FEBS Lett, 1995 Oct 23, 374(1), 82 - 4 NADPH-sulfite reductase flavoprotein from Escherichia coli: contribution to the flavin content and subunit interaction; Eschenbrenner M et al.; The flavoprotein component (SiR-FP) of the sulfite reductase of E . coli is an octamer of the 66 kDa alpha subunit . It was shown to be cleaved in two peptide fragments . The 23 kDa fragment has been purified as a polymer of 8-10 subunits . It corresponds to the N-terminal part of the native protein and was shown to contain essentially FMN as cofactor . The 43 kDa fragment is monomeric . It contains exclusively FAD and remains able to catalyze efficiently NADPH-dependent reductions . One can conclude that each alpha-chain of SiR-FP is composed of two distinct domains, one binding FAD and the other FMN and that the FMN-binding domains cooperate for a head-to-head subunit interaction. FEBS Lett, 1995 Oct 23, 374(1), 72 - 6 Reconstitution of the metal-tetracycline/H+ antiporter of Escherichia coli in proteoliposomes including F0F1-ATPase; Someya Y et al.; The tetracycline resistance gene (tetA) was cloned downstream of the lac promoter . When expression of the tetA gene in E . coli cells carrying the lac Iq gene was induced with isopropyl beta-D-thiogalactopyranoside, the tetracycline resistance protein (TetA) was overproduced, amounting to about 30% of the integral cytoplasmic membrane protein . Essentially pure TetA protein could be obtained by solubilization with 1.25% n-octyl-beta-D-glucopyranoside and one-step purification by DEAE Sepharose CL-6B column chromatography . The TetA protein was incorporated into proteoliposomes with F0F1-ATPase . The proteoliposomes exhibited {3H}tetracycline transport dependent on ATP hydrolysis . The specific activity was about 2 nmol/mg protein/min . The proteoliposomes also showed H+ efflux coupled with tetracycline influx . Tetracycline/H+ antiport by proteoliposomes reconstituted with the Ser-65-->Cys mutant TetA protein was inhibited by N-ethylmaleimide . These results proved for the first time that the tetracycline/H+ antiport is only mediated by the TetA protein. Arch Intern Med, 1995 Oct 23, 155(19), 2077 - 84 Adult hemolytic-uremic syndrome . A review of 37 cases; Melnyk AM et al.; BACKGROUND: Adult hemolytic-uremic syndrome is a serious, poorly understood disease with a high and variable mortality . We studied several demographic, clinical, and treatment variables, related them to outcome, and developed a new classification . METHODS: We analyzed data from 37 patients admitted from 1981 to 1991 who fulfilled four criteria (age > 16 years, microangiopathic hemolytic anemia, creatinine level > 150 mumol/L {> 1.7 mg/dL}, and no artificial heart valve) . Three outcome variables were studied (survival vs death, recurrence vs no recurrence, and chronic renal failure vs no chronic renal failure) . RESULTS: Eleven (30%) of the patients died, 10 (27%) needed dialysis, five (14%) developed chronic renal failure, and nine (24%) had recurrent episodes . Patients who presented with colitis did not die or have recurrences, but they developed chronic renal failure as often as other patients . Patients with hemolytic-uremic syndrome secondary to other diseases had the worst survival and the most recurrences . Those without any triggering factor (primary cases) were in between . In multivariate analysis, hemolytic-uremic syndrome secondary to colitis, a higher white blood cell count at admission, and a high maximum mean arterial pressure were associated with good survival prognosis . CONCLUSIONS: The persistence of the trigger of adult hemolytic-uremic syndrome sets the stage for outcome . If the trigger is transient (such as Escherichia coli colitis), the disease will not recur and is rarely lethal . If no trigger is apparent (primary hemolytic-uremic syndrome) or the trigger persists (systemic lupus erythematosus and cancer), the syndrome has a high mortality and often recurs . We suggest a new classification: (1) extrinsic hemolytic-uremic syndrome: (a) toxic, (b) infectious; (2) intrinsic hemolytic-uremic syndrome: (a) primary, (b) secondary . The use of this classification, combined with simple data obtained at presentation and a further division of the cause as transient or persistent and irreversible, may improve the selection of therapy. Int J Cancer, 1995 Oct 20, 64(5), 336 - 41 Characterization of monoclonal antibodies against stromelysin-3 and their use to evaluate stromelysin-3 levels in breast carcinoma by semi-quantitative immunohistochemistry; Santavicca M et al.; Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator . Expression of recombinant ST3 forms in Escherichia coli from cDNA constructs indicated that high levels of expression were achieved when the ST3 pro-domain was deleted . The putative mature form of ST3 thus produced and recovered from bacterial inclusion bodies was used to prepare monoclonal antibodies (MAbs) against ST3 by immunization of BALB/C mice . Ten hybridomas producing MAbs against ST3 were obtained and analyzed for their ability to detect endogenous ST3 in breast cancer and in conditioned media from human fibroblasts . One of these MAbs (5ST-4A9) was found to be suitable for the routine detection of ST3 on breast cancer tissue sections, thus opening the possibility to evaluate ST3 prognostic value in breast cancer using semi-quantitative immunohistochemistry. Cell, 1995 Oct 20, 83(2), 257 - 67 Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs; Liu SH et al.; Clathrin polymerization into a polyhedral vesicle coat drives receptor sorting at cellular membranes during endocytosis and organelle biogenesis . To study clathrin self-assembly, we expressed the C-terminal third of the clathrin heavy chain in bacteria . The recombinant fragment trimerized, bound clathrin light chains, and morphologically resembled the hub domain of the triskelion-shaped clathrin molecule . Self-assembly of recombinant hubs demonstrated a regulatory role for clathrin light chains and for the distal portions of triskelion legs in clathrin coat formation . Deletion mutagenesis of the hub localized a domain mediating light chain binding and clathrin self-assembly and mapped a transferable trimerization domain . These studies define molecular interactions controlling clathrin self-assembly and establish a recombinant system for future analysis. Cell, 1995 Oct 20, 83(2), 237 - 45 Small peptides activate the latent sequence-specific DNA binding function of p53; Hupp TR et al.; Normal cells contain p53 protein in a latent state that can be activated for sequence-specific transcription by low levels of UV radiation without an increase in protein levels . Microinjection of cells with an antibody specific to the C-terminal negative regulatory domain can activate the function of p53 as a specific transcription factor in the absence of irradiation damage, suggesting that posttranslational modification of a negative regulatory domain in vivo is a rate-limiting step for p53 activation . Small peptides derived from the negative regulatory domain of p53 have been used as biochemical tools to distinguish between allosteric and steric mechanisms of negative regulation of p53 tetramer activity . Presented is the development of a highly specific peptide activation system that is consistent with an allosteric mechanism of negative regulation and that forms a precedent for the synthesis of novel low molecular mass modifiers of the p53 response. Cell, 1995 Oct 20, 83(2), 227 - 35 A role for a small stable RNA in modulating the activity of DNA-binding proteins; Retallack DM et al.; The 10Sa RNA, encoded by the E . coli ssrA gene, appears to modulate action of some DNA-binding proteins . When ssrA is inactivated, lacZ expression from the lac operon, as well as galK from a gal operon fused to a phage lambda promoter, is reduced from that observed in bacteria wild-type for ssrA . These differences are not observed if the relevant repressor is inactive, suggesting that in the absence of 10Sa RNA binding of LacI and lambda cI repressors is enhanced . Gel mobility shifts show that 10Sa RNA binds these repressors and that an excess of 10Sa RNA competes for binding of lambda cI with a DNA fragment containing the OR2 repressor-binding sequence . Similar observations were made in studies of the E . coli LexA repressor and phage P22 C1 transcription activator proteins . These results suggest that direct interaction with 10Sa RNA may explain this modulation of protein-DNA interactions. Cell, 1995 Oct 20, 83(2), 219 - 26 Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B . subtilis; Hofmeister AE et al.; We present biochemical evidence for an intercellular signal transduction pathway in B . subtilis . This pathway governs the conversion of the proprotein pro-sigma E to mature transcription factor sigma E . Proteolytic processing is mediated by the membrane protein SpollGA and is triggered by the inferred extracellular signal protein SpollR . A factor in conditioned medium from B . subtilis cells engineered to produce SpollR during growth triggered processing in protoplasts of B . subtilis cells that had been engineered to produce SpollGA and pro-sigma E . The factor was also detected in, and partially purified from, extracts of SpollR-producing cells of E . coli . We speculate that SpollGA is both a receptor and a protease and the SpollR interacts with SpollGA on the outside of the cytoplasmic membrane, activating the intracellular protease domain of SpollGA. J Mol Biol, 1995 Oct 20, 253(2), 358 - 69 6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding; Burgisser DM et al.; 6-Pyruvoyl tetrahydropterin synthase (PTPS) is an enzyme involved in tetrahydrobiopterin biosynthesis, the cofactor for several aromatic amino acid monooxygenases and the nitric oxide synthases . The crystal structure of PTPS was recently solved and showed a homohexameric enzyme composed of a dimer of trimers . A transition metal binding site formed by the three histidine residues 23, 48 and 50 was found in each subunit . We showed by metal analysis and reconstitution