Biochem Biophys Res Commun, 1995 Apr 6, 209(1), 327 - 34 Drosophila glutathione S-transferase D27: functional analysis of two consecutive tyrosines near the N-terminus; Lee HC et al.; The Drosophila glutathione S-transferase D27 (GST D27) has been purified and characterized after direct expression of the intronless gstD27 gene in E . coli . The GST D27 has both conjugation activity against the common substrate 1-chloro-2,4-dinitrobenzene and peroxidase activity against cumene hydroperoxide . Its pH optimum is 8.5 in 0.125 M bis-tris propane buffer at 22 degrees C . It is more thermal labile than the human GST121 . The GST D27 has two tyrosines at positions 3 and 4 . Both of them appear to be important but neither of them is essential for the enzyme activity . Thus, other residues may also participate in catalysis . The two tyrosines of GST D27 could also be important in binding to GSH or S-hexyl GSH . Results from in vitro biochemical analyses were confirmed by the in vivo activity-based CDNB growth inhibition analyses . Our results clearly indicate that the Drosophila GST D isozymes are different from any of the known mammalian GSTs. Biochem Biophys Res Commun, 1995 Apr 6, 209(1), 198 - 204 A cell division inhibitor SulA of Escherichia coli directly interacts with FtsZ through GTP hydrolysis; Higashitani A et al.; E . coli SulA is an SOS-inducible protein that inhibits cell division . FtsZ is a protein that plays a central role in bacterial cell division . Using purified SulA protein that was fused to the maltose binding protein, we demonstrate in vitro that SulA interacts with FtsZ to form a stable complex . The reaction requires GTP and Mg ion . GDP and GTP gamma S cannot substitute for GTP, which suggests that hydrolysis of GTP is required for the reaction . The complex is formed in a molar ratio of approximately one to one of the two proteins . It is likely that the complex formation represents the in vivo mechanism by which SulA inhibits cell division. Biochem Biophys Res Commun, 1995 Apr 6, 209(1), 126 - 30 Mutational study at Ser300 position of the Escherichia coli lactose repressor; Miura-Onai S et al.; We have previously reported that a Ser300Asn mutant of the Escherichia coli lactose repressor protein produced a temperature-sensitive phenotype . In order to analyze the structure-function relationship of the lactose repressor protein, we conducted 18 amino acid substitutions at this Ser 300 site by using in vitro mutagenesis . The substitutions at this position that exhibited repressors with the wild-type phenotype in vivo were Gly, Ala, Ile, Thr, Met and Cys; the other 10 substitutions examined (Leu, Val, Tyr, Trp, Asp, Glu, Gln, His, Lys and Arg) resulted in the lacI- phenotype . In addition, the Ser300Phe mutation resulted in the lacIts phenotype, while the Ser300Pro mutation resulted in lacIts,s . It seems likely that the Ser300 position plays an important role in oligomer formation and inducer binding. Biochem Biophys Res Commun, 1995 Apr 6, 209(1), 117 - 25 Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli; Ratajczak T et al.; Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose . Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC . The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity . With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a kcat/KM value of 5.1 x 10(5) M-1s-1 at 5 degrees C . The isomerase activity was inhibited by cyclosporin A with an IC50 value of 1030 nM . These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin . Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca2+ and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein. Biochim Biophys Acta, 1995 Apr 4, 1261(2), 321 - 4 Cloning and sequence analysis of the cDNA for bovine mitochondrial translational initiation factor 2; Ma J et al.; The complete sequence of the cDNA encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained by library screening followed by 3'-RACE PCR . The open reading frame for bovine IF-2mt encodes a protein of 727 amino acids . The sequence of bovine IF-2mt exhibits 85% identity to human IF-2mt, but only 38% identity to yeast IF-2mt and 39% identity to Escherichia coli IF-2 alpha. Biochim Biophys Acta, 1995 Apr 4, 1261(2), 233 - 42 Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases; Ciesla J et al.; Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA . The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein . The missing region was obtained by screening a rat genomic library . The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da . Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues . Six of these differences are in the first 21 amino acids from the amino-end . The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature . The three species differed completely at only four sites . Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS . The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein . Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed . The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool. Biochim Biophys Acta, 1995 Apr 4, 1229(1), 49 - 58 The involvement of NADP(H) binding and release in energy transduction by proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli; Bizouarn T et al.; Proton-translocating transhydrogenase was solubilised and purified from membranes of Escherichia coli . Consistent with recent evidence {Hutton, M., Day, J., Bizouarn, T . and Jackson, J.B . (1994) Eur . J . Biochem . 219, 1041-1051}, at low pH and salt concentration, the enzyme catalysed rapid reduction of the NAD+ analogue AcPdAD+ by a combination of NADH and NADPH . At saturating concentrations of NADPH, the dependence of the steady-state rate on the concentrations of NADH and AcPdAD+ indicated that, with respect to these two nucleotides, the reaction proceeds by a ping-pong mechanism . High concentrations of either NADH or AcPdAD+ led to substrate inhibition . These observations support the view that, in this reaction, NADP(H) remains bound to the enzyme: AcPdAD+ is reduced by enzyme-bound NADPH, and NADH is oxidised by enzyme-bound NADP+, in a cyclic process . When this reaction was carried out with {4A-2H}NADH replacing {4A-1H}NADH, the rate was decreased by 46%, suggesting that the H- transfer steps are rate-limiting . In simple 'reverse' transhydrogenation, the reduction of AcPdAD+ was slower with {4B-2H}NADPH than with {4B-1H}NADPH when the reaction was performed at pH 8.0, but there was no deuterium isotope effect at pH 6.0 . This indicates that H- transfer is rate-limiting at pH 8.0 and supports our earlier suggestion that NADP+ release from the enzyme is rate-limiting at low pH . The lack of a deuterium isotope effect in the reduction of thio-NADP+ by NADH at low pH is also consistent with the view that NADPH release from the enzyme is slow under these conditions . A steady-state rate equation is derived for the reduction of AcPdAD+ by NADPH plus NADH, assuming operation of the cyclic pathway . It adequately accounts for the pH dependence of the enzyme, for the features described above and for kinetic characteristics of E . coli transhydrogenase described in the literature. Biochemistry, 1995 Apr 4, 34(13), 4428 - 33 Proton transfer in cytochrome bo3 ubiquinol oxidase of Escherichia coli: second-site mutations in subunit I that restore proton pumping in the mutant Asp135-->Asn; Garcia-Horsman JA et al.; The ubiquinol oxidase, cytochrome bo3, of Escherichia coli is a member of the respiratory heme-copper oxidase family and conserves energy from the reduction of dioxygen to water by translocation of protons across the bacterial membrane . Mutation of an aspartic acid residue (Asp135) to asparagine in subunit I of this enzyme was previously found to impair proton translocation {Thomas et al . (1993) Biochemistry 32, 10923-10928} . This residue is located in an interhelical "loop" between transmembranous helices II and III, which contains six well-conserved residues (Asn124, Pro128, Gly132, Asp135, Pro139, and Asn142) . Site-directed mutagenesis was performed to study the function of this entire domain . Nonconservative mutations of Asn124 and Asn142 also resulted in a loss of proton translocation, whereas their conservative substitution to glutamine had no effect . Mutations in eight other positions within this domain did not affect proton translocation . Introduction of an acidic group at positions 139 or 142, but not at eight other tested positions, restored proton pumping in the Asp135-->Asn mutated protein . These results suggest that the C-terminal part of the domain may be alpha-helical and that the entire "loop" plays an important structural and functional role as part of an input channel of the proton translocation machinery. Biochemistry, 1995 Apr 4, 34(13), 4421 - 7 Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase; Wilks A et al.; A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter . The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies . The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase . A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain . Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover . The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein . The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system . Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking. Biochemistry, 1995 Apr 4, 34(13), 4412 - 20 Melibiose permease of Escherichia coli: large scale purification and evidence that H+, Na+, and Li+ sugar symport is catalyzed by a single polypeptide; Pourcher T et al.; As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography . Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids . The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin . After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography . Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence . Purified permeases, reconstituted in liposomes, display H(+)-, Na(+)-, or Li(+)-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide. Biochemistry, 1995 Apr 4, 34(13), 4358 - 68 Effect of aminofluorene and (acetylamino)fluorene adducts on the DNA replication mediated by Escherichia coli polymerases I (Klenow fragment) and III; Doisy R et al.; N-(Deoxyguanosin-C8-yl)-2-(acetylamino)fluorene (dG-C8-AAF) and N-(deoxyguanosin-C8-yl)-2-aminofluorene (dG-C8-AF) are the two major DNA adducts induced by the chemical carcinogen 2-(acetylamino)fluorene (AAF) . Molecular modeling shows that, in the DNA double helix, dG-C8-AF can maintain an anti-structure and normal base pairing, while dG-C8-AAF favors a syn-structure and causes base displacement . In the phi X174 RF DNA-Escherichia coli transfection system, it has been found that dG-C8-AF is 7-10-fold less lethal than dG-C8-AAF; these results suggest that these two kinds of DNA adducts may have different effects on DNA replication and that they may be repaired by different pathways . We have investigated the effects of these two kinds of adducts on DNA polymerase III holoenzyme (pol III-H) and DNA polymerase I Klenow fragment (pol I-Kf) mediated DNA synthesis by using carcinogen-modified M13 single-stranded DNA hybridized with 32P-labeled primer as templates . We have found that pol III-H and pol I-Kf replicate through dG-C8-AF with 92% and 62% frequency, respectively; in contrast, these two enzymes replicate through dG-C8-AAF with only 38% and 25% frequency, respectively . AF-adducted DNA shows a more profound sequence specificity in blocking DNA synthesis than AAF-adducted DNA, and the sequence specificities in blocking DNA synthesis for both kinds of adducts differ for pol III-H and pol I-Kf. Biochemistry, 1995 Apr 4, 34(13), 4331 - 41 Crystal structure of recombinant pea cytosolic ascorbate peroxidase; Patterson WR et al.; The crystal structure of recombinant pea cytosolic ascorbate peroxidase has been refined to an R = 0.19 for data between 8.0 and 2.2 A resolution and magnitude of F > or = 2 sigma(magnitude of F) . The refined model consists of four ascorbate peroxidase monomers consisting of 249 residues per monomer assembled into two homodimers, with one heme group per monomer . The ascorbate peroxidase model confirms that the pea cytosolic enzyme is a noncovalent homodimer held together by a series of ionic interactions arranged around the 2-fold noncrystallographic dimer axis . As expected from the high level of sequence identity (33%), the overall fold of the ascorbate peroxidase monomer closely resembles that of cytochrome c peroxidase . The average root mean square