDCBA operator DNA used, shifting from primarily 2:1 for 40- and 33-bp TrpEDCBA DNA to primarily 1:1 for 29-, 26-, and 20-bp TrpEDCBA DNAs . In addition, the stability of the repressor-operator complex to electrophoresis is reduced for DNA lengths of 33, 29, and 26 bp . Based on the binding data and footprinting patterns for two hybrid 40-bp TrpEDCBA/lac half-binding site DNAs, it appears that repressor associates tightly to the specific trp half-site, whereas the nonspecific half of the DNA is more loosely bound . These results suggest that repressor dimer-dimer interaction may be an important feature in the trp repressor-operator interaction. J Biol Chem, 1993 Nov 5, 268(31), 23148 - 56 Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli . A functionally active neurotrophic factor; Becerra SP et al.; Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells . Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F . R., Chader, G . J., Johnson, L . V., and Tombran-Tink, J . (1993) Proc . Natl . Acad . Sci . U . S . A . 90, 1526-1530) . We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli . The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs . Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein . After solubilization, BH was highly purified by gel filtration and cation exchange chromatography . The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct . We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system . Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF . Thus, unfolded and refolded in vitro BH retained a potent biological activity . In parallel experiments, protease inhibition assays were performed . Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity. J Biol Chem, 1993 Nov 5, 268(31), 23139 - 47 Molecular characterization and outer membrane association of a Chlamydia trachomatis protein related to the hsp70 family of proteins; Raulston JE et al.; One route by which Chlamydia trachomatis is internalized into host endometrial epithelial cells is receptor-mediated endocytosis . Although this implies an adhesin-receptor interaction exists, specific chlamydial surface molecules have not been identified . We are investigating potential adhesin molecules using an in vitro functional assay to select for chlamydial recombinant Escherichia coli expressing an adherent phenotype . We have previously shown that E . coli JM109(pPBW58) attaches to epithelial cells by a specific process paralleling C . trachomatis and expresses at least three plasmid-encoded proteins (18, 28, and 82 kDa; Schmiel, D . H., Knight, S . T., Raulston, J . E., Choong, J., Davis, C . H., and Wyrick, P . B . (1991) Infect . Immun . 59, 4001-4012) . In this report, we demonstrate that (i) the 82-kDa protein is associated with the outer membrane of both E . coli JM109-(pPBW58) and C . trachomatis serovar E elementary bodies; (ii) the plasmid-encoded protein is identical to the native chlamydial protein by mass, charge, antigenicity, and partial proteolytic peptide profiles; (iii) a highly homologous protein is present in C . trachomatis biovariant lymphogranuloma venereum; (iv) the 82-kDa protein is not covalently linked by disulfide bonds to other protein species in either E . coli JM109(pPBW58) or C . trachomatis; (v) sequence analysis of the open reading frame indicates this protein is a relative of the heat shock 70 family of proteins; and (vi) the inferred amino acid sequence contains a contiguous 73-amino acid region having 51% identity with the extracellular sperm receptor binding domain in Strongylocentrosus purpuratus (Foltz, K . R., Partin, J . S., and Lennarz, W . J . (1993) Science 259, 1421-1425) . The potential involvement of an hsp70 protein in attachment may provide new insight on adherence mechanisms by obligate intracellular pathogens. J Biol Chem, 1993 Nov 5, 268(31), 23132 - 8 Function of the active-site lysine in Escherichia coli serine hydroxymethyltransferase; Schirch D et al.; Serine hydroxymethyltransferase has a conserved lysine residue (Lys-229) that forms the internal aldimine with pyridoxal 5'-phosphate . In other pyridoxal 5'-phosphate enzymes investigated so far, this conserved lysine residue also plays a catalytic role as a base that removes the alpha-proton from the amino acid substrate . Three mutant forms of Escherichia coli serine hydroxymethyltransferase (K229Q, K229R, and K229H) were constructed, expressed, and purified . The absorbance spectra, rapid reaction kinetics, and thermal denaturation of the mutant analogs were studied . Only the K229Q mutant serine hydrox