of apo-PTPS that Zn(II) was the bound transition metal and responsible for the enzymatic activity . Site-directed mutagenesis of each of these three histidine residues resulted in a complete loss of metal binding and enzymatic activity . The three residues, Cys42, His89 and Glu133, located close to the metal binding site, were previously postulated to be involved in the catalytic reaction . We altered these residues and found a complete loss of enzymatic activity for the mutant C42A . The two mutants, H89N and E133Q, showed 4.3% and 1.3% enzymatic activity, respectively, but had similar KM values for the substrate as compared to wild-type PTPS . Based on these results we propose a model of the substrate fitted into the active site and we described a novel intersubunit catalytic triad motif composed of the amino acid residues Cys42, His89 and Asp88 . Different from most other catalytic triads that catalyse the hydrolysis of an amide or ester bond, the catalytic triad in the active site of PTPS seems to be involved in the deprotonation of the substrate's side-chain carbons . Our model also proposes Zn(II) as the coordination site for the two substrate side-chain hydroxy groups as well as the involvement of Glu133 as putative stereospecific proton server. J Mol Biol, 1995 Oct 20, 253(2), 333 - 46 High-resolution structure of the catalytic domain of avian sarcoma virus integrase; Bujacz G et al.; Retroviral integrase (IN) functions to insert retroviral DNA into the host cell chromosome in a highly coordinated manner . IN catalyzes two biochemically separable reactions: processing of the viral DNA ends and joining of these ends to the host DNA . Previous studies suggested that these two reactions are chemically similar and are carried out by a single active site that is characterized by a highly conserved constellation of carboxylate residues, the D,D(35)E motif . We report here the crystal structure of the isolated catalytic domain of avian sarcoma virus (ASV) IN, solved using multiwavelength anomalous diffraction data for a selenomethionine derivative and refined at 1.7 A resolution . The protein is a crystallographic dimer with each monomer featuring a five-stranded mixed beta-sheet region surrounded by five alpha-helices . Based on the general fold and the arrangement of catalytic carboxylate residues, it is apparent that ASV IN is a member of a superfamily of proteins that also includes two types of nucleases, RuvC and RNase H . The general fold and the dimer interface are similar to those of the analogous domain of HIV-1 IN, whose crystal structure has been determined at 2.5 A resolution . However, the ASV IN structure is more complete in that all three critical carboxylic acids, Asp64, Asp121 and Glu157, are ordered . The ordered active site and the considerably higher resolution of the present structure are all important to an understanding of the mechanism of retroviral DNA integration, as well as for designing antiviral agents that may be effective against HIV. J Mol Biol, 1995 Oct 20, 253(2), 313 - 32 The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein; Aboul-ela F et al.; The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation . Tat is introduced to the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop encoded by the 5' leader sequence found on all HIV-1 mRNAs . We have used multidimensional heteronuclear NMR to determine the structure of the TAR RNA in the presence of the ADP-1 polypeptide, a 37-mer that carries the minimal RNA recognition region of the Tat protein and closely mimics Tat binding specificity . In the presence of a variety of ligands, including ADP-1, related basic peptides and the amino acid derivative argininamide, the bulge region of TAR undergoes a local conformational rearrangement and forms a more stable structure . The structure of TAR in the bound form has been determined from over 1000 NMR-derived constraints . The U23 residue at the 5' end of the bulge is positioned near G26 and A27 in the major groove, rather than stacked on A22 as in the free TAR . U23 and G26 are brought into close proximity by contacts to the guanidinium group and side-chain amide group of a common arginine residue . However, the interaction of this guanidinium group with TAR is not the only source of binding specificity . Besides NOEs to the arginine residue participating in the conformational change, ADP-1 shows additional intermolecular NOEs to TAR, suggesting that there are multiple points of contacts between TAR RNA and residues from the basic and core regions of Tat . These structural results provide important clues towards the identification of small molecular mass and/or peptidomimetic inhibitors of the essential Tat-TAR interaction. J Mol Biol, 1995 Oct 20, 253(2), 304 - 12 Effect of folding on the export of ribose-binding protein studied with the genetically isolated suppressors for the signal sequence mutation; Song T et al.; Ribose-binding protein (RBP) has a bilobate structure and functions in the periplasm of Escherichia coli . Mutations that affect the folding of RBP were isolated as intragenic suppressors for the export-defective signal sequence mutation . Of 13 different mutational changes found in the mature region, 12 were located in the several peptides forming the N-domain, and one in the C-domain . Translocation kinetics of mutant proteins were analyzed by pulse-labeling and chase experiments, showing the recovery of precursor processing in the range of 42 to 70% . Folding properties of seven mutant RBPs purified were investigated in vitro by means of tyrosine fluorescence . The stability of the mutant proteins, estimated by equilibrium analysis in the presence of denaturant, were reduced by 2.1 to 5.1 kcal/mol of changes in free energy of unfolding . All the mutant proteins showed retardation in folding rate by 4.4 to 63-fold compared to wild-type while unfolding was little affected . The only exception was the L129Q that has a change in the C-domain resulting in unstability due to faster unfolding . Our approach took advantage of an involvement of the folding process in protein export, which was genetically employed to dissect the folding pathway of RBP . As a result, amino acid residues that are specifically involved in the folding pathway of RBP were identified . Most of them are concentrated in one of the subdomains, suggesting that the folding event in the N-domain of RBP is crucial in the rate-determining step. J Mol Biol, 1995 Oct 20, 253(2), 266 - 76 Evidence for coupling of folding and function in trp repressor: physical characterization of the superrepressor mutant AV77; Reedstrom RJ et al.; The fine-control of gene expression in the trp repressor system is achieved through the thermodynamic linkage of multiple equilibria involving the trp repressor protein (TR), tryptophan (L-Trp) and DNA . We have undertaken studies of superrepressor mutants of TR as a means of dissecting the coupled equilibria that contribute to repressor function . Unlike all the other tested super-repressors that exhibit differences from wild-type TR DNA binding affinity or stoichiometry, the AV77 superrepressor (an alanine to valine substitution at position 77: AV77TR) has been indistinguishable from TR in vitro . The present studies using a variety of biophysical measurements comparing TR and AV77TR provide strong evidence that the helix-turn-helix (HTH) region of apoTR exists in a partially folded conformation . Far UV CD spectra of the two proteins reveal a 10% increase in helical content for the apoAV77TR compared to apoTR . Moreover, urea denaturation studies demonstrate that apoAV77TR is more stable to denaturation than apoTR . ApoTR binds large amounts of 1,8-ANS, a hydrophobic fluorescence probe used to detect protein folding intermediates, with high affinity, where apoAV77TR exhibits only marginal binding of this ligand . While the tryptophan affinities of the two proteins as measured by titration calorimetry are quite similar, the thermodynamic signatures are distinct, with a much reduced unfavorable entropic contribution for AV77TR . Finally, the allosteric effect of L-Trp on oligomerization is abolished by the AV77 mutation . Taken together these data support previous calorimetric studies implicating coupling of folding and L-Trp binding for TR . Moreover, they are consistent with NMR observations indicating partial disorder in the HTH region of apoTR . Based upon the distinct biophysical properties of TR and AV77TR, we propose a model in which folding of the HTH region accompanies ligand binding in TR . In this model distinct protein-protein interactions of the apo- and holoTR link this conformational change to apparent operator affinities, thereby modulating TR function in vivo. J Mol Biol, 1995 Oct 20, 253(2), 243 - 58 The amino terminal domain of HIV-1 Rev is required for discrimination of the RRE from nonspecific RNA; Daly TJ et al.; The ability of HIV-1 Rev to successfully discriminate between specific Rev-responsive elements (RRE) and nonspecific binding sites in the presence of excess nonspecific RNA was examined using filter binding, gel shift, and gel filtration techniques, using purified M4 Rev mutant protein and endoproteinase Lys-C cleaved wild-type Rev . The M4 Rev displayed a slightly reduced binding affinity to the RRE, as well as a tenfold decrease in its ability to discriminate the RRE from non-specific RNA compared to the wild-type Rev . Gel shift and gel filtration chromotography data also showed decreased ability of the mutant to multimerize in the absence or presence of the RRE . The Lys-C cleaved Rev, which lacks the amino-terminal 20 amino acids of the protein, displayed less ability to discriminate the RRE from nonspecific RNA compared to either the wild-type or the M4 mutant Rev and appeared unable to form protein-protein interactions, yet still bound sense and antisense RNA species with high affinity (Kd was in the nanomolar concentration range) . A 40 amino acid peptide containing the arginine-rich RRE binding domain of Rev was also observed to interact with both the RRE and antisense RNA fragments with a binding constant of about 1 x 10(-9) M . However, the peptide displayed almost no ability to discriminate between the RRE and a comparably sized antisense RRE . The loss in ability to discriminate correct from incorrect binding sites correlates with overall decreases in the alpha-helical character of the protein and perturbations within the amino terminus . The amino terminus of Rev is likely to maintain the conformational integrity of the arginine rich RRE binding domain which is required for specific RNA binding site discrimination or stabilization of specific Rev-RRE interactions. J Biol Chem, 1995 Oct 20, 270(42), 25127 - 32 Functional analysis of the propeptide of subtilisin E as an intramolecular chaperone for protein folding . Refolding and inhibitory abilities of propeptide mutants; Li Y et al.; The amino-terminal propeptide, consisting of 77 amino acid residues, is known to be required as an intramolecular chaperone to guide the folding of mature subtilisin E, a serine protease, into active mature enzyme . Many mutations within the pro-sequence have been shown to abolish the production of active subtilisin E (Kobayashi, T., and Inouye, M . (1992) J . Mol . Biol . 