differences for 137 helical alpha-carbon atoms between the four ascorbate peroxidase monomers and cytochrome c peroxidase and for 249 topologically equivalent alpha-carbon atoms are 0.9 and 1.3 A, respectively . The active site structures are also the same, including the hydrogen-bonding interactions between the proximal His ligand, a buried Asp residue, and a Trp residue, whose indole ring is parallel to and in contact with the proximal His ligand just under the heme ring . This proximal Trp residue is thought to be the site of free radical formation in cytochrome c peroxidase compound I and is also essential for enzyme activity . The corresponding Trp in ascorbate peroxidase, Trp179, occupies exactly the same position . The most interesting, and possibly functionally important, difference between the two peroxidases is the presence of a cation binding site in ascorbate peroxidase located approximately 8 A from the alpha-carbon atom of Trp179. Biochemistry, 1995 Apr 4, 34(13), 4287 - 98 Crystallographic and enzymatic investigations on the role of Ser558, His610, and Asn614 in the catalytic mechanism of Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2p); Hendle J et al.; Dihydrolipoamide acetyltransferase (E2p) is the structural and catalytic core of the pyruvate dehydrogenase multienzyme complex . In Azotobacter vinelandii E2p, residues Ser558, His610', and Asn614' are potentially involved in transition state stabilization, proton transfer, and activation of proton transfer, respectively . Three active site mutants, S558A, H610C, and N614D, of the catalytic domain of A . vinelandii E2p were prepared by site-directed mutagenesis and enzymatically characterized . The crystal structures of the three mutants have been determined at 2.7, 2.5, and 2.6 A resolution, respectively . The S558A and H610C mutants exhibit a strongly (200-fold and 500-fold, respectively) reduced enzymatic activity whereas the substitution of Asn614' by aspartate results in a moderate (9-fold) reduced activity . The decrease in enzymatic activity of the S558A and H610C mutants is solely due to the absence of the hydroxyl and imidazole side chains, respectively, and not due to major conformational rearrangements of the protein . Furthermore the sulfhydryl group of Cys610' is reoriented, resulting in a completely buried side chain which is quite different from the solvent-exposed imidazole group of His610' in the wild-type enzyme . The presence of Asn614' in A . vinelandii E2p is exceptional since all other 18 known dihydrolipoamide acyltransferase sequences contain an aspartate in this position . We observe no difference in conformation of Asp614' in the N614D mutant structure compared with the conformation of Asn614' in the wild-type enzyme . Detailed analysis of all available structures and sequences suggests two classes of acetyltransferases: one class with a catalytically essential His-Asn pair and one with a His-Asp-Arg triad as present in chloramphenicol acetyltransferase {Leslie, A . G . W . (1990) J . Mol . Biol . 213, 167-186} and in the proposed active site models of Escherichia coli and yeast E2p. Biochemistry, 1995 Apr 4, 34(13), 4267 - 75 Evidence by site-directed mutagenesis supports long-range electron transfer in mouse ribonucleotide reductase; Rova U et al.; Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone . The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity . The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A . Therefore, an electron pathway was suggested between the active site and the tyrosyl radical . Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand . To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine . All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center . In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein . Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique . However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer. Biochemistry, 1995 Apr 4, 34(13), 4246 - 56 Old Yellow enzyme: aromatization of cyclic enones and the mechanism of a novel dismutation reaction; Vaz AD et al.; The origin of charge transfer bands that develop on reaction of Old Yellow Enzyme with alpha,beta-unsaturated cyclic ketones such as 3-oxodecalin-4-ene (ODE, numbered according to the convention for steroids), 3-oxodecalin-4-ene-10-carboxaldehyde (ODEC), and 2-cyclohexenone is shown to be due to the aromatization of ODE and ODEC to 3-hydroxy-6,7,8,9-tetrahydronaphthalene (HTN) and of 2-cyclohexenone to phenol . The aromatization of ODEC to HTN is stereospecific and involves the trans dehydrogenation of the 1 beta, 2 alpha hydrogens . The aromatization occurs under aerobic as well as anaerobic conditions . With the exception of ODEC under aerobic conditions, the aromatization of these substrates is accompanied by a dismutation reaction in which the olefinic bond of a second molecule of each substrate is reduced to give the saturated cyclic ketone . Molecular oxygen may serve as the electron acceptor with ODEC and some other substrates under aerobic reaction conditions . The dismutation reaction involves an overall sequence of hydride transfer from one substrate molecule to the beta-carbon of a second substrate molecule along with a solvent proton uptake by the alpha-carbon . 19-Nortestosterone is aromatized to beta-estradiol; however, other 3-oxo-delta 4-steroids such as progesterone, testosterone, and androstene-3,17-dione bind tightly to the enzyme but are not aromatized . The NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds is limited to aldehydes and ketones . alpha,beta-Unsaturated acids, esters, amides, and nitriles are not reduced . The reduction of the olefinic bond of ODE or cinnamaldehyde by NADPH occurs by an overall sequence of hydride transfer from the reduced pyridine nucleotide to the beta-carbon of the alpha,beta-unsaturated carbonyl compound and a solvent proton uptake by the alpha-carbon . The 4-pro-R hydride of NADPH is transferred in the reduction reaction . Structure-function relationships in the NADPH-dependent reduction of alpha,beta-unsaturated aldehydes or ketones indicate that increasing alkyl substitution at the beta-carbon results in marked decrease in the rate of reduction of the olefinic bond, consistent with a steric hindrance to hydride transfer at the beta-carbon. Biochemistry, 1995 Apr 4, 34(13), 4238 - 45 Dependence of the phosphorylation of alkaline phosphatase by phosphate monoesters on the pKa of the leaving group; Han R et al.; The hydrolysis and transphosphorylation reactions of a series of phosphate monoesters, ROPO3(2)-(R = 2,4-dinitrophenyl, 4-nitrophenyl, phenyl, glucose-1, glycerol-1, methyl, ethyl, and dodecyl), catalyzed by Escherichia coli alkaline phosphatase and a mutant enzyme, Ser102Cys, have been studied at alkaline pH using the rates of change in the 31P NMR signals of substrate, the hydrolysis product (inorganic phosphate), and the transphosphorylation product (O-Tris phosphate) as the assay . The kcat at pH 8.0 for the wild-type enzyme is approximately 30 s-1 and is independent of the nature of the R group, when the pKa of the leaving group is < 10 . Under these conditions the rate of phosphorylation is much faster than dissociation of inorganic phosphate, 15-60 s-1 . If the pKa of the leaving group is between 10 and 15, phosphorylation and dissociation of the product phosphate both contribute to the rate limit . If the pKa of the leaving group is > 15, phosphorylation is rate limiting . A Bronsted plot of log kcat vs pKa of the leaving group for those substrates for which phosphorylation is rate limiting yields a beta lg of approximately -0.6 . In contrast to the wild-type enzyme, the log kcat values for the S102C mutant enzyme catalyzing the hydrolysis of phosphate esters are linearly dependent on the pKa's of the leaving group throughout the range of pKa from 4 to 16 . Phosphorylation of C102 is the rate controlling step, and kcat is independent of the Tris concentration as predicted for rate limiting phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Apr 4, 34(13), 4231 - 7 Chemical modification and site-directed mutagenesis studies of rat 3-hydroxyisobutyrate dehydrogenase; Hawes JW et al.; Rat 3-hydroxyisobutyrate dehydrogenase shares sequence homology with the short-chain alcohol dehydrogenases . Site-directed mutagenesis and chemical modifications were used to examine the roles of cysteine residues and other residues conserved in this family of enzymes . It was found that a highly conserved tyrosine residue, Y162 in 3-hydroxyisobutyrate dehydrogenase, does not function catalytically as it may in other short-chain alcohol dehydrogenases . Of the six cysteine residues present in 3-hydroxyisobutyrate dehydrogenase, only cysteine 215 was found to be critical to catalysis . C215A and C215D mutant enzymes were catalytically inactive but produced CD spectra identical to wild-type enzyme . C215S mutant enzyme displayed a lowered Vmax than wild-type enzyme, but Km values were similar to those of wild-type enzyme . The C215S mutant enzyme was inactivated by treatment with phenylmethanesulfonyl fluoride but was not inactivated by treatment with iodoacetate, whereas the wild-type enzyme was inactivated by treatment with iodoacetate but not inactivated by treatment with phenylmethanesulfonyl fluoride . The present data suggest that 3-hydroxyisobutyrate dehydrogenase differs in mechanism from other short-chain alcohol dehydrogenases studied to date and that cysteine 215 has a critical function in catalysis, possibly as a general base catalyst. Biochemistry, 1995 Apr 4, 34(13), 4220 - 4 Major contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: mutation and rescue; Carlow DC et al.; The crystal structure of an inhibitory complex formed between Escherichia coli cytidine deaminase and the transition-state analog 3,4-dihydrouridine indicates the presence of a short H-bond between Glu-104 and the inhibitor . To test the possibility that analogous H-bonds might play a significant role in stabilizing the hydrated substrate in the transition state for deamination, we replaced Glu-104 by alanine . Compared with the wild-type enzyme, the mutant enzyme's affinities for substrate cytidine and product uridine were found to have increased, whereas kcat for deamination of cytidine had been reduced by 8 orders of magnitude . By its presence, the carboxymethyl group of Glu-104 appears to minimize the activation barrier for deamination, not only by stabilizing the altered substrate in the transition state but also by destabilizing the enzyme-substrate and enzyme-product complexes . In the presence of added formate ion, but not in the presence of bulkier carboxylic acids, the low catalytic activity of the mutant enzyme was enhanced substantially. Biochemistry, 1995 Apr 4, 34(13), 4202 - 10 Kinetics of human soluble and membrane-bound catechol O-methyltransferase: a revised mechanism and description of the thermolabile variant of the enzyme; Lotta T et al.; Human soluble (S) and membrane-bound (MB) catechol O-methyltransferase (COMT, EC 2.1.1.6) enzymes have been expressed at sufficiently high levels in Escherichia coli and in baculovirus-infected insect cells to allow kinetic characterization of the enzyme forms . The use of tight-binding inhibitors such as entacapone enabled the estimation of actual enzyme concentrations and, thereby, comparison of velocity parameters, substrate selectivity, and regioselectivity of the methylation of both enzyme forms . Kinetics of the methylation reaction of dopamine, (-)-noradrenaline, L-dopa, and 3,4-dihydroxybenzoic acid was studied in detail . Here, the catalytic number (Vmax) of S-COMT was somewhat higher than that of MB-COMT for all four substrates . The Km values varied considerably, depending on both substrate and enzyme form . S-COMT showed about 15 times higher Km values for catecholamines than MB-COMT . The distinctive difference between the enzyme forms was also the higher affinity of MB-COMT for the coenzyme S-adenosyl-L-methionine (AdoMet) . The average dissociation constants Ks were 3.4 and 20.2 microM for MB-COMT and S-COMT, respectively . Comparison between the kinetic results and the atomic structure of S-COMT is presented, and a revised mechanism for the reaction cycle is discussed . Two recently published human COMT cDNA sequences differed in the position of S-COMT amino acid 108, the residue being either Val-108 {Lundstrom et al . (1991) DNA Cell . Biol . 10, 181-189} or Met-108 {Bertocci et al . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 1416-1420}.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1995 Apr 3, 14(7), 1430 - 8 Signal transfer through three compartments: transcription initiation of the Escherichia coli ferric citrate transport system from the cell surface; Harle C et al.; Transport of ferric citrate into cells of Escherichia coli K-12 involves two energy-coupled transport systems, one across the outer membrane and one across the cytoplasmic membrane . Previously, we have shown that ferric citrate does not have to enter the cytoplasm of E . coli K-12 to induce transcription of the fec ferric citrate transport genes . Here we demonstrate that ferric citrate uptake into the periplasmic space between the outer and the cytoplasmic membranes is not required for fec gene induction . Rather, FecA and the TonB, ExbB and ExbD proteins are involved in induction of the fec transport genes independent of their role in ferric citrate transport across the outer membrane . The uptake of ferric citrate into the periplasmic space of fecA and tonB mutants via diffusion through the porin channels did not induce transcription of fec transport genes . Point mutants in FecA displayed the constitutive expression of fec transport genes in the absence of ferric citrate but still required TonB, with the exception of one FecA mutant which showed a TonB-independent induction . The phenotype of the FecA mutants suggests a signal transduction mechanism across three compartments: the outer membrane, the periplasmic space and the cytoplasmic membrane . The signal is triggered upon the interaction of ferric citrate with FecA protein . It is postulated that FecA, TonB, ExbB and ExbD transfer the signal across the outer membrane, while the regulatory protein FecR transmits the signal across the cytoplasmic membrane to FecI in the cytoplasm . FecI serves as a sigma factor which facilitates binding of the RNA polymerase to the fec transport gene promoter upstream of fecA.(ABSTRACT TRUNCATED AT 250 WORDS) Gene, 1995 Apr 3, 155(2), 231 - 5 Cloning of a cDNA encoding a human DNA-binding protein similar to ribosomal protein S1; Eklund EA et al.; We report the cloning of a human complementary DNA that encodes a protein which exhibits 36% identity and 62% similarity to Escherichia coli ribosomal protein S1 (rpS1), including conservation of four copies of an RNA-binding domain . This clone was obtained by ligand-screening a lambda gt11 expression library with a DNA probe derived from the CYBB gene promoter . Electrophoretic mobility shift and Southwestern blot assays confirm DNA binding activity of the protein, which exhibits preferential binding to single-stranded and double-stranded DNA and a low binding affinity for RNA . Hence, the rpS1 protein domain previously identified as an RNA-binding motif can also serve as a DNA-binding domain. Gene, 1995 Apr 3, 155(2), 225 - 30 Overproduction and rapid purification of human fast skeletal beta troponin T using Escherichia coli expression vectors: functional differences between the alpha and beta isoforms; Wu QL et al.; Troponin T (TpnT), an essential component of the Ca(2+)-regulatory troponin complex, is involved in protein-protein interactions with other thin-filament proteins during muscle contraction in vertebrate striated muscle (VSM) . The isoforms of TpnT are encoded by members of a multigene family which, by alternate splicing, produces a complex pattern of isoproteins in VSM . The functional domains of TpnT are only tentatively identified and structure-function analysis on this protein is limited due to the heterogeneity of the multiple isoforms . We reasoned that the overproduction and purification of a single TpnT species in Escherichia coli would provide an insight into these studies, besides being useful in crystallizing the protein . We cloned the human fast skeletal beta TpnT-encoding cDNA (beta TpnTf) in three expression vectors . Overexpression was achieved in an E . coli BL21 (DE3) lysogen using a T7 RNA polymerase promoter-based vector, pET17b . The unfused recombinant protein was purified by a simple and rapid procedure in a biologically active and immunoreactive form . This is the first successful synthesis of a complete beta TpnTf polypeptide from any species using an in vitro expression system . Purified human beta TpnTf, a predominant fetal form, was less Ca(2+)-sensitive and exhibited considerably reduced affinity for troponin C and tropomyosin, as compared to the rabbit fast skeletal alpha TpnT, a predominant adult isoform . These results provide a biochemical correlate to the age-related differences in Ca2+ sensitivity of tension development in vertebrate fast skeletal muscles. FEBS Lett, 1995 Apr 3, 362(2), 205 - 9 Sequence specificity for removal of uracil from U.A pairs and U.G mismatches by uracil-DNA glycosylase from Escherichia coli, and correlation with mutational hotspots; Nilsen H et al.; The rate of removal of uracil from different positions in double-stranded DNA by uracil-DNA glycosylase from Escherichia coli varied more than 15-fold . Consensus sequences for good and poor removal were 5'-(A/T)UA(A/T)-3' and 5'-(G/C)U(T/G/C)-3', respectively . In general, the sequence context surrounding U was more important for the rate of removal than whether U was present in U.A pairs or U.G mispairs . Rates of removal of U from sites of amber mutations in the lacI gene, where mutation frequencies and deamination rates were known, indicated that the observed variation in removal is biologically significant. FEBS Lett, 1995 Apr 3, 362(2), 175 - 9 Properties of N-terminus truncated and C-terminus mutated muscle acylphosphatases; Taddei N et al.; Enzymatic activity and structure of N-terminus truncated and C-terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and 1H NMR spectroscopy, respectively . The N-terminus truncated mutant lacked the first six residues (delta 6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C-terminus substituted mutants (R97Q and Y98Q, respectively) . All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in E . coli . The delta 6 deletion mutant elicited a reduced specific activity and a native-like structure . The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg-97 in the stabilisation of the active site correct conformation, most likely via back-bone and side chain interactions with Arg-23, the residue involved in phosphate binding by the enzyme . This study also suggests a possible involvement of Tyr-98 in the stabilisation of the acylphosphatase overall structure. FEBS Lett, 1995 Apr 3, 362(2), 171 - 4 Reassembly of Synechocystis sp . PCC 6803 F1-ATPase from its over-expressed subunits; Steinemann D et al.; Subunits alpha, beta, and gamma of the F1-part of cyanobacterial F0F1-ATPase have been cloned into expression vectors . Over-expressed subunit beta was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts . Recombinant alpha and gamma subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded . The correct folding and functional integrity of the alpha and beta subunits was monitored by their ability to bind nucleotides . Active cyanobacterial F1-ATPase was assembled from its purified subunits alpha, beta, gamma, delta and epsilon . The reassembled enzyme reconstituted ATP synthesis in F1-depleted thylakoid membranes of Synechocystis sp . PCC 6803 and hydrolyzed ATP. EMBO J, 1995 Apr 3, 14(7), 1446 - 52 FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter; Muskhelishvili G et al.; The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase . The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register . Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established . We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon . This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers . The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein-protein interactions . We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly . Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase. J Gen Virol, 1995 Apr, 76 ( Pt 4), 975 - 8 Rice ragged stunt Oryzavirus genome segment 9 encodes a 38 600 Mr structural protein; Uyeda I et al.; The complete nucleotide sequence of rice ragged stunt virus genome segment 9 (S9) was determined . The S9 segment is 1132 nucleotides long and has a long open reading frame starting from the first AUG codon at nucleotide position 14-16 and terminating at a UAG codon located at 1028-1030, which could encode a polypeptide with an Mr of 38 600 (P9) . The encoded polypeptide has no sequence homology to polypeptides of any other plant reoviruses published previously . An immunological study demonstrated that P9 was the smallest of the structural proteins . The P9 polypeptide was expressed as a fusion protein with maltose binding protein in Escherichia coli . Antisera to purified virions and to the fusion protein reacted with both the bacterially expressed polypeptide and the smallest polypeptide of the purified virus in immunoblotting analyses. J Gen Virol, 1995 Apr, 76 ( Pt 4), 907 - 15 Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo; Ritzenthaler C et al.; The putative 38 kDa movement protein (P38) gene, located on the RNA2 of grapevine fanleaf nepovirus (GFLV), was cloned in Escherichia coli and expressed as a fusion protein fused to six histidines (6HisP38) . This protein was purified and used to produce a specific polyclonal antiserum from which immunoglobulins were isolated by immunoaffinity against the recombinant 6HisP38 . Western immunoblot analyses on GFLV RNA2 in vitro maturation products showed that the antibodies were specific for P38 protein . This protein was detected as early as 18 h post-inoculation in GFLV-infected Chenopodium quinoa protoplasts and accumulated to very high levels . Tissue-prints and time course experiments on infected C . quinoa plants confirmed that P38 is present at a high level late in infection and is a final maturation product of the GFLV RNA2 polyprotein in vivo . P94 and P66 intermediates of maturation and polyprotein P2 were also detected in vivo but in very low concentrations . No significant difference was observed in the relative amounts of P66 and P94 detected in vivo, contrary to what occurs in vitro . Subcellular fractionation studies showed that P38, although mainly cytosolic, is also found in association with cell wall and membranes . Thought to be the GFLV movement protein, P38 would thus behave in an 'atypical' manner. J Gen Virol, 1995 Apr, 76 ( Pt 4), 837 - 44 The human immunodeficiency virus type 1 Nef protein functions as a protein kinase C substrate in vitro; Coates K et al.; The human immunodeficiency virus type 1 Nef protein was expressed in Escherichia coli as a C-terminal fusion with glutathione S-transferase (GST) . The ability of GST-Nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified GST-Nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines . In the presence of {gamma32P}ATP, phosphorylation of Nef occurred predominantly on serine residues . Studies with protein kinase inhibitors suggested that protein kinase C (PKC) was involved in Nef phosphorylation . This was supported further by the demonstration that purified PKC was also able to phosphorylate Nef in the absence of cell extract . Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-terminal GST or 6-histidine tag in Spodoptera frugiperda insect cells by recombinant baculoviruses . In extracts from Jurkat T cells and U937 monocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro . Phosphorylation of this Nef-associated protein was inhibited by heparin and is thus likely to be mediated by casein kinase II . The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components. J Gen Virol, 1995 Apr, 76 ( Pt 4), 819 - 26 Mutations in the human papillomavirus type 16 E2 protein identify a region of the protein involved in binding to E1 protein; Storey A et al.; Papillomavirus DNA replication is primarily dependent upon two viral gene products, E1 and E2 . Work with bovine papillomavirus has shown that the E2 protein can bind directly to the E1 protein and enhance the binding of E1 to the viral origin of replication . However, little is known about the mechanism of interaction between E1 and E2 proteins . In this study we have analysed in detail the association between human papillomavirus type 16 (HPV-16) E1 and E2 proteins . Using a purified glutathione S-transferase-HPV-16 E1 fusion protein from Escherichia coli and E2 proteins produced by in vitro transcription-translation, we have developed a rapid and simple method for investigating the association between E1 and E2 in vitro . The binding of E2 to E1 was found to be dependent on sequences in the N-terminal activation domain of the E2 protein . Truncated forms of E2, including a putative repressor form of E2 encoding the DNA binding domain, failed to associate with E1 in this assay . The region of E2 required for efficient binding to E1 was then localized using mutants in the activation domain of E2 . These results demonstrated that only a short region of E2 was required for association with E1 . This region of E2 was found to be highly conserved amongst all papillomaviruses, suggesting a conservation of E2 function and a common mechanism of interaction between these virally encoded proteins. Neurobiol Dis, 1995 Apr, 2(2), 79 - 85 Re-expression of the intermediate filament nestin in reactive astrocytes; Lin RC et al.; The intermediate filament nestin is highly expressed in multipotential stem cells of the developing central nervous system (CNS) . During neuro- and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g . neurofilaments and glial fibrillary acidic protein (GFAP) . In this study, we demonstrate that nestin expression is re-induced in reactive astrocytes in the lesioned adult brain . Following ischaemic and mechanical lesioning, a strong and sustained expression of nestin was noted in GFAP-positive cells surrounding the lesion site . Lesion experiments in transgenic mice carrying the lacZ gene under control of regulatory sequences from the nestin gene suggested that the upregulation of nestin in reactive astrocytes is mediated via the same sequences that control nestin expression during CNS development . These observations and recent data on the co-expression of glial and neuronal marker antigens in reactive astrocytes point to a close relationship between proliferating astrocytes and neuroepithelial precursor cells . Cell Mol Neurobiol, 1995 Apr, 15(2), 283 - 8 Analogue of aspartate and glutamate active at synapses are attractants for Escherichia coli; Lake EM et al.; 1 . This research was carried out to compare Escherichia coli bacteria with animals in their response to L-aspartate and L-glutamate and their analogues . 2 . Various analogues of aspartate and glutamate known to be neurotransmitters at synapses were shown to be attractants for E . coli . 3 . The amino acid sequences of the animal receptors and the bacterial receptor, however, have no detectable relationship . Based on the amino acid sequence, evolutionarily the two systems appear not to be related. J Bacteriol, 1995 Apr, 177(7), 1908 - 10 TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli; Schlosser A et al.; The corrected sequence of the trkH gene of Escherichia coli predicts that the TrkH protein is a hydrophobic membrane protein of 483 amino acid residues, of which 41% are identical to those of the homologous and functionally analogous TrkG protein . These two proteins form the transmembrane component of the Trk system for the uptake of K+ . Each protein alone is sufficient for high-level Trk activity . When Trk is assembled with the TrkG protein, Rb+ and K+ are transported with a Km near or below 1 mM; however, the Vmax for Rb+ is only about 7% of that for K+ . When Trk is formed with TrkH, the affinities for both for K+ and Rb+ are somewhat lower, and the Vmax for Rb+ is only 1% of that for K+ transport . The kinetics of transport in strains with wild-type alleles at trkG and at trkH suggest that both products participate in transport. J Bacteriol, 1995 Apr, 177(7), 1906 - 7 Export of periplasmic galactose-binding protein in Escherichia coli depends on the chaperone SecB; Powers EL et al.; The efficient export of galactose-binding protein to the periplasm of Escherichia coli is shown to be dependent on the presence of the cytosolic chaperone SecB. J Bacteriol, 1995 Apr, 177(7), 1896 - 9 Transcription termination in the Escherichia coli dnaA gene is not mediated by the internal DnaA box; Perez-Roger I et al.; DnaA protein is a DNA-binding protein which recognizes a 9-bp consensus sequence called the DnaA box . By binding to DnaA boxes, DnaA protein regulates initiation of chromosomal replication and transcription of several genes . The dnaA gene contains two DnaA boxes, one located in the regulatory region and one within the structural gene . In this paper, we explore the role of the internal DnaA box in dnaA expression because it has been proposed that the DnaA box-DnaA protein complex can block transcribing RNA polymerase . Firstly, we analyzed the degree of derepression of the dnaA gene, measured as beta-galactosidase activity of a dnaA-lacZ fusion inserted onto the bacterial chromosome, produced by an extra copy number of the dnaA DnaA boxes carried by multicopy plasmids . Secondly, we analyzed repression produced by elevated levels of DnaA protein on single-copy dnaA-lacZ fusions containing or not containing the internal DnaA box . Our results indicate that the internal DnaA box does not play a regulatory role in dnaA expression. J Bacteriol, 1995 Apr, 177(7), 1883 - 7 Enzymatic properties of Escherichia coli peptide deformylase; Meinnel T et al.; Since its discovery in crude extracts in the late sixties, Escherichia coli peptide deformylase activity could not be further characterized because of an apparent extreme instability . We show that this behavior was caused by an inadequate activity assay, involving substrate concentration inhibition and substrate precipitation in crude extracts . The homogeneous protein, as it was previously purified (T . Meinnel and S . Blanquet J . Bacteriol . 175:7737-7740, 1993), had actually retained its initial activity . The influence on the deformylation reaction of several factors was studied and used to improve the activity assay . Pure peptide deformylase proves to act only on peptide substrates with an N-formylmethionyl moiety . In agreement with the occurrence of zinc in the enzyme, peptide deformylase activity is inhibited by 1,10-phenanthroline. J Bacteriol, 1995 Apr, 177(7), 1860 - 3 Differences between inner membrane and peptidoglycan-associated PBP1B dimers of Escherichia coli; Zijderveld CA et al.; Earlier studies revealed that PBP1B of Escherichia coli occurred as a monomeric as well as a dimeric form (C.A.L . Zijderveld, M.E.G . Aarsman, T . den Blaauwen, and N . Nanninga, J . Bacteriol . 173:5740-5746, 1991) . In this study, the dimer of PBP1B was further analyzed . It appeared that the dimeric form could be divided into two classes . One class, which cofractionated with the cell wall fraction, could be artificially cross-linked to peptidoglycan, indicating a close association with the latter . This class of PBP1B dimers was sensitive to beta-mercaptoethanol . The second class, like the monomeric form of PBP1B, could be isolated with the inner membrane fraction . This dimeric form dissociated in the presence of zinc in combination with beta-mercaptoethanol. J Bacteriol, 1995 Apr, 177(7), 1742 - 50 Genetic definition of the Escherichia coli zwf "soxbox," the DNA binding site for SoxS-mediated induction of glucose 6-phosphate dehydrogenase in response to superoxide; Fawcett WP et al.; In Escherichia coli K-12, transcription of zwf, the gene for glucose 6-phosphate dehydrogenase, is subject to growth rate-dependent regulation and is activated by SoxS in response to superoxide stress . To define genetically the site of SoxS activation, we undertook a detailed deletion analysis of the zwf promoter region . Using specifically targeted 5' and 3' deletions of zwf sequences, we localized the SoxS activation site to a 21-bp region upstream of the zwf promoter . This minimal "soxbox" was able to confer paraquat inducibility when placed upstream of a normally unresponsive gnd-lacZ protein fusion . In addition, we used these findings as the basis for resecting unnecessary sequences from the region upstream of the promoters of two other SoxS-regulated genes, sodA and nfo . Like the zwf soxbox, the regions required for SoxS activation of sodA and nfo appear to lie just upstream or overlap the -35 hexamers of the corresponding promoters . Importantly, the sequence boundaries established here by deletion analysis agree with the primary SoxS recognition sites of zwf, sodA, and nfo that we previously identified in vitro by gel mobility shift and DNase I protection assays with a purified MalE-SoxS fusion protein. J Bacteriol, 1995 Apr, 177(7), 1712 - 8 Transcription activation at the Escherichia coli uhpT promoter by the catabolite gene activator protein; Merkel TJ et al.; Transport and utilization of sugar phosphates in Escherichia coli depend on the transport protein encoded by the uhpT gene . Transmembrane induction of uhpT expression by external glucose 6-phosphate is positively regulated by the promoter-specific activator protein UhpA and the global regulator catabolite gene activator protein (CAP) . Activation by UhpA requires a promoter element centered at -64 bp, relative to the start of transcription, and activation by CAP requires a DNA site centered at position -103.5 . This DNA site binds the cyclic AMP-CAP complex in vitro, and its deletion from the promoter reduces transcription activity to 7 to 9% of the wild-type level . Ten uhpT promoter derivatives with altered spacing between the DNA site for CAP and the remainder of the promoter were constructed . Their transcription activities indicated that the action of CAP at this promoter is dependent on proper helical phasing of promoter elements, with CAP binding on the same face of the helix as RNA polymerase does . Five CAP mutants defective in transcription activation at class I and class II CAP-dependent promoters but not defective in DNA binding or DNA bending (positive control mutants) were tested for the ability to activate transcription . These CAPpc mutants exhibited little or no defect in transcription activation at uhpT, indicating that CAP action at uhpTp involves a different mechanism than that which is used for its action at other classes of CAP-dependent promoters. J Bacteriol, 1995 Apr, 177(7), 1692 - 8 Primary products of break-induced recombination by Escherichia coli RecE pathway; Silberstein Z et al.; Alternative models for break-induced recombination predict different distributions of primary products . The double-stranded break-repair model predicts a noncrossover product and equimolar amounts of two crossover products . The one-end pairing model predicts two crossover products, but not necessarily in equimolar amounts, and the single-stranded annealing model predicts deletion of the fragment between the pairing sequences . Depending on the structure of the recombining substrate(s) and the nature of the resectioning step that precedes strand annealing, the single-stranded annealing mechanism would yield only one or both crossover products . We tested these predictions for the RecE recombination pathway of Escherichia coli . Nonreplicating intramolecular recombination substrates with a double-stranded break (DSB) within one copy of a direct repeat were released from chimera lambda phage by in vivo restriction, and the distribution of primary circular recombination products was determined . Noncrossover products were barely detectable, and the molar ratio of the two crossover products was proportional to the length ratio of the homologous ends flanking the DSB . These results suggest an independent pairing of each end with the intact homolog and argue against the double-stranded break-repair model . However, the results do not distinguish alternative pairing mechanisms (strand invasion and strand annealing) . The kinetics of heteroduplex formation and heteroduplex strand polarity were investigated . Immediately following the DSB induction, heteroduplex formation was done by pairing the strands ending 3' at the break . A slow accumulation of the complementary heteroduplex made by the pairing of the strands ending 5' at the break (5' heteroduplexes) was observed at a larger stage . The observed bias in heteroduplex strand polarity depended on DSB induction at a specific site . The 5' heteroduplexes may have been generated by reciprocal strand exchange, pairing that is not strand specific, or strand-specific pairing induced at random breaks. J Bacteriol, 1995 Apr, 177(7), 1670 - 82 Inhibition and restart of initiation of chromosome replication: effects on exponentially growing Escherichia coli cells; Bernander R et al.; Escherichia coli strains in which initiation of chromosome replication could be specifically blocked while other cellular processes continued uninhibited were constructed . Inhibition of replication resulted in a reduced growth rate and in inhibition of cell division after a time period roughly corresponding to the sum of the lengths of the C and D periods . The division inhibition was not mediated by the SOS regulon . The cells became elongated, and a majority contained a centrally located nucleoid with a fully replicated chromosome . The replication block was reversible, and restart of chromosome replication allowed cell division and rapid growth to resume after a time delay . After the resumption, the septum positions were nonrandomly distributed along the length axis of the cells, and a majority of the divisions resulted in at least one newborn cell of normal size and DNA content . With a transient temperature shift, a single synchronous round of chromosome replication and cell division could be induced in the population, making the constructed system useful for studies of cell cycle-specific events . The coordination between chromosome replication, nucleoid segregation, and cell division in E . coli is discussed. Mol Cell Biol, 1995 Apr, 15(4), 1907 - 14 Tetracycline-reversible silencing of eukaryotic promoters; Deuschle U et al.; A tetracycline-controlled transrepressor protein has been engineered to silence transcriptional activities of eukaryotic promoters that are stably integrated into the chromatin of human cells . By fusing the KRAB domain of human Kox1 to the Tet repressor derived from Tn10 of Escherichia coli, a tetracycline-controlled hybrid protein (TetR-KRAB) was generated and constitutively expressed in HeLa cells . The TetR-KRAB protein binds to tet operator (tetO) sequences in the absence but not in the presence of tetracycline . When TetR-KRAB bound to tetO sequences upstream of the immediate-early promoter-enhancer of human cytomegalovirus (CMV), the expression of a CMV-driven luciferase reporter construct (ptetO7-CMV-L) was repressed in transient transfection experiments . This silencing was found to operate on different promoters and from tetO sequences placed more than 3 kb from the transcriptional start site . We constructed a stable, doubly transfected cell line (TIS-10) carrying a chromosomally integrated ptetO7-CMV-L reporter construct and expressing the TetR-KRAB protein . Upon addition of tetracycline, luciferase expression was induced more than 50-fold above the baseline level, with half-maximal induction by 2 days . Furthermore, a protein of around 110 kDa was found to coimmunoprecipitate with the TetR-KRAB fusion protein . This protein might play a role as an adaptor protein mediating the silencing exerted by the TetR-KRAB protein . The TetR-KRAB silencing system should be useful as a genetic switch for regulating the expression of chromosomally integrated heterologous and endogenous genes present in mammalian genomes. J Neurochem, 1995 Apr, 64(4), 1721 - 7 A protease inhibitor of the serpin family is a major protein in carp perimeningeal fluid: II . cDNA cloning, sequence analysis, and Escherichia coli expression; Huang CJ et al.; A cDNA clone, pCP9, has been isolated from a common carp liver cDNA library by immunoscreening with polyclonal antiserum raised against purified bighead carp alpha 1-antitrypsin . This clone is 1,396 bp in length and has an open reading frame encoding a protein of 410 amino acid residues . The deduced amino acid sequence shows moderate homology to human alpha 1-antitrypsin (38%), guinea pig contrapsin (35%), human alpha 1-antichymotrypsin (34%), and human proteinase C inhibitor (31%), all members of the serine protease inhibitor (serpin) family . To confirm further that the cDNA clone was derived from the authentic carp alpha 1-antitrypsin gene, the presumptive mature protein of pCP9 was expressed in Escherichia coli . The molecular mass of the recombinant protein matched that predicted from the nucleotide sequence . This recombinant protein, which was recognized by antiserum against native alpha 1-antitrypsin, was capable of formation of serpin-enzyme complexes with trypsin, chymotrypsin, and elastase . Therefore, we conclude that the protein encoded by the pCP9 clone is indeed carp alpha 1-antitrypsin . Expression of alpha 1-antitrypsin in brain was confirmed by reverse transcription and polymerase chain reaction performed on mRNA derived from both common carp and bighead carp brain. Infect Immun, 1995 Apr, 63(4), 1473 - 7 Independent down-regulation of central and peripheral tumor necrosis factor production as a result of lipopolysaccharide tolerance in mice; Faggioni R et al.; Lipopolysaccharide (LPS) induces a variety of central and peripheral effects that are largely mediated by cytokines, including tumor necrosis factor (TNF) . Peripheral (intravenous {i.v.}) administration of LPS (2.5 micrograms per mouse) induced TNF levels in the serum and spleen but not in the brain, while central (intracerebroventricular {i.c.v.}) administration of LPS induced TNF production both in the brain and in the periphery . Mice challenged with LPS after LPS pretreatment (35 micrograms per mouse, intraperitoneally, as a single dose on day -3 or as a 4-day treatment on days -5 to -2) were unresponsive in terms of induction of serum TNF . When peripherally LPS-tolerant mice (where LPS pretreatment was given intraperitoneally) were challenged with an i.c.v . dose of LPS, brain (but not serum) TNF was still produced, meaning that the LPS-tolerant state was confined to the periphery . However, if LPS pretreatment was given i.c.v . (35 micrograms, as a single dose), the brain, like the periphery, became LPS tolerant in terms of TNF production . We investigated how tolerance to LPS affected two of its actions, decrease in food intake and induction of serum corticosterone (CS) . After an i.v . challenge in peripherally LPS-tolerant mice, no decrease in food intake was observed, but this response was still elicited by an i.c.v . challenge . LPS tolerance reduced the CS response to i.v . and i.c.v . challenge . These results suggest that LPS-induced decrease in food intake might be a fully central effect, while the increase of serum CS might be due to both central and peripheral actions. Infect Immun, 1995 Apr, 63(4), 1394 - 9 Urease-specific monoclonal antibodies prevent Helicobacter felis infection in mice; Blanchard TG et al.; Experiments were performed to determine the antigenic specificity of a monoclonal antibody (immunoglobulin A {IgA} 71) previously demonstrated to neutralize the ability of Helicobacter felis to colonize mice . Immunoprecipitation of radiolabeled H . felis outer membrane proteins with IgA 71 revealed specificity for a 62-kDa protein . Another of our monoclonal antibodies, IgG 40, precipitated a protein of similar molecular weight . IgA 71 but not IgG 40 also precipitated purified recombinant H . pylori urease . The antigenic specificity of both antibodies was confirmed to be urease by the ability of each to select Escherichia coli clones expressing the H . felis urease genes . The two antibodies were shown to bind nonoverlapping epitopes in a competition enzyme-linked immunosorbent assay . Both IgA 71 and IgG 40 could effectively neutralize H . felis infectivity by incubating the bacteria with the antibodies prior to oral administration to naive mice . The mechanism of protection does not appear to be inhibition of urease activity, as IgA 71 does not inhibit the conversion of urea to ammonia by H . pylori urease in vitro . These results support a protective role for the secretory humoral immune response in Helicobacter immunity and provide further evidence that the urease enzyme can serve as a protective antigen. Infect Immun, 1995 Apr, 63(4), 1356 - 61 Identification of an immunologically important hypervariable domain of major outer surface protein A of Borrelia burgdorferi; McGrath BC et al.; The gene for the major outer surface protein A (OspA) from several clinically obtained strains of Borrelia burgdorferi, the cause of Lyme disease, has been cloned, sequenced, and expressed in Escherichia coli by using a T7-based expression system (J . J . Dunn, B . N . Lade, and A . G . Barbour, Protein Expr . Purif . 1:159-168, 1990) . All of the OspAs have a single conserved tryptophan at residue 216 or, in some cases, 217; however, the region of the protein flanking the tryptophan is hypervariable, as determined by a moving-window population analysis of ospA from 15 European and North American isolates of B . burgdorferi . Epitope-mapping studies using chemically cleaved OspA and a TrpE-OspA fusion have indicated that this hypervariable region is important for immune recognition . Biophysical analysis, including fluorescence and circular dichroism spectroscopy, have indicated that the conserved tryptophan is buried in a hydrophobic environment . Polar amino acid side chains flanking the tryptophan are likely to be exposed to the hydrophilic solvent . The hypervariability of these solvent-exposed amino acid residues may contribute to the antigenic variation in OspA . To test this, we have performed site-directed mutagenesis to replace some of the potentially exposed amino acid side chains in the B31 protein with the analogous residues of a Borrelia garinii strain, K48 . The altered proteins were then analyzed by Western blot (immunoblot) with monoclonal antibodies which bind OspA on the surface of the intact B31 spirochete . Our results indicate that specific amino acid changes near the tryptophan can abolish the reactivity of OspA to these monoclonal antibodies, which is an important consideration in the design of vaccines based on recombinant OspA. Infect Immun, 1995 Apr, 63(4), 1263 - 9 Loss of the O4 antigen moiety from the lipopolysaccharide of an extraintestinal isolate of Escherichia coli has only minor effects on serum sensitivity and virulence in vivo; Russo TA et al.; The O-specific antigen in extraintestinal isolates of Escherichia coli is believed to be an important virulence factor . To assess its role in the pathogenic process, proven isogenic derivatives with either a complete (CP921) or nearly complete (CP920) deficiency of the O4 antigen were obtained by TnphoA'1-mediated transposon mutagenesis of an O4/K54/H5 blood isolate (CP9) . By utilizing a previously reported isogenic K54 capsule-deficient derivative (CP9.137), additional isogenic derivatives deficient in both the K54 capsular antigen and either all (CP923) or nearly all (CP922) of the O4 antigen were also constructed . These strains and their wild-type parent were evaluated in vitro for serum sensitivity and in vivo by intraperitoneal challenge of outbred mice . The complete or nearly complete loss of the O4 antigen (CP920 and CP921) resulted in only a minor increase in serum sensitivity . In contrast, CP9.137 had a significant increase in serum sensitivity, and CP922 and CP923 were extremely serum sensitive . When tested in vivo, the complete or nearly complete loss of the O4 antigen resulted in a small but significant increase (P < or = 0.05), not the expected decrease, in virulence compared with its wild-type parent . In contrast, CP9.137 and CP922 were significantly less virulent (P < or = 0.05) . These studies do not exclude a role for the O4 antigen moiety of lipopolysaccharide in the pathogenesis of extraintestinal E . coli infection; however, they demonstrate that the O4 antigen plays only a minor role in serum resistance in vitro and that its loss does not diminish and perhaps enhances the virulence of CP9 in vivo after intraperitoneal challenge. Infect Immun, 1995 Apr, 63(4), 1183 - 7 Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide; Perez-Perez GI et al.; The mechanism by which Helicobacter pylori, which has little or no invasive activity, induces gastric-tissue inflammation and injury has not been well characterized . We have previously demonstrated that water-extracted proteins of H . pylori are capable of activating human monocytes by a lipopolysaccharide (LPS)-independent mechanism . We have now compared activation of macrophages by purified LPS from H . pylori and from Escherichia coli . LPS was prepared by phenol-water extraction from H . pylori 88-23 and from E . coli O55 . THP-1, a human promyelomonocytic cell line, and macrophages derived from rat bone marrow each were incubated with the LPS preparations, and cell culture supernatants were assayed for production of tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2), and nitric oxide . THP-1 cells