226, 931-933) . Here we report characterization, refolding, and inhibitory abilities of six single amino acid substitution mutations (Ile-67-->Val, Ile-48-->Thr, Gly-44-->Asp, Lys-36-->Glu, Ala-30-->Thr, and Pro-15-->Leu) and a nonsense mutation (N59-mer) at the codon for Lys-18 . These mutant propeptides were expressed in Escherichia coli using a T7 expression system and were purified to homogeneity . Surprisingly, Lys-36-->Glu, Ala-30-->Thr and Pro-15-->Leu were found to still function as a chaperone for in vitro refolding of denatured subtilisin BPN' with 60, 80, and 54% efficiency compared to the wild-type propeptide, respectively . The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively . The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively, almost identical to the Ki value exhibited by the wild-type propeptide (1.4 x 10(-9) M) . In contrast, Ile-67-->Val and Gly-44-->Asp were able to refold denatured subtilisin BPN' with only 18 and13% efficiencies and had Ki values of 10 and 11 x 10(-9) M, respectively . The Ile-48-->Thr mutant propeptide was unable to refold denatured subtilisin BPN' and gave a 100-fold higher Ki (118 x 10(-9) M) than the wild-type propeptide . The N59-mer propeptide extending from Leu-19 to Met-78 was unable to function as a chaperone . Like the wild-type propeptide, none of the mutant propeptides had secondary structures as judged by their circular dichroism spectra . The present results demonstrate that the ability of the propeptide as a chaperone to refold the denatured protein is well correlated with its ability as a competitive inhibitor for the active enzyme . This supports the notion that the secondary and tertiary structures of the propeptide are identical or highly homologous between the renatured propeptide-subtilisin complex and the inhibitory complex formed between the propeptide and the active enzyme. J Biol Chem, 1995 Oct 20, 270(42), 24884 - 90 The ligand binding domain of the human retinoic acid receptor gamma is predominantly alpha-helical with a Trp residue in the ligand binding site; Lupisella JA et al.; Retinoic acid exerts its many biological effects by interaction with a nuclear protein, the retinoic acid receptor (RAR) . The details of this interaction are unknown due mainly to the lack of sufficient quantities of pure functional receptor protein for biochemical and structural studies . We have recently subcloned the D and E domains of human RAR gamma for expression in Escherichia coli . Using nickel-chelation affinity chromatography with a polyhistidine amino-terminal tail, purification of the DE peptide with a pI of 5.18 was accomplished to greater than 98% purity . Scatchard analysis and fluorescence quenching techniques using the purified protein indicate a very high percentage of functional molecules ( > 95%) with a Kd for retinoic acid (t-RA) of 0.6 +/- 0.1 nM . Circular dichroism spectra of the purified domains predict a predominantly alpha-helical structure (approximately 56%) with little beta sheet present . No significant changes in these structural characteristics were observed upon binding of t-RA . Inspection of the amino acid sequence within these domains identified a single tryptophan residue at position 227 . Modeling the amino acid sequence in this region as an alpha-helical structure indicates that this tryptophan is adjacent to alanine 234, which corresponds to alanine 225 in RAR beta that has previously been linked to the ligand binding site . Fluorescence of this tryptophan was quenched in a dose-dependent manner on the addition of t-RA, confirming that Trp-227 is within the ligand binding site . Tryptophan fluorescence quenching analysis also demonstrates that a single retinoic acid molecule is bound per receptor and suggests that receptor-ligand interactions occur within the amino-terminal portion of the predominantly alpha-helical ligand binding domain. J Biol Chem, 1995 Oct 20, 270(42), 24674 - 7 Identification of a gene encoding a yeast histone H4 acetyltransferase; Kleff S et al.; A collection of yeast temperature-sensitive mutants was screened by an enzymatic assay to find a mutant defective in the acetylation of histone H4 . The assay used a fractionated cell extract and measured acetylation of a peptide corresponding to amino acids 1-28 of H4 . There are at least two activities in this fraction that acetylate the peptide . A mutation, hat1-1, that eliminates one of the activities was identified and mapped to a locus near the centromere of chromosome XVI . The HAT1 gene was cloned and found to encode a protein of 374 amino acids . Analysis of the peptide used in the assay demonstrated that the HAT1 enzyme acetylates lysine 12 of histone H4 . hat1 mutants have no obvious growth defects or phenotypes other than the enzyme defect itself . The HAT1 protein expressed in Escherichia coli gave histone acetyltransferase activity in vitro, demonstrating that HAT1 is the structural gene for the enzyme. J Biol Chem, 1995 Oct 20, 270(42), 24658 - 61 Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase; Lambalot RH et al.; Holo-acyl carrier protein synthase (ACPS) transfers the 4'-phosphopantetheine (4'-PP) moiety from coenzyme A (CoA) to Ser-36 of acyl carrier protein (ACP) in Escherichia coli . This post-translational modification renders holo-ACP capable of acyl group activation via thioesterification of the cysteamine thiol of 4'-PP . We have purified E . coli ACPS to near homogeneity by exploiting the ability to refold ACPS and reconstitute its activity after elution from an apo-ACP affinity column under denaturing conditions . N-terminal sequencing of ACPS allowed us to identify dpj, an essential gene of previously unknown function, as the structural gene for ACPS . We report herein the 70,000-fold purification of wild-type ACPS and the overproduction and initial characterization of recombinant ACPS from E . coli. Arch Biochem Biophys, 1995 Oct 20, 323(1), 79 - 86 Recombinant human dihydroorotate dehydrogenase: expression, purification, and characterization of a catalytically functional truncated enzyme; Copeland RA et al.; An N-terminally truncated cDNA for human dihydroorotate dehydrogenase (DHODase) was placed under the control of the inducible T7 lac promoter in a pyrimidine auxotrophic strain of Escherichia coli lacking the endogenous enzyme . Induction of gene expression rescued growth in media lacking exogenous pyrimidines . The recombinant enzyme was purified to homogeneity from detergent extracts of bacterial membranes by two chromatographic steps . The purity of the resulting enzyme was judged to be > 95% based on SDS-PAGE with Coomassie staining . The enzyme displays an apparent molecular weight of ca . 40 kDa on SDS-PAGE and ca . 120 kDa on native size-exclusion chromatography, suggesting that the native enzyme is multimeric . Recombinant DHODase displayed a specific activity and Km for dihydroorotate that were similar to those for the enzymes from bovine and human liver tissue . The pH dependence of the activity of the recombinant enzyme was likewise similar to that of the enzyme from human liver and may indicate the involvement of a critical histidine residue in catalytic turnover; only eight histidine residues remain in the truncated version of DHODase used here . The catalytic activity of the recombinant enzyme is inhibited in a dose-dependent fashion by the histidine-selective modifying agent diethylpyrocarbonate . These results further suggest a potential role for histidine in enzyme turnover . Brequinar sodium, an experimental drug which has been shown to be a nanomolar noncompetitive inhibitor of mammalian DHODases, inhibited the activity of the purified recombinant enzyme with a Ki value similar to that for enzyme derived from human liver tissue . The recombinant DHODase thus displays enzymatic behavior similar to the 50-kDa full-length human liver enzyme, illustrating that the catalytically essential structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enzyme that was expressed here. Arch Biochem Biophys, 1995 Oct 20, 323(1), 19 - 26 Sites of electron transfer to membrane-bound copper and hydroperoxide-induced damage in the respiratory chain of Escherichia coli; Rodriguez-Montelongo L et al.; Previous studies in Escherichia coli as a model system for peroxide toxicity (L . Rodriguez-Montelongo, L . C . De la Cruz-Rodriguez, R . N . Farias, and E . M . Massa, 1993, Biochim . Biophys . Acta 1144, 77-84) have shown that electron flow through the respiratory chain supports a membrane-associated Cu(II)/Cu(I) redox cycle involved in irreversible impairment of the respiratory system by tert-butyl hydroperoxide (t-BOOH) . In this paper, E . coli mutants deficient in specific respiratory chain components have been used to determine the sites of copper reduction and the targets inactivated by t-BOOH . Two sites of electron transfer to membrane-bound copper were identified: one in the region between NADH and ubiquinone supported by NADH as electron donor and another localized between ubiquinone and the cytochromes supported by electrons coming from NADH, succinate, or D-lactate . Electron flow through the former site in the presence of t-BOOH led to inactivation of NADH dehydrogenase II, whereas electron flow through the latter site in the presence of the hydroperoxide led to damage of ubiquinone . In agreement with the above in vitro results with isolated membranes, copper-dependent inactivation of NADH dehydrogenase and ubiquinone was demonstrated in E . coli cells exposed to t-BOOH . It is proposed that the t-BOOH-induced damage is a consequence of t-butylalkoxy radical generation through a Fenton-type reaction mediated by redox cycling of membrane-bound copper at those two loci of the respiratory chain. Arch Biochem Biophys, 1995 Oct 20, 323(1), 164 - 8 Purification and characterization of a fully active recombinant tobacco cytosolic NADP-dependent isocitrate dehydrogenase in Escherichia coli: evidence for a role for the N-terminal region in enzyme activity; Galvez S et al.; The recently isolated full-length NADP-dependent isocitrate dehydrogenase (ICDH) cDNA encoding the tobacco cytosolic isoenzyme has been cloned into the expression vector pET8c and used to transform Escherichia coli strain BL21 (DE3) . The recombinant protein was purified to electrophoretic homogeneity and used to raise polyclonal antibodies . Its kinetic properties were found to be identical to those of the cytosolic ICDH isoenzyme purified from tobacco cell cultures . The recombinant and the endogenous bacterial ICDH could be easily distinguished by their different behaviors during anion-exchange column chromatography and immunological response . An incomplete ICDH-encoding cDNA clone, encoding a protein lacking the first 36 amino acids at the N-terminus, was cloned into the expression vector pKK233-2 and used to transform ICDH-lacking E . coli cells (strain 2004) . The truncated, recombinant ICDH produced by the bacteria was found to be inactive. Arch Biochem Biophys, 1995 Oct 20, 323(1), 155 - 63 Expression of spinach nitrite reductase in Escherichia coli: site-directed mutagenesis of predicted active site amino acids; Bellissimo DB et al.; Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled {Fe4S4}-siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia . An expression system which produced enzymatically active spinach nitrite reductase (NiR) in Escherichia coli was developed in order to study the structure-function relationships of the coupled active site using site-directed mutagenesis . The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a beta-galactosidase fusion containing five additional amino acids at the N-terminus . The expressed NiR in aerobic cultures was mostly insoluble and inactive . After optimizing growth conditions, active NiR represented 0.5-1.0% of the total protein . E . coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis . The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis . The absorption spectrum of the E . coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A278/A386 = 1.9 . The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum . The specific activity for the methyl viologen-dependent reduction of nitrite of E . coli-expressed NiR was 100 U/mg and the Km determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme . These results indicate that the E . coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state.(ABSTRACT TRUNCATED AT 250 WORDS) Virology, 1995 Oct 20, 213(1), 97 - 108 Molecular biological characterization of the human foamy virus reverse transcriptase and ribonuclease H domains; Kogel D et al.; Foamy viruses form a separate group of retroviruses encoding a pol protein with at least four domains based on comparative sequence alignments . The polymerase and ribonuclease H domains of the human foamy virus (HFV) pol gene were expressed in Escherichia coli either individually or in combination . The histidine-tagged HFV fusion proteins were subsequently purified to near homogeneity by affinity Ni2+ chelate column chromatography . The polymerase and RNase H activities were characterized by performing conventional DNA polymerase and ribonuclease H assays and in situ gel assays . Six purified recombinant HFV proteins were enzymatically active either individually as DNA polymerase and ribonuclease H or as combined domains . The HFV enzymatic activities were characterized with respect to cation preferences and pH optima . Western blots with antibodies against the RNase H domain, in situ reverse transcriptase (RT), and RNase H gel assays showed that in HFV-infected cells pol proteins of 120 and 80 kDa were detectable . A novel activity band of 60 kDa was found in situ RT gel assays . Recombinant RNase H protein additionally purified by fast performance liquid chromatography was capable of removing the primer for minus-strand DNA synthesis when labeled tRNA(Lys1,2) model substrates were used . Specific cleavages occurred at the phosphodiester bonds one to three nucleotides 5' of the RNA-DNA junction . The results revealed biochemical properties of the HFV pol gene products that define functional domains of the HFV pol gene that are distinct but comparable to other retroviruses. Virology, 1995 Oct 20, 213(1), 46 - 56 Characterization of DNA binding properties of the immediate-early gene product of equine herpesvirus type 1; Kim SK et al.; The equine herpesvirus type 1 (EHV-1) immediate-early (IE) gene encodes a phosphoprotein that is essential for the activation of transcription from viral early and late promoters and that regulates the transcription from its own promoter . Employment of EHV-1 IE promoter DNA probes and glutathione S-transferase fusion proteins harboring truncated portions of the IE gene product in gel shift assays, super shift assays with anti-IE monoclonal antibodies, and DNase I footprinting analyses revealed: (1) amino acid residues 422 to 597 within the 1487-amino-acid IE protein are sufficient for sequence-specific DNA binding; (2) the IE protein binds to EHV-1 DNA at sequences from -11 to +14 that overlap the transcription initiation site (+1); (3) the conserved pentanucleotide 5'-ATCGT-3' in the IE promoter located at nucleotides (nt) -6 to -2, relative to the transcription initiation site (+1), is critical for IE protein binding; (4) a weak binding site for the IE protein is also present at nt -92 to -82 of the IE gene within the sequence (-86)ATCGA(-82) in which four of the five nt in the consensus binding sequence are conserved; (5) the IE protein binds to sequences in EHV-1 early and late promoters that contain a degenerate version of the consensus sequence 5'-ATCGT-3'; and (6) mutation of the C or G nt in the pentanucleotide 5'-ATCGT-3' prevents sequence-specific binding of the IE protein, whereas mutation of each of the other three nt only reduces binding . These results suggest that the IE protein can recognize the sites which differ slightly from the proposed consensus sequence . Overall, these findings suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene . In addition, the finding that the DNA binding domain of the IE protein maps within amino acids 422 to 597, a domain conserved in the IR2 early protein that is a truncated form of the IE protein, suggests that the IR2 protein plays a role in the regulation of the IE gene expression. Biochim Biophys Acta, 1995 Oct 19, 1269(1), 19 - 24 The influence of antioxidants and cycloheximide on the level of nitric oxide in the livers of mice in vivo; Mikoyan VD et al.; When injected into mice prior to the NO generation increase induced with lipopolysaccharide (LPS) from Escherichia coli, exogenous antioxidants diethyldithiocarbamate (DETC) or phenazan (sodium 3.5-di-tert-butyl-4-oxiphenylpropionate) as well as the inhibitor of protein biosynthesis, cycloheximide (CHI) attenuated the NO production in mouse liver in vivo . These data demonstrated the key role of free radicals, which were likely, active oxygen species, in the synthesis of inducible NO-synthase (iNOS) responsible for the NO production in this organ . Similar effects of phenazan and CHI were observed in livers of mice treated with gamma-irradiation or LPS + Fe(2+)-citrate, which suggested that these treatments also induced 1NOS synthesis through initiating the action of active oxygen species . The rate of NO synthesis was estimated by accumulation of paramagnetic mononitrosyl iron complexes with DETC (MNIC-DETC) detected using the EPR method . The formation of MNIC-DETC complexes was found in the brain of mice pre-treated with LPS + Fe(2+)-citrate which seemed to be due to iNOS synthesis stimulated by this treatment. Nature, 1995 Oct 19, 377(6550), 627 - 30 Asymmetric segregation of the homeodomain protein Prospero during Drosophila development; Hirata J et al.; Asymmetric divisions that produce two distinct cells play fundamental roles in generating different cell types during development . In the Drosophila central nervous system, neural stem cells called neuroblasts divide unequally into another neuroblast and a ganglion mother cell which is subsequently cleaved into neurons . Correct gene expression of ganglion mother cells requires the transcription factor Prospero . Here we demonstrate the asymmetric segregation of Prospero on neuroblast division . Prospero synthesized in neuroblasts is retained in the cytoplasm and at mitosis is exclusively partitioned to ganglion mother cells, in which it is translocated to the nucleus . Differential segregation of Prospero was also found in the endoderm . We have identified a region in Prospero that is responsible for this event . The region shares a common motif with Numb, which also shows unequal segregation . We propose that asymmetric segregation of transcription factors is an intrinsic mechanism for establishing asymmetry in gene expression between sibling cells. Biochim Biophys Acta, 1995 Oct 19, 1245(2), 181 - 