showed maximal activation by the LPS molecules after cell differentiation was induced by phorbol 12-myristate 13-acetate . Maximal TNF-alpha and PGE2 production occurred by 6 and 18 h, respectively, in both types of cells . In contrast, NO was produced by rat bone marrow-derived macrophages only and was maximal at 18 h . The minimum concentration of purified LPS required to induce TNF-alpha, PGE2, and NO responses in both types of cells was 2,000- to 30,000-fold higher for H . pylori than for E . coli . Purified LPS from three other H . pylori strains with different polysaccharide side chain lengths showed a similarly low level of activity, and polymyxin B treatment markedly reduced activity as well, suggesting that activation was a lipid A phenomenon . These results indicate the low biological activity of H . pylori LPS in mediating macrophage activation. Mutat Res, 1995 Apr, 334(2), 161 - 5 Development of a rat cell line containing stably integrated copies of a lambda/lacI shuttle vector; Wyborski DL et al.; A rat embryo cultured cell line was generated that carries stably integrated copies of a lambda/lacI shuttle vector, containing the lacI gene as a mutational target . After the desired treatment of the cells, this vector can be rapidly and efficiently recovered from the cell DNA by in vitro packaging and then screened for mutations in the lacI gene, using bacterial detection systems . The vector is identical to that integrated into the Big Blue transgenic mouse, which was developed for in vivo mutation analysis . Characterization of the cell line by fluorescence in situ hybridization showed that the phage DNA is integrated at two distinct sites on separate chromosomes at approximately 50-70 copies per cell and the cell line is polyploid . The rescue efficiency is approximately 100,000 pfu/micrograms of genomic DNA . To examine the ability of the cell line to detect mutations in the lacI gene, the cells were treated with 100 micrograms/ml of the direct-acting alkylating agent N-methyl-N-nitrosourea (MNU) for 30 min at 37 degrees C and grown to confluence . The shuttle vector was rescued from untreated and mutagen treated cells, and spontaneous and induced mutant frequencies were determined to be 4.0 x 10(-5) and 92.7 x 10(-5), respectively . The cell line can be used to detect mutations in the lacI gene, followed by recovery of mutants for sequence analysis . The cell line may be valuable for short-term in vitro mutagenesis studies, oncogene and tumor suppressor studies, and DNA repair studies. J Virol, 1995 Apr, 69(4), 2602 - 4 The H4 subunit of vaccinia virus RNA polymerase is not required for transcription initiation at a viral late promoter; Wright CF et al.; Chromatography of RNA polymerase purified from vaccinia virions and from vaccinia virus-infected HeLa cells resulted in the separation of populations active for early and late transcription . An RNA polymerase population immunodepleted for the vaccinia virus H4 gene peptide could support late transcription. J Virol, 1995 Apr, 69(4), 2565 - 73 Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus; Kim HJ et al.; The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV) . To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences . Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system . Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences . The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml . The results of this study are significant in three respects . (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system . (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein . (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field . The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect. J Virol, 1995 Apr, 69(4), 2101 - 9 RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1; Clever J et al.; The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript . Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro . Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative . The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli . In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts . Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag . Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops . Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus . Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo. Nat Struct Biol, 1995 Apr, 2(4), 281 - 6 Bipartite structure of the alpha-lactalbumin molten globule; Wu LC et al.; Molten globules are thought to be general intermediates in protein folding . Apparently conflicting studies have failed to clarify whether one of the best characterized molten globules, that of alpha-lactalbumin, resembles an expanded native-like protein or a nonspecific collapsed polypeptide . Here we show that the molten globule properties of alpha-lactalbumin are largely confined to one of its two domains . The alpha-helical domain forms a helical structure with a native-like tertiary fold, while the beta-sheet domain is largely unstructured . Molten globules thus possess a native-like backbone topology, but this topology does not necessarily encompass the entire polypeptide chain . Our studies indicate that molten globules provide an approximate solution to, and considerable simplification of the protein folding problem. Mol Membr Biol, 1995 Apr-Jun, 12(2), 209 - 15 SecA-dependence of the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein is a function of sequence context; Saaf A et al.; We have analysed the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein when placed in different sequence contexts and under conditions when the function of the SecA protein is inhibited . The results show that the degree of SecA-dependence varies with sequence context: while translocation of the large loop in its normal context is only minimally affected by SecA inhibition, translocation is much more sensitive to SecA inhibition when the loop is placed in the context of other inner membrane proteins . Conversely, when the large MalF loop is replaced by segments from other proteins, translocation of those segments is again very sensitive to SecA inhibition . Thus, SecA-dependence is not an all-or-none phenomenon and is not only a simple function of, e.g . the length of a translocated segment or the hydrophobicity of the flanking transmembrane segments. Liver, 1995 Apr, 15(2), 104 - 9 Endotoxin inactivating action of plasma in patients with liver cirrhosis; Fukui H et al.; The endotoxin inactivating action of plasma was evaluated in 62 patients with cirrhosis and 10 healthy subjects . Endotoxin from E . coli 0111:B4 was added to each plasma sample to a final concentration of 250 pg/ml and the percentage loss of endotoxin activity by incubation (37 degrees C for 1 h) was calculated as the endotoxin inactivating rate . The plasma endotoxin inactivating rate in cirrhotics was significantly greater than that in healthy subjects, although patients with Child C cirrhosis and marked hyperbilirubinemia had a significantly lower endotoxin inactivating rate than other cirrhotics . The plasma endotoxin inactivating rate was positively correlated to serum HDL-cholesterol levels . In patients with Child A and Child B cirrhosis, the endotoxin inactivating rate was positively correlated to the endotoxin binding capacity of plasma albumin . The present results support the assumption that the plasma of cirrhotics has a high endotoxin inactivating capacity . Its decrease may augment endotoxicity in patients with advanced liver cirrhosis. J Photochem Photobiol B, 1995 Apr, 28(1), 87 - 92 Evidence of photoenzymatic repair due to the phrA gene in a phrB mutant of Escherichia coli K-12; Dorrell N et al.; The role of the phrA gene in the genetic control of photoreactivation in Escherichia coli has been a matter of some controversy . It has been proposed that the gene has no significant physiological role in photoreactivation . However, we have previously sequenced a restriction fragment thought to contain the phrA gene and shown it to contain a putative gene . When this gene, termed the putative phrA gene, was transformed into a phrAphrB mutant, a photoreactivable response above that of the phrAphrB mutant was observed . It has been suggested that the photorepair seen in phrB mutants is due to Type III photoreactivation, which is independent of temperature and fluence rate effects . Here we have shown that the photorecovery associated with the phrA gene is dependent on both temperature and fluence rate . This suggests that the photorecovery is not due to Type III photoreactivation but to an enzymatic reaction caused by an unknown photoactive protein, the phrA gene product, which acts on lesions other than pyrimidine dimers, possibly pyrimidine (6-4) pyrimidone photoproducts . We therefore propose that the phrA gene be reaccepted and its role in photoreactivation in Escherichia coli acknowledged. Immunology, 1995 Apr, 84(4), 662 - 8 Characterization of a progesterone-binding, three-domain antibody fragment (VH/K) expressed in Escherichia coli; He M et al.; The heavy chain variable region (VH) and the kappa light chain of the anti-progesterone monoclonal antibody (mAb) DB3, have been expressed as a single-chain three-domain polypeptide, designated VH/K, and secreted into the periplasmic space of Escherichia coli (E . coli) . The linker sequence was derived from the VH-CH1 elbow region . The C kappa domain provides a sensitive detection tail for Western blotting and enzyme-linked immunosorbent assay (ELISA) . Periplasmic extracts of transformed E . coli contained material that bound progesterone and related steroids with similar specificity and affinity to DB3, and displayed the DB3 idiotype and kappa chain epitopes . Reference to the crystal structure of DB3 suggests that all the characteristics of the combining site interaction with steroids are retained in the bacterially expressed material . Western blotting demonstrated material with a molecular weight equivalent to three domains after reduction, but six domains in the unreduced state, suggesting that the VH/K polypeptide is assembled in the periplasm as a disulphide-bridged dimer . The VH/K construct provides a novel route to expression of antibody combining sites in E . coli for antibody engineering. Genetics, 1995 Apr, 139(4), 1533 - 44 Ectopic integration of transforming DNA is rare among neurospora transformants selected for gene replacement; Miao VP et al.; In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules . Such "cotransformants" are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation . We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination . Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid . Transformants were initially selected for hph (hygromycinR), allowed to conidiate to generate homokaryons and then selected for either Am- (gene replacements) or hph . Surprisingly, most am replacement strains were hygromycinS (124/140) and carried no extraneous DNA (116/140) . Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur . To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph . The initial selection did not appreciably help (or hinder) recovery of strains with replacements. Genetics, 1995 Apr, 139(4), 1483 - 94 The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli; Cao Y et al.; The mechanism of recA polA lethality in Escherichia coli has been studied . Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA . An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing . Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I . The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants . The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature . recF+ is essential for this suppression pathway . recJ and recQ mutations have minor but significant adverse effects on the suppression . The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent . lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold . lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type . These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants. Exp Neurol, 1995 Apr, 132(2), 194 - 208 Transgenic neural plate contributes neuronal cells that survive greater than one year when transplanted into the adult mouse central nervous system; Uchida K et al.; Neural plate cells from the early embryo may have a number of important advantages as donor material for the delivery of foreign genes into the diseased adult central nervous system (CNS) . Mesencephalic neural plate from transgenic GT4-2 mice was used as a source of marked donor cells to determine whether transgene-expressing embryonic CNS progenitor cells can be used as donor material for implantation into the adult mouse brain . Transgenic mouse embryos from this line express the Escherichia coli beta-galactosidase (beta-gal) gene throughout early CNS development . At the early somite stage (Embryonic Day 8.5), mesencephalic neural plate tissue from heterozygous embryos was dissected out and either transferred into culture for characterization or immediately implanted into the striatum or lateral ventricle of adult wild-type CD-1 mice . Explants of neural plate tissue possessed intense beta-gal activity and produced extensive outgrowth of neurofilament-positive processes after 6 days in vitro . Many beta-gal-positive cells migrated away from the explanted tissue mass . Grafts of transgenic neural plate tissue in the normal adult mouse striatum, sampled 2 weeks to 1 year after implantation, possessed healthy beta-gal-positive cells . More detailed analysis of grafts 3 months after implantation indicated that most beta-gal-positive cells were also immunoreactive for neurofilament and microtubule-associated proteins, two neuron-specific markers . In addition, extensive neurofilament-positive axonal tangles were evident within the grafts among the beta-gal-positive cells . Electron microscopic (EM) findings of implanted tissue stained with Bluo-Gal revealed many beta-gal-positive neurons received synaptic contacts from other cells . A few donor-derived astrocytes were also found in the grafts by EM analysis . No obvious signs of immunological rejection, or of significant decrease in graft volume, were observed at any age . Some beta-gal-positive cells were observed to lie up to 230 microns away from the main graft mass in both striatal and intraventricular implantations . These data suggest that the neural plate can contribute a long-surviving population of neuronal and astrocytic cells when transplanted into the adult CNS. Plant Mol Biol, 1995 Apr, 28(1), 61 - 72 Narbonin, a novel 2S protein from Vicia narbonensis L . seeds: cDNA, gene structure and developmentally regulated formation; Nong VH et al.; cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced . The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M(r) ca . 33 100) and an initiation methionine . The deduced amino acid sequence lacks a transient N-terminal signal peptide . The genomic clones do not contain any intron . No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries . The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins . However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein . Southern analysis suggests the existence of a small narbonin gene family . Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max . In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds. Biophys J, 1995 Apr, 68(4 Suppl), 180S - 184S; discussion 184S-185S Helicase-catalyzed DNA unwinding: energy coupling by DNA motor proteins; Moore KJ et al.; DNA helicases catalyze the unwinding of double-stranded (ds) DNA to yield the single-stranded (ss) DNA intermediates required in DNA replication, recombination, and repair . DNA helicases couple the free energy of nucleoside triphosphate (NTP) binding and hydrolysis to separate the two complementary DNA strands while also translocating vectorially along the DNA substrate . As such, helicases are functionally DNA motor proteins . The functional form of helicases generally appears to be oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites that are required for translocation and DNA unwinding . The affinity of ss- versus dsDNA for these multiple DNA binding sites is modulated allosterically by NTP binding, hydrolysis, and product release, which is central to helicase-catalyzed DNA unwinding . The mechanistic details of the DNA unwinding, translocation, and NTPase reactions are only starting to emerge . We discuss energy coupling by DNA helicases in general, and by the dimeric E . coli Rep helicase in particular, focusing on the similarities of these enzymes to classical motor proteins. Am J Vet Res, 1995 Apr, 56(4), 481 - 5 Ceftiofur distribution in serum and milk from clinically normal cows and cows with experimental Escherichia coli-induced mastitis; Erskine RJ et al.; Eight Holstein cows, 4 inoculated intracisternally in 1 quarter of the mammary gland with Escherichia coli and 4 noninfected controls, were administered ceftiofur sodium (3 mg/kg of body weight, IV, q 12 hours) for 24 hours, beginning at 14 hours after inoculation of infected cows . All challenge-exposed cows became infected, with mean +/- SEM peak log10 bacterial concentration in milk of 5.03 +/- 0.69 colony-forming units/ml . The infection resulted in systemic signs (mean peak rectal temperature, 41.5 +/- 0.3 C; anorexia; signs of depression) and local inflammation (mean peak albumin concentration in milk, 7.89 +/- 1.71 mg/ml) . Ceftiofur was detectable in milk from all challenge-exposed cows, compared with only 1 of 4 noninfected cows, and the mean period after inoculation that ceftiofur was detectable in milk was longer (P < 0.05) in infected (147.7 +/- 27.5 hours) than noninfected cows (1.3 +/- 1.3 hours) . However, maximal ceftiofur concentration attained in milk for all cows was 0.28 microgram/ml, and was 0.20 microgram/ml or less for all but 2 milk samples collected for 10 days after challenge exposure . Mean serum concentration of ceftiofur peaked at 1.0 +/- 0.3 microgram/ml and 0.7 +/- 0.1 microgram/ml for infected and noninfected cows, respectively . After each ceftiofur dose, mean peak and trough concentrations of ceftiofur in serum did not differ between groups; however, concentration of ceftiofur in serum was higher at 7 hours after each dose in noninfected cows, suggesting more rapid clearance of the drug in infected cows.(ABSTRACT TRUNCATED AT 250 WORDS) Sangre (Barc), 1995 Apr, 40(2), 147 - 51 {Production of specific antibodies against isoforms of the beta subunit of human Na,K-ATPase}; Lecuona E et al.; The study of the differential and functional tissular distribution of the isoforms of Na,K-ATPase, an enzyme located on the plasma membrane of animal cells, must be performed with the aid of antibodies capable of specifically recognising beta 1 and beta 2 subunits . In order to facilitate the study of the functions and distribution of such isoforms, proteins were generated using pET series plasmid construction and transformation in E coli bacteria, and their production was promoted with IPTG, these being the antigens that induce antibodies specific for each isoform . The antisera thus obtained were used in Western Blot assays with tissue microsomes from different animal species; specifically, beta 1 and beta 2 isoforms of Na,K-ATPase could be recognised . A panel of polyclonal antibodies was obtained which proved capable of recognising these isoforms in different animal species, as well as their deglycosylated isoform . Such antibodies can be used as an efficient tool for functional and tissue distribution studies of the two isoforms. Nippon Ika Daigaku Zasshi, 1995 Apr, 62(2), 150 - 60 {Endotoxin-induced lung injury . The roles of leukocytes and oxidants, and the efficacies of steroids and antioxidants}; Matsuda T et al.; Previous work in our laboratory has shown the relation between leukocytes (WBC) and the generation of oxidants in endotoxin (LPS) shock . The purpose of this study was to find out if WBC-derived oxidants can produce acute lung injury in guinea pigs given LPS . We alos evaluated the efficacies of steroids and antioxidants against the initial changes in LPS-induced lung injury . One group of guinea pigs (200-250 g, male) received 0.7 mg/kg (LD50, 24 hrs.) of E . coli LPS in the peritoneal cavity (group I) . The animals in group II received 30 mg/kg of methylprednisolone (MP), followed by intraperitoneal LPS . The animals in group III were given 30 mg/kg of 2-aminomethyl-4-tert-butyl-6-propionylphenol hydrochloride (ONO-3144), a known as antioxidant (OH radical scavenger), and then an injection of LPS . The animals were killed at following time course: 30, 60 or 180 minutes after the LPS injection . Hematological examinations (WBC counts), total cell counts and differential counts in bronchoalveolar lavage (BAL) fluid were done along with light microscopic studies . Superoxide dismutase (SOD) activity, catalase activity and malonaldehyde (MDA) produced as a result of lipid peroxidation in the lung were measured and correlated with histological changes . Survival ratios of the three groups were compared . The results obtained were: 1) Significant leukopenia occurred in all groups . 2) In group I, WBC, especially eosinophils, were recovered by BAL and the total cell number of the BAL fluid had increased by 180 minutes after LPS injection, but MP or ONO-3144 treatment inhibited the migration of WBC (eosinophils and neutrophils) into alveolar lumen after LPS injection . 3) Histologic examinations revealed diffuse edema, hemorrhage, and marked leukocyte infiltration in the alveoli in group I, but not in group II or III . 4) SOD activity in all group diminished below the control level . Catalase activity had significantly increased by 180 minutes after LPS injection in group I, but not in group II or III . MDA had increased remarkably by 60 minutes after injection of LPS in group I, but MP or ONO-3144 treatment prevented such increases . 5) Animals in group II and III survived significantly longer than those in the other group . In conclusion, these findings suggest that LPS provokes WBC-mediated vascular damage in the lung and steroids or antioxidants can minimize the injury and prevent edema formation . Steroids might be useful for achieving quantifiable changes in LPS-induced WBC chemotaxis to the lung and for decreasing oxidant-induced lung injury. Microbiology, 1995 Apr, 141 ( Pt 4), 959 - 60 Filling the gap between hns and adhE in Escherichia coli K12; Danchin A et al.; As a consequence of the absence of a general coordination process in the sequencing of the Escherichia coli K12 genome, to completely sequence the genome in a reasonable time it is important to fill in gaps between known regions . We report the sequence of the hns-adh region, at 27 min on the genome. Microbiology, 1995 Apr, 141 ( Pt 4), 1037 - 44 Biochemical genetics of a natural population of Escherichia coli: seasonal changes in alleles and haplotypes; Pupo GM et al.; The level of diversity, degree of enzyme polymorphism, effective population size, and the relative roles of drift and selection were examined in a cross-section of a natural Escherichia coli population based on random samples of haplotypes of E . coli isolated from sewage . The population studied contained E . coli strains derived from a human population of approximately 16,000 individuals, as well as from other sources . Three sample sets were taken between May and August . Each set consisted of 100 E . coli clones . Six enzyme loci {GPI (5 alleles), GPD (5 alleles), PGD (10 alleles), ADH (8 alleles), IDH (6 alleles), PGM (6 alleles)} were surveyed electrophoretically for each clone; 159 different haplotypes were obtained and it is likely that all possible combinations are present in the population sampled . The large numbers of different haplotypes observed were distributed as a series of four genetically similar families of clones . The large estimated effective population size (Ne = 10(10)) means that the observed large and highly significant changes in allele frequencies with time are not due to genetic drift . Selection, though not necessarily at the loci studied, is considered the only likely explanation. Microbiology, 1995 Apr, 141 ( Pt 4), 1007 - 16 Streptomyces peucetius daunorubicin biosynthesis gene, dnrF: sequence and heterologous expression; Filippini S et al.; The dnrF gene, responsible for conversion of aklavinone to epsilon-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin (Dnr) gene cluster of Streptomyces peucetius ATCC 29050, close to drrAB, one of the anthracycline-resistance genes . The dnrF gene was sequenced and should encode a protein of 489 amino acids with a molecular mass of 52 kDa . The deduced DnrF protein shows significant similarities with bacterial FAD- and NADPH-dependent hydroxylases either required to introduce hydroxyl groups into polycyclic aromatic polyketide antibiotics or involved in catabolism of aromatic compounds . Heterologous expression of dnrF in Streptomyces lividans TK23 and in Escherichia