6 The role of nitric oxide in hemodynamic and metabolic alterations induced by prostaglandin F2 alpha in the perfused rat liver; Weidenbach H et al.; In the liver prostaglandins have been shown to be potent regulators of portal blood flow, carbohydrate metabolism and bile secretion . It is not known whether these effects represent a direct action of prostaglandins, and it has been suggested that nitric oxide (NO) might be a critical mediator for prostaglandin induced hepatic events . We have studied whether nitric oxide formation or inhibition alters the action of prostaglandin F2 alpha (PG F2 alpha) in a single-pass liver perfusion model . The liver of untreated rats (constitutive NO-synthase) or after pretreatment with endotoxin (inducible form of NO-synthase) was perfused at a constant pressure via the portal vein . Effluate were collected in 1-min intervals and bile in 5-min intervals . In both groups the addition of PG F2 alpha (10 microM) to the perfusate for 5 min resulted in a significant increase of glucose and lactate production, and in a significant decrease in portal blood flow (-0.56 +/- 0.04 ml/g per min), in bile flow (-60.7%) and in bile acid release (-60.6%) . Inhibition of NO synthase by adding NG-monomethyl-L-arginine (L-NMMA, 100 microM) to the perfusate did not affect any of the alterations induced by PG F2 alpha . Substitution of the endogenous substrate for the NO synthase L-arginine (500 microM) in the perfusate completely prevented the hemodynamic alterations induced by PG F2 alpha in endotoxin pretreated livers and limited the flow reduction (0.15 +/- 0.04 ml/g per min) in the untreated group . The substitution of L-arginine in the perfusate of endotoxin pretreated livers raised nitrite (from 1.5 +/- 0.3 to 3.6 +/- 0.7 nmol/g per min) and urea release (from 65 +/- 25 to 294 +/- 68 nmol/g per min), but had no effect on any of the other metabolic parameters and bile secretion . We conclude that PG F2 alpha increases glucose and lactate production in the perfused rat liver and decreases portal flow bile secretion . The metabolic effects induced by PG F2 alpha appear to be independent of NO mediation and hemodynamic alterations . Portal flow alone can be influenced by endogenous NO formation. Biochemistry, 1995 Oct 17, 34(41), 13627 - 34 {125I}margatoxin, an extraordinarily high affinity ligand for voltage-gated potassium channels in mammalian brain; Knaus HG et al.; Monoiodotyrosine margatoxin ({125I}MgTX) specifically and reversibly labels a maximum of 0.8 pmol of sites/mg of protein in purified rat brain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions . This Kd value was confirmed by kinetic experiments (Kd of 0.07 pM), competition assays employing native margatoxin (MgTX) (Ki of 0.15 pM), and receptor saturation studies (Kd of 0.18 pM) . Thus, this toxin represents the highest affinity, reversible radioligand for any membrane-bound receptor or ion channel described to date . {125I}MgTX binding in this system is modulated by charybdotoxin (Ki of 5 pM), kaliotoxin (Ki of 1.5 pM), and the agitoxins I and II (Ki's of 0.1 and 0.3 pM, respectively), in a noncompetitive manner . Moreover, alpha-dendrotoxin displayed a Ki value of 0.5 pM . Iberiotoxin was without any effect, suggesting that the receptor site is likely to be associated with a voltage-gated K+ channel complex . {125I}MgTX binding is inhibited by cations that are established blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+) . The monovalent cations Na+ and K+ stimulate binding at low concentrations before producing complete inhibition as their concentrations are increased . Stimulation of binding results from an allosteric interaction that decreases Kd, whereas inhibition is due to an ionic strength effect . Affinity labeling of the binding site in rat brain synaptic plasma membranes employing {125I}MgTX and the bifunctional cross-linking reagent, disuccinimidyl suberate, causes specific and covalent incorporation of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74,000 . Deglycosylation studies reveal an M(r) for the core polypeptide of the MgTX receptor of 63,000.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Oct 17, 34(41), 13622 - 6 Interaction of substrate and effector binding sites in the ArsA ATPase; Zhou T et al.; The ars operon of plasmid R773 confers resistance to antimonials and arsenicals in Escherichia coli by encoding an ATP-dependent extrusion system for the oxyanions . The catalytic subunit, the ArsA protein, is an ATPase with two nucleotide binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein . The ArsA ATPase is allosterically activated by tricoordinate binding of As(3+) or Sb(3+) to three cysteine thiolates . Previous measurements suggested that the intrinsic fluorescence of tryptophans might be useful for examining binding of Mg2+ ATP and antimonite . In the present study an increase in intrinsic tryptophan fluorescence was observed upon addition of Mg2+ ATP . This enhancement was reversed by addition of antimonite . The ArsA protein contains four tryptophan residues: Trp159, Trp253, Trp522, and Trp524 . The first two were altered to tyrosine residues by site-directed mutagenesis . Cells expressing both the arsAW159Y and arsAW253Y mutations retained resistance to arsenite, and the purified W159Y and W253Y proteins retained ATPase activity . While the intrinsic tryptophan fluorescence of the W253Y protein responded to addition of Mg2+ ATP, intrinsic tryptophan fluorescence in the purified W159Y protein was no longer enhanced by substrate . These results suggest that Trp159 is conformationally coupled to one or both of the nucleotide binding sites and provides a useful probe for the interaction of effector and substrate binding sites. Biochemistry, 1995 Oct 17, 34(41), 13582 - 93 Interaction of the UvrABC nuclease system with a DNA duplex containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE; Zou Y et al.; Oligonucleotides containing site-specifically-modified N2-guanine (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments . These substrates were used in reactions with the Escherichia coli UvrABC nuclease system . The interaction of the UvrA2 and UvrA2B complexes with these four stereoisomers was probed using DNase I footprinting and gel mobility shift assays . DNase I digestion of substrates containing each stereoisomer of BPDE displayed a unique pattern which was consistent with the known structure of these DNA adducts . UvrA and UvrA2B appeared to interact very similarly with all four substrates . Binding of UvrA2 to these substrates produced a 33-bp footprint, and the UvrB--DNA complex resulted in footprint of 24 bp . The UvrABC nuclease system produced bimodal incisions at the eighth phosphate 5' and the fifth, sixth, or seventh phosphate 3' to the modified guanine . The variation of the 3' incision site was linked to the stereochemistry and orientation of the BPDE adduct . For example, the 3' incision of the 50-bp duplex containing (-)-trans-BPDE-N2-guanine was inhibited at the fifth phosphate . UvrABC nuclease incision kinetics revealed a hierarchy of specificity . The intercalative cis isomers were incised more efficiently than the corresponding trans isomers which lie in the minor groove . The (+) enantiomers were incised more efficiently than the (-) form for both cis and trans isomers . These observations reveal that UvrABC nuclease recognition and incision are directly influenced by the conformation of the DNA adduct. Biochemistry, 1995 Oct 17, 34(41), 13545 - 53 The major, N2-Gua adduct of the (+)-anti-benzo{a}pyrene diol epoxide is capable of inducing G-->A and G-->C, in addition to G-->T, mutations; Jelinsky SA et al.; Mutations induced by the (+)-anti-diol epoxide of benzo{a}pyrene {(+)-anti-B{a}PDE} were collected in the supF gene of the Escherichia coli plasmid pUB3 . pUB3 was reacted with (+)-anti-B{a}-PDE and then either (1) transformed immediately into E . coli or (2) heated at 80 degrees C for 10 min and then cooled prior to transformation--the latter to probe mechanism {Rodriguez & Loechler (1993) Biochemistry 32, 1759} . Qualitatively, heating did not affect the mutagenic pattern, except at the major base substitution hotspot in supF, G115, where principally G-->T mutations were obtained prior to heating, while after heating, G-->A and G-->C mutations became statistically significantly more prevalent . Several studies have suggested that a heat-induced chemical transformation of a (+)-anti-B{a}PDE adduct at G115 (e.g., into an apurinic site) is not likely to explain the change in mutational pattern . The most likely model is that (+)-anti-B{a}P-N2-Gua is initially trapped in a metastable conformation giving principally G-->T mutations, while heating induces a change to a stable conformation(s) resulting in G-->T, A, and C mutations . This suggests that adduct conformational complexity is at the root of adduct mutational complexity . To investigate this model, a plasmid (B{a}P-G115-pRE1) with (+)-anti-B{a}P-N2-Gua in the G115 sequence context is constructed using adduct site-specific techniques.