coli demonstrated that the gene encodes a NADPH-dependent hydroxylase catalysing the hydroxylation of aklavinone to yield epsilon-rhodomycinone . The enzyme is inactive on anthracyclines glycosylated at position C-7 and its activity decreases to a different extent with other substrate modifications, indicating that DnrF has a significant substrate specificity. Biosci Biotechnol Biochem, 1995 Apr, 59(4), 756 - 8 Two distinct species of corn cystatin in corn kernels; Abe M et al.; Besides corn cystatin I (CC-I) we found, by cloning, another molecular species of cystatin, named corn cystatin II (CC-II), which was found by screening with CC-I cDNA as a probe . The dissimilarity in amino acid sequence between CC-I and CC-II was distinct around the N-terminal region . Enzymologically, CC-II protein expressed in Escherichia coli was a stronger inhibitor of cathepsin L than CC-I. Biosci Biotechnol Biochem, 1995 Apr, 59(4), 735 - 7 Construction of a plasmid for high level expression of Escherichia coli phosphoenolpyruvate carboxylase; Terada K et al.; The minimum size DNA fragment (3011 bp) containing the entire phosphoenolpyruvate carboxylase {EC 4.1.1.31} gene of E . coli was cloned into a modified plasmid vector of high copy-number . The gene expression was directed by its own promoter and the content of the enzyme reached about 30% of total soluble protein of the transformed cells. Biosci Biotechnol Biochem, 1995 Apr, 59(4), 728 - 30 A DNA region that complements on Escherichia coli cysG mutation in Thiobacillus ferrooxidans; Sugio T et al.; Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space . The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T . ferrooxidans AP19-3 . When pTHS1 was used to transform sulfite reductase deficient E . coli mutants, strain AT2455 (cysG), JM246 (cysI), and AT2427 (cysJ), it complemented only the E . coli cysG mutation . Since cysG codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase is present in a T . ferrooxidans 1.3 kb DNA fragment on pTHS1. Plant Physiol, 1995 Apr, 107(4), 1323 - 32 Maize ribosome-inactivating protein (b-32) . Homologs in related species, effects on maize ribosomes, and modulation of activity by pro-peptide deletions; Hey TD et al.; The ribosome-inactivating protein (RIP) from maize (Zea mays L.) is unusual in that it is produced in the endosperm as an inactive pro-form, also known as b-32, which can be converted by limited proteolysis to a two-chain active form, alpha beta RIP . Immunological analysis of seed extracts from a variety of species related to maize showed that pro/alpha beta forms of RIP are not unique to maize but are also found in other members of the Panicoideae, including Tripsacum and sorghum . Ribosomes isolated from maize were quite resistant to both purified pro- and alpha beta maize RIPs, whereas they were highly susceptible to the RIP from pokeweed . This suggests that the production of an inactive pro-RIP is not a mechanism to protect the plant's own ribosomes from deleterious action of the alpha beta RIP . RIP derivatives with various pro-segments removed were expressed at high levels in Escherichia coli . Measurement of their activity before and after treatment with subtilisin Carlsberg clearly identified the 25-amino acid intradomain insertion, rather than the N- or C-terminal extensions, as the major element responsible for suppression of enzymatic activity . A RIP with all three processed regions deleted had activity close to that of the native alpha beta form. Lett Appl Microbiol, 1995 Apr, 20(4), 240 - 2 Increased resistance of Escherichia coli to acrylic acid and to copper ions after cold-shock; Whiting GC et al.; The effects of cold-shock on the resistance of plasmid-free and plasmid-carrying Escherichia coli to acrylate and copper ions have been tested . Such shock, produced by transfer from 37 to 5 degrees C, with 60 min incubation at the lower temperature, significantly enhanced the resistance of all the tested strains to both inhibitors . Such resistances may have arisen because the inhibitory agents are less able, due to porin changes, to penetrate into the organisms after cold-shock . It is more likely, however, that inhibitor penetration is unaffected but that cold-shocked organisms are better able to repair the damage caused (e.g . to membranes, DNA or cellular enzymes) by the inhibitors. FEMS Microbiol Lett, 1995 Apr 1, 127(3), 187 - 93 Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II; Piedade J et al.; The degradation of mRNA plays a central role in the control of protein synthesis . In Escherichia coli, the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay . We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tetr cassette . This is the first rnb absolute deletion mutant that shows the complete absence of rnb-specific mRNA . This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism. FEMS Microbiol Lett, 1995 Apr 1, 127(3), 175 - 80 The recA gene of Chlamydia trachomatis: cloning, sequence, and characterization in Escherichia coli; Hintz NJ et al.; The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant . The cloned gene restored resistance to methyl methanesulfonate in E . coli recA mutants . The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins . In E . coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings . The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E . coli homologue . As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated. J Submicrosc Cytol Pathol, 1995 Apr, 27(2), 235 - 49 Surface coat of sheep pulmonary intravascular macrophages is reconstituted following brefeldin A-mediated endocytosis; Singh B et al.; Pulmonary intravascular macrophages (PIMs) of sheep have a globular coat on their surface which is mobilized by heparin and halothane, and is implicated in the endocytosis of tracer particles and Escherichia coli lipopolysaccharide . Brefeldin A (BFA) was used in vivo to investigate the effects of a pre-Golgi secretion blocker on the integrity of surface coat, and to know whether PIMs produce coat globules . Sheep were injected intravenously with BFA to reach a one time concentration of 2-5 microgram/ml of plasma, and euthanised at 10, 30, 45, 120 and 180 min (n = 1) post-treatment . Lungs were fixed in situ with 2% glutaraldehyde and 2.5% paraformaldehyde for 30 min . Lung tissues were processed for routine ultrastructural examination including treatment with tannic acid (0.1%), and for the acid phosphatase (ACPase) cytochemistry to identify Golgi complex and enzyme-rich endocytic vesicles . Surface coat was endocytosed by the PIMs within 10 min of BFA treatment through long ACPase-negative endocytic channels and degraded in the acid hydrolase-rich lysosomes . Golgi complex membranes were tubulated and were associated with prominent microtubules, centrioles and secretory vesicles . However, trans-Golgi network was not affected by BFA administration . The coat of PIMs was reconstituted within three hours of BFA-induced endocytosis, in concurrence with signs of enhanced biosynthetic activity . It seems that PIMs do not synthesize the globules, and rather sequester from the plasma to organize a surface coat . It is possible that PIMs contribute a membrane anchor or a receptor to facilitate reconstitution of the coat to perform multiple rounds of globule-mediated cell functions. Plant Cell Physiol, 1995 Apr, 36(3), 435 - 9 Expression in Escherichia coli of the extrinsic 18-kDa protein of photosystem II of spinach; Kuwabara T et al.; A cDNA encoding the precursor for the 18-kDa protein of PSII of spinach was expressed in Escherichia coli . When the cell lysate was incubated at 7 degrees C, the precursor was degraded by proteases of E . coli to a polypeptide of 18 kDa (P18) that consisted of the mature protein moiety plus the last four residues of the transit peptide . P18 was able to reconstitute the water-oxidizing complex of NaCl-treated PSII membranes supplemented with the 23-kDa protein . Moreover, P18 was cleaved by the prolyl endoproteinase of spinach specifically at the Pro-12-Leu-13 bond, as was the authentic 18-kDa protein . These properties of P18 indicate that the present expression system is potentially useful for studies of the substrate specificity of the endoproteinase, as well as of the structure-function relationships of the 18-kDa protein. Eur J Biochem, 1995 Apr 1, 229(1), 91 - 8 Arg21 is the preferred kexin cleavage site in parathyroid-hormone-related protein; Diefenbach-Jagger H et al.; Parathyroid-hormone-related protein (PTHrP) contains several potential sites for proteolytic processing . Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post-translational processing of PTHrP . We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1-141) might be of physiological significance . Cleavage products were identified by N-terminal sequencing . Kex2 preferentially cleaved PTHrP(1-141) carboxy to the triplet arginine site Arg-Arg-Arg21 with a Km of 3.3 +/- 1.7 microM and a kcat of 6 +/- 1.2 s-1 . Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21 . In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(1-34) was lower . No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites . Low levels of cleavage also took place carboxy to Lys-Arg97, Lys-Arg105, Arg-Arg106 and Thr-Arg108 . Cleavage carboxy to Lys-Arg105, the best of these minor sites, occurred with a Km of 8.4 +/- 2.7 microM and a kcat of 0.8 +/- 0.2 s-1 . These studies indicate that the preferred Kex2 cleavage site in PTHrP(1-141) is carboxy to Arg-Arg-Arg21, which effectively destroys its parathyroid-hormone-like biological activity . Cleavage of this site by Kex2-related mammalian convertases in vivo may be an important mechanism for full elaboration of the non-parathyroid-hormone-like paracrine actions of PTHrP in a tissue-specific manner. Eur J Biochem, 1995 Apr 1, 229(1), 83 - 90 Structural domains of streptokinase involved in the interaction with plasminogen; Rodriguez P et al.; Two fragments of recombinant streptokinase, comprising amino acids Val143-Lys293 (17-kDa rSK) or Val143-Lys386 (26-kDa rSK), were cloned and expressed in Escherichia coli, purified to homogeneity and their interactions with plasmin(ogen) were evaluated . Both 17-kDa rSK and 26-kDa rSK bound to plasminogen with a 1:1 stoichiometry and with affinity constants of 3.0 x 10(8) M-1 and 12 x 10(8) M-1, respectively, as compared to 6.3 x 10(8) M-1 for the binding of intact recombinant streptokinase to plasminogen . Binding of 17-kDa rSK to plasminogen-Sepharose was displaced by addition of increasing concentrations of recombinant streptokinase, whereas bound recombinant streptokinase was not displayed by 17-kDa rSK . In equimolar mixtures of plasminogen and 26-kDa rSK, the appearance of amidolytic activity as monitored with a chromogenic substrate, was significantly delayed compared to the equimolar mixture with recombinant streptokinase (60% of the maximal activity after 30 min, compared to maximum activity within < or = 2 min) . In contrast, no amidolytic activity was generated in equimolar mixtures of plasminogen and 17-kDa rSK . Plasminogen was rapidly activated by catalytic amounts (1:100 molar ratio) of recombinant streptokinase (60-70% within 10-15 min), whereas only 4% of the plasminogen was activated within 60 min with 26-kDa rSK, and no plasmin was generated with 17-kDa rSK . Complexes of plasmin with 17-kDa rSK or with 26-kDa rSK were very rapidly inhibited by alpha 2-antiplasmin (apparent second-order inhibition rate constant of approximately 2 x 10(7) M-1 s-1), whereas the complex with recombinant streptokinase was resistant to inhibition . With 26-kDa rSK, inhibition by alpha 2-antiplasmin resulted in dissociation of the complexes and recycling of functionally active 26-kDa rSK to other plasminogen molecules; 17-kDa rSK, in contrast, remained associated with the plasmin-alpha 2-antiplasmin complex . These findings suggest that different regions of the streptokinase molecule are involved in binding to plasminogen, in active-site exposure, and in impairment of the inhibition of plasmin by alpha 2-antiplasmin . Thus, the 17-kDa region spanning Val143-Lys293 in streptokinase mediates its binding to plasminogen but does not induce activation . Furthermore, this region does not interfere with the inhibition of the complex with plasmin by alpha 2-antiplasmin. Eur J Biochem, 1995 Apr 1, 229(1), 194 - 200 The structures of oligosaccharide bisphosphates isolated from the lipopolysaccharide of a recombinant Escherichia coli strai