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Oct 17, 34(41), 13491 - 501 Methionine-393 is an axial ligand of the heme b558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli; Kaysser TM et al.; The cytochrome bd oxidase is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli . The complex is composed of two subunits (I and II) and three heme prosthetic groups (heme b558, heme b595, and a chlorin, called heme d) . Both subunits are located within the bacterial cytoplasmic membrane, and each has multiple putative transmembrane helices . Heme b558 is a six-coordinate, low-spin heme component of the oxidase which has been shown to be contained within subunit I and has been implicated in the oxidation of the substrate, ubiquinol-8, in the cytoplasmic membrane . Previous site-directed mutagenesis studies identified His186, predicted to be near the periplasmic side of transmembrane helix D of subunit I, as one of the axial ligands of heme b558 . Since mutagenesis of none of the other histidines in subunit I perturbs heme b558, it was concluded that this heme cannot have bis(histidine) ligation . In this work, the properties of 14 mutants are reported, including substitutions for each of 10 methionine residues within subunit I . Among this set of mutants, only the replacement of M393 perturbs heme b558 . Replacement of M393 by leucine results in the conversion of heme b558 to a high-spin state . Surprisingly, the M393L mutation does not eliminate enzymatic activity, and the mutant oxidase has sufficient turnover to support aerobic growth of the cells . The addition of imidazole to the purified M393L oxidase converts heme b558 back to a low-spin configuration . The data strongly suggest that the sixth axial ligand of heme b558 is methionine-393, and that this heme, therefore, has histidine-methionine ligation . The results are consistent with recent cryogenic near-infrared magnetic circular dichroism spectra that also indicate histidine-methionine ligation of heme b558. Biochemistry, 1995 Oct 17, 34(41), 13472 - 6 Identification of an essential cysteinyl residue in the ArsC arsenate reductase of plasmid R773; Liu J et al.; The ArsC protein encoded by the arsenical resistance operon of plasmid R773 catalyzes the reduction of arsenate to arsenite in Escherichia coli . The reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism . The ArsC arsenate reductase has two cysteinyl residues, Cys12 and Cys106 . By a combination of random and site-specific mutagenesis, Cys12 was altered to four other amino acid residues . Cells expressing any of those arsC genes were sensitive to arsenate . The ArsCC12S protein was purified and found to be catalytically inactive . Cys106 was altered separately to seryl, glycyl, and valyl residues . Cells expressing arsCC106S, arsCC106G, and arsCC106V genes retained arsenate resistance, and the purified C106S and C106G proteins had reductase activity . Both wild-type ArsC and C106S proteins were inactivated by iodoacetate . In the native enzyme only Cys12 was alkylate by iodoacetate; Cys106 was alkylated only if the enzyme was first denatured . In the presence of the substrate, arsenate, or competitive inhibitors, phosphate or sulfate, the rate of alkylation was reduced . Reductase activity was inhibited by N-ethylmaleimide and could be protected by arsenate . These results suggest Cys12 is an active-site residue essential for catalysis by the arsenate reductase. Biochemistry, 1995 Oct 17, 34(41), 13437 - 42 E461H-beta-galactosidase (Escherichia coli): altered divalent metal specificity and slow but reversible metal inactivation; Martinez-Bilbao M et al.; beta-galactosidase (Escherichia coli) with a His substituted for Glu-461 retained about 10% of its normal activity in the absence of divalent metals but was inactivated rather than activated by Mg2+, Mn2+, Zn2+, Ni2+, Cu2+, and Co2+ . Since Zn2+, Ni2+, Cu2+, and Co2+ do not interact with wild type beta-galactosidase while Mg2+ and Mn2+ activate and Ca2+ binds but has no effect on wild type beta-galactosidase activity, the substituted enzyme has very different divalent metal interactions . A much larger amount of Mg2+ than of the other divalent metal ions was needed to inactivate the substituted enzyme at pH 7 (half-maximal activity was at 12.5 mM Mg2+ while the half-maximal activities with the other metals were at micromolar levels) compared to the amount of Mg2+ needed to activate the wild type enzyme . The inactivation of E461H-beta-galactosidase caused by Mg2+ took about 20 min . Reactivation by removal of the divalent metal took about 60 min . Interaction with Mg2+ was about 10(7)-fold stronger at pH 9 than at pH 7, and inactivation occurred in less than 2 min at higher pH values . "Galactosylation" (k2, cleavage of the glycosidic bond) seemed to be rate-limiting for E461H-beta-galactosidase at pH values above 6 with both o-nitrophenyl beta-D-galactopyranoside and p-nitrophenyl beta-D-galactopyranoside in both the presence and absence of Mg2+ . Mg2+ caused decreases (about 50-fold) of the k2 values of E461H-beta-galactosidase (apparent pKa was about 6.8).(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Oct 17, 34(41), 13431 - 6 Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase; Osterman AL et al.; Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli . The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site . The fragmented enzyme was purified and analyzed . The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer . The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme . These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305 . In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies . Small quantities of soluble N-terminal peptide could be purified . This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure . Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T . brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169) . Kinetic analysis of a chimeric enzyme composed of T . brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170. Biochemistry, 1995 Oct 17, 34(41), 13423 - 30 Role of the carboxyl terminal MATEE sequence of spermidine/spermine N1-acetyltransferase in the activity and stabilization by the polyamine analog N1,N12-bis(ethyl)spermine; Coleman CS et al.; Purified recombinant spermidine/spermine N1-acetyltransferase (SSAT) was found to be unstable in the absence of polyamines, but the loss of activity could be prevented or reversed by the addition of the polyamine analog and potential antitumor agent N1,N12-bis(ethyl)spermine (BE-3-4-3), which is known to be a potent inducer of SSAT in mammalian cells . Addition of BE-3-4-3 prevented the loss of SSAT activity and the digestion of the protein by the proteases trypsin, Lys-C, or Glu-C . In the absence of BE-3-4-3, this digestion occurred at the sequence Lys141Arg142Arg143 for trypsin or Lys-C and at the sequence Glu151Glu152 for Glu-C . When these sites were altered by mutation to residues which are not substrates for these proteases, cleavage in the absence of BE-3-4-3 occurred at residues Lys161, Lys166, and Glu162 . These results indicate that the structure of SSAT contains a region that binds to the polyamine analog, BE-3-4-3, and that binding alters the configuration of the protein to prevent protease access to the region from amino acid residue 141 to the carboxyl terminal end (residue 171) of the SSAT . In order to determine the nature of the regulatory sites, specific mutations were made in the SSAT amino acid sequence, and the activity of the resulting SSAT protein and the sensitivity to proteases in the presence and absence of BE-3-4-3 was determined . The results indicate that the carboxyl terminal domain, MATEE, is critical for activity and for protection by BE-3-4-3.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Oct 17, 34(41), 13334 - 42 Phospholipid binding and lecithin-cholesterol acyltransferase activation properties of apolipoprotein A-I mutants; Holvoet P et al.; Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space of Escherichia coli, were studied . The rate of turbidity decrease following mixing of apo A-I(delta Ala190-Gln243) with dimyristoylphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding . The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants {6.3 nm for apo A-I(delta Leu44-Leu126), 6.1 nm for apo A-I(delta Glu139-Leu170), and 6.5 nm for apo A-I(delta Ala190-Gln243)} than for intact apo A-I (5.0 nm) . The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I . Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(delta Leu44-Leu126), 6 per apo A-I(delta Glu139-Leu170), and 4 per apo A-I(delta Ala190-Gln243) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Oct 17, 34(41), 13312 - 9 Designing subtilisin BPN' to cleave substrates containing dibasic residues; Ballinger MD et al.; The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing two consecutive basic (dibasic) residues . Mutants were designed on the basis of both the structure of subtilisin BPN' and considerations of sequence differences between it and eukaryotic homologs, Kex2, PC2, and furin, which are known to cleave dibasic substrates . These eukaryotic proteases have high sequence homology to one another but differ substantially from subtilisin BPN' in loops that interact with the substrate . When these loops were grafted into subtilisin BPN', the mutated enzyme could not be expressed, presumably due to destabilization of the folded enzyme . We noted that several neutral residues in subtilisin BPN' (Gly 166, Ser 33, and Asn 62) that are positioned to interact with a dibasic substrate are acidic residues at analogous positions in Kex2 . Mutating these residues individually to either Glu or Asp in subtilisin BPN' resulted in systematic shifts in substrate specificity (kcat/Km) toward basic residues and away from the natural preference for hydrophobic substrates . A combination mutant, where Asn 62 was changed to Asp and Gly 166 was changed to Asp (N62D/G166D), had a larger than additive shift in specificity toward dibasic substrates . This unexpectedly large change was confirmed by detailed analysis with a variety of synthetic substrates . Additional substrate determinants were revealed by sorting a library of phage particles (substrate phage) containing five contiguous randomized residues . This method identified a particularly good substrate (Asn-Leu-Met-Arg-Lys) that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Oct 17, 34(41), 13289 - 304 Solution structure of the DNA-binding protein Sac7d from the hyperthermophile Sulfolobus acidocaldarius; Edmondson SP et al.; The Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius are a heterogeneous mixture of small, thermostable, nonspecific DNA-binding proteins . One of these proteins, Sac7d, has been overexpressed in Escherichia coli to provide a homogeneous preparation for structure, stability, and function studies . We present here essentially complete sequence-specific 1H NMR assignments for Sac7d, a delineation of secondary structural elements, and the high-resolution solution structure obtained from a full relaxation matrix refinement . The final structure provides an excellent fit to the NMR data with an NOE R-factor of 0.27 for backbone NOEs . The structure has a compact globular fold with 82% of the sequence involved in regular secondary structure: an antiparallel two-stranded beta-ribbon with a tight turn, followed by a short 3(10) helix, an antiparallel three-stranded beta-sheet, another short 3(10) helix, and finally four turns of alpha-helix . The amphipathic alpha-helix packs across the hydrophobic face of the three-stranded beta-sheet in an open-faced sandwich arrangement with at least one turn of the helix exposed beyond the sheet . The hydrophobic face of the beta-ribbon packs against a corner of the twisted beta-sheet . The single tryptophan responsible for the 88% fluorescence quenching upon DNA binding is exposed on the surface of the three-stranded beta-sheet . Lysines 5 and 7, whose monomethylation may be associated with enhanced thermostability, are highly solvent exposed along the inner edge of the two-stranded ribbon . The structure of Sac7d differs in many respects from that reported for the homologous native Sso7d {Baumann et al . (1994) Nature Struct . Biol . 1, 808} with a backbone RMSD greater than 3.0 A, largely due to the packing and length of the C-terminal alpha-helix which may be important in Sac7d DNA binding. Biochemistry, 1995 Oct 17, 34(41), 13272 - 7 Fructose-1,6-bisphosphatase: arginine-22 is involved in stabilization of the T allosteric state; Lu G et al.; A comparison of the X-ray crystallographic structures of the R and T allosteric states {Ke, H . M., Liang, J.-Y., Zhang, Y., & Lipscomb, W . N . (1991) Biochemistry 30, 4412-4420} of the pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) reveals major changes in the quaternary structure of the enzyme upon the binding of the allosteric inhibitor AMP . This change in quaternary structure involves the breaking of one set of interactions that stabilize the R state and the formation of another set of interactions that stabilize the T state of the enzyme . In particular, the interactions of Arg-22 with nearby amino acid residues are quite different in the R and T states of the enzyme . Although the crystallographic data suggest that intersubunit interactions such as those involving Arg-22 are important for stabilization of the R and/or T states, the X-ray structures do not provide direct evidence concerning the functional role of specific amino acid residues . Therefore, site-specific mutagenesis has been used to probe the function of Arg-22 in pig kidney fructose-1,6-bisphosphatase . The replacement of Arg-22 by Ala results in a mutant enzyme with enhanced catalytic efficiency compared to the wild-type, as indicated by a kinetic analysis showing a slightly lower Km and increased Vmax compared to the wild-type enzyme . In addition, the substitution enhances both substrate inhibition and the affinity of the inhibitor fructose 2,6-bisphosphate . Moreover, the replacement of Arg-22 by Ala results in a more than 10-fold loss of the ability of AMP to inhibit the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Carbohydr Res, 1995 Oct 16, 276(1), 199 - 208 Enzymic glycosylation of (+/-)-(3,5/4,6)-3,6-diazido-4,5- dihydroxycyclohexene . A way to prepare stereochemically pure and enzyme resistant, basic pseudo-disaccharides as competitive enzyme inhibitors; Lehmann J et al.; By beta-D-galactosylation of (+/-)-(3,5/4,6)-3,6-diazido-4,5-dihydroxycyclohexene, pure (+)-3,5/4,6)-3,6-diazido-4-O-(beta-D-galactopyranosyl)-5-hydrox ycyclohexene (3) was obtained . The diamine (+)-(1,3/2,6)-3,6-diamino-1-O-(beta-D-galactopyranosyl)-2-hydroxycycl ohexane, derived from compound 3 by catalytic hydrogenation, is stable against enzymic cleavage and competitively inhibits beta-D-galactosidase from Escherichia coli with a Ki-value of 5.5 mM . Sigmatropic rearrangement of 3 in methanolic solution partially led to an unseparable mixture of the regioisomers (3,5/4,6)-3,4-diazido-5-O-(beta-D-galactopyranosyl)-6-hydroxycy clohexene and (3,5/4,6)-3,4-diazido-6-O-(beta-D-galactopyranosyl)-5-hydroxycy clohexene . Catalytic hydrogenation thereof yielded an equally unseparable mixture of the diamines (1,3/2,4)-1,2-diamino-4-O-(beta-D-galactopyranosyl)-4-hydroxycyclohex ane and (1,3/2,4)-1,2-diamino-4-O-(beta-D-galactopyranosyl)-3-hydroxycyclohex ane, inhibiting beta-D-galactosidase competitively with K(i) 0.9 mM. Gene, 1995 Oct 16, 164(1), 9 - 15 Versatile, multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: overproduction of human and murine cytokines; Mertens N et al.; We describe the construction, expression characteristics and some applications of a versatile dual-promoter expression plasmid for heterologous gene expression in Escherichia coli which contains both lambda pL and PT7 promoters . Furthermore, the plasmid is optimized to allow the expression of mature coding sequences without compromising the strength of the highly efficient PT7 or of the T7g10 ribosome-binding site . The effect of the the naturally occurring RNA loops at both the 5' and 3' ends of the T7g10 mRNA on expression was also examined . A double T7 RNA polymerase transcription terminator was inserted to ensure more reliable transcription termination and a higher expression level of the preceding gene . Further improvements involve a clockwise orientation of the promoters to minimize read-through transcription from plasmid promoters, a largely extended multiple cloning site, an antisense phage T3 promoter and a phage f1-derived, single-stranded replication origin . Variants of this plasmid allow for the production of fusion proteins with part of T7g10, a hexahistidine peptide and an enterokinase recognition site . The potential of these expression vectors is demonstrated by comparing the expression levels of a number of mammalian cytokines (human tumor necrosis factor, human immune interferon, human and murine interleukins 2, murine interleukin 4 and murine fibroblast interferon), using these expression plasmids. Gene, 1995 Oct 16, 164(1), 65 - 9 Purification of the KpnI DNA methyltransferase and photolabeling of the enzyme with S-adenosyl-L-methionine; Finta C et al.; An Escherichia coli strain overproducing the KpnI DNA methyltransferase (M.KpnI) was constructed by cloning the kpnIM gene downstream from the inducible T7 phage luminal diameter 10 promoter . A method involving three chromatographic steps has been developed to purify M.KpnI to homogeneity . The purified enzyme has a pH optimum around 7.3 and is inhibited by salts . M.KpnI can be photolabeled by UV-irradiation of the enzyme in the presence of S-adenosyl-L-{methyl-3H}methionine ({methyl-3H}AdoMet) . Photolabeling results from a specific interaction between M.KpnI and AdoMet, as indicated by the dependence of photolabeling on native enzyme conformation and by the inhibitory effect of the AdoMet analogs, sinefungin and S-adenosyl-L-homocysteine (AdoHcy). Gene, 1995 Oct 16, 164(1), 59 - 63 Template secondary structure can increase the error frequency of the DNA polymerase from Thermus aquaticus; Loewen PC et al.; Amplification of portions of the intergenic spacer between the katE gene and cryptic cel operon of Escherichia coli was accomplished by the polymerase chain reaction using the DNA polymerase from Thermus aquaticus . Nine different segments were amplified and cloned without error, but one 83-bp fragment was amplified with a high error rate such that 32 of 34 selected clones had three or more nucleotide changes from the expected sequence . The changes were all located in two 9-bp segments immediately adjacent to the 3'-ends of the two primers . Moving the end points of the primers to increase the spacing between them resulted in the isolation of significantly fewer error-containing products . It is proposed that stem-loop structures in the template immediately downstream from the primers interfere with an early stage of elongation and cause misincorporation . This is supported by the observation that destabilisation of one of the stem-loop structures reduced the frequency of errors. Gene, 1995 Oct 16, 164(1), 55 - 8 A versatile low-copy-number cloning vector derived from plasmid F; Shi J et al.; We have constructed a cloning vector based on plasmid mini-F for use in Escherichia coli . Plasmid pZC320 consists of the ori-2 replication unit of F that confers very low copy number (lcn), and includes the sop partition functions to insure stable plasmid maintenance in the absence of selection . A multiple cloning site (MCS) containing 16 unique restriction sites is located within the 5' end of the lacZ alpha gene . Expression of lacZ alpha is under the control of the wild-type lactose operator/promoter (lacOP) region and is efficiently repressed by the lacI repressor . Clones containing inserts can be detected using the blue/white screen for beta-galactosidase (beta Gal) . A T7 promoter allows transcription of cloned inserts in the presence of T7 RNA polymerase . We have demonstrated the use of this lcn vector for cloning the regulated tetracycline-resistance genes from Tn10, which confer only low-level resistance when present at high copy number. Gene, 1995 Oct 16, 164(1), 129 - 32 Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification; Prestidge RL et al.; The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth . In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism . We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble . By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E . coli, from where it can be easily purified in high yield . 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant. FEBS Lett, 1995 Oct 16, 373(3), 310 - 2 Crystallization and crystallographic investigations of the small subunit of mouse ribonucleotide reductase; Nielsen BB et al.; The R2 protein component of mouse ribonucleotide reductase has been obtained from overproducing Escherichia coli bacteria . It has been crystallized using NaCl as precipitant . The crystals are orthorhombic, space group C222(1), with cell dimensions a = 76.9 A, b = 108.9 A, c = 92.7 A and diffract to at least 2.5 A . The asymmetric unit of the crystals contains one monomer . Rotation and translation function searches using a model based on the weakly homologous E . coli R2 gave one significant peak . Rotation about a crystallographic 2-fold axis parallel to the a-axis produces an R2 dimer with dimer interactions very similar to those found for E . coli R2. FEBS Lett, 1995 Oct 16, 373(3), 296 - 8 Expression and refolding of a high-affinity receptor binding domain from rat alpha 1-macroglobulin; Nielsen KL et al.; A recombinant version of the receptor binding domain of rat alpha 1-macroglobulin (RBDv) consisting of residues 1319-1474 has been expressed in E . coli . Competition experiments with 125I-labelled methylamine treated human alpha 2-macroglobulin reveal that the alpha 1-macroglobulin-RBDv exhibit the same high affinity for the alpha 2-macroglobulin receptor as the entire 40 kDa light chain from rat alpha 1-macroglobulin . It is therefore concluded, that all determinants for receptor interaction reside in the C-terminal approx . 150 residues of the alpha-macroglobulin subunit. EMBO J, 1995 Oct 16, 14(20), 5141 - 7 Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication; Takechi S et al.; Initiation of in vitro ColE2 DNA replication requires the plasmid-specified Rep protein and DNA polymerase I but not RNA polymerase and DnaG primase . The ColE2 Rep protein binds specifically to the origin where replication initiates . Leading-strand synthesis initiates at a unique site in the origin and lagging-strand DNA synthesis terminates at another unique site in the origin . Here we show that the primer RNA for leading-strand synthesis at the origin has a unique structure of 5'-ppApGpA . We reconstituted the initiation reaction of leading-strand DNA synthesis by using purified proteins, the ColE2 Rep protein, Escherichia coli DNA polymerase I and SSB, and we showed that the ColE2 Rep protein is a priming enzyme, primase, which is specific for the ColE2 origin . The ColE2 Rep protein is unique among other primases in that it recognizes the origin region and synthesizes the primer RNA at a fixed site in the origin region . Specific requirement for ADP as a substrate and its direct incorporation into the 5' end of the primer RNA are also unique properties of the ColE2 Rep protein. EMBO J, 1995 Oct 16, 14(20), 5085 - 93 Both ambient temperature and the DnaK chaperone machine modulate the heat shock response in Escherichia coli by regulating the switch between sigma 70 and sigma 32 factors assembled with RNA polymerase; Blaszczak A et al.; In Escherichia coli individual sigma factors direct RNA polymerase (RNAP) to specific promoters . Upon heat shock induction there is a transient increase in the rate of transcription of approximately 20 heat shock genes, whose promoters are recognized by the RNAP-sigma 32 rather than the RNAP-sigma 70 holoenzyme . At least three heat shock proteins, DnaK, DnaJ and GrpE, are involved in negative modulation of the sigma 32-dependent heat shock response . Here we show, using purified enzymes, that upon heat treatment of RNAP holoenzyme the sigma 70 factor is preferentially inactivated, whereas the resulting heat-treated RNAP core is still able to initiate transcription once supplemented with sigma 32 (or fresh sigma 70) . Heat-aggregated sigma 70 becomes a target for the joint action of DnaK, DnaJ and GrpE proteins, which reactivate it in an ATP-dependent reaction . The RNAP-sigma 32 holoenzyme is relatively stable at temperatures at which the RNAP-sigma 70 holoenzyme is inactivated . Furthermore, we show that formation of the RNAP-sigma 32 holoenzyme is favored over that of RNAP-sigma 70 at elevated temperatures . We propose a model of negative autoregulation of the heat shock response in which cooperative action of DnaK, DnaJ and GrpE heat shock proteins switches transcription back to constitutively expressed genes through the simultaneous reactivation of heat-aggregated sigma 70, as well as sequestration of sigma 32 away from RNAP. EMBO J, 1995 Oct 16, 14(20), 4976 - 84 Isolation of a psaF-deficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit; Farah J et al.; The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood . Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cw15-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy . This psaF mutant still assembles a functional PSI complex and is capable of photoautotrophic growth . However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex . These results contrast with those obtained previously with a cyanobacterial psaF-, psaJ- double mutant where no phenotype was apparent. EMBO J, 1995 Oct 16, 14(20), 4939 - 48 A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor; Stoller G et al.; Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo . We isolated a peptidyl-prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome . This association was abolished by adding at least 1.5 M LiCl . Sequencing the N-terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor . A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases . Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied . In its activity towards oligopeptide substrates, the trigger factor resembles the FK506-binding proteins (FKBPs) . Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc-Ala-Xaa-Pro-Phe-4-nitroanilides is reminiscent of FKBPs . However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM . In vitro, the trigger factor catalysed the proline-limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far. Structure, 1995 Oct 15, 3(10), 1097 - 108 Crystal structure of thioredoxin-2 from Anabaena; Saarinen M et al.; BACKGROUND: Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases . The structures of thioredoxins from a number of species, including man and Escherichia coli, are known . Cyanobacteria, such as Anabaena, contain two thioredoxins that exhibit very different activities with target enzymes and share little sequence similarity . Thioredoxin-2 (Trx-2) from Anabaena resembles chloroplast type-f thioredoxin in its activities and the two proteins may be evolutionarily related . We have undertaken structural studies of Trx-2 in order to gain insights into the structure/function relationships of thioredoxins . RESULTS: Anabaena Trx-2, like E . coli thioredoxin, consists of a five-stranded beta sheet core surrounded by four alpha helices . The active site includes a conserved disulfide ring (in the sequence 31WCGPC35) . An aspartate (E . coli) to tyrosine (Trx-2) substitution alters the position of this disulfide ring relative to the central pleated sheet . However, loss of this conserved aspartate does not affect the disulfide geometry . In the Trx-2 crystals, the N-terminal residues make extensive contacts with a symmetry-related molecule with hydrogen bonds to residues 74-76 mimicking thioredoxin-protein interactions . CONCLUSIONS: The overall three-dimensional structure of Trx-2 is similar to E . coli thioredoxin and other related disulfide oxido-reductases . Single amino acid substitutions around the protein interaction area probably account for the unusual enzymatic activities of Trx-2 and its ability to discriminate between substrate and non-substrate peptides. Structure, 1995 Oct 15, 3(10), 1087 - 95 The solution structure of the Mu Ner protein reveals a helix-turn-helix DNA recognition motif; Strzelecka TE et al.; BACKGROUND: The Mu Ner protein is a small (74 amino acids), basic, DNA-binding protein found in phage Mu . It belongs to a class of proteins, the cro and repressor proteins, that regulate the switch from the lysogenic to the lytic state of the phage life cycle . There is no significant sequence identity between Mu Ner and the cro proteins of other phages, despite their functional similarity . In addition, there is no significant sequence identity with any other DNA-binding proteins, with the exception of Ner from the related phage D108 and the Nlp protein of Escherichia coli . As the tertiary structures of Mu Ner and these two related proteins are unknown, it is clear that a three-dimensional (3D) structure of Mu Ner is essential in order to gain insight into its mode of DNA binding . RESULTS: The 3D solution structure of Mu Ner has been solved by 3D and 4D heteronuclear magnetic resonance spectroscopy . The structure consists of five alpha helices, two of which comprise a helix-turn-helix (HTH) motif . Analysis of line broadening and disappearance of crosspeaks in a 1H-15N correlation spectrum of the Mu Ner-DNA complex suggests that residues in these two helices are most likely to be in contact with the DNA . CONCLUSIONS: Like the functionally analogous cro proteins from phages lambda and 434, the Mu Ner protein possesses a HTH DNA recognition motif . The Ner protein from phage D108 and the Nlp protein from E . coli are likely to have very similar tertiary structures due to high amino-acid-sequence identity with Mu Ner. Structure, 1995 Oct 15, 3(10), 1051 - 9 Detergent structure in tetragonal crystals of OmpF porin; Pebay-Peyroula E et al.; BACKGROUND: The high-resolution structures of five porins have been solved by X-ray crystallography including the trigonal crystal form of the trimeric OmpF porin from Escherichia coli . In an accompanying article, the structure of the tetragonal form of OmpF porin is presented . In contrast to the trigonal crystal form, the protein surfaces normally in contact with lipids in the membrane are exposed and interact with amphiphiles in the tetragonal crystal . Thus, the tetragonal form can be used to investigate protein-detergent interactions . RESULTS: Using single-crystal neutron diffraction studies and two different detergents (one of them deuterated in its hydrophobic moiety), details of the amphiphile-protein interactions are revealed . Detergent molecules bind to the so-called hydrophobic zone that surrounds the OmpF porin trimer and which is exposed to lipid in the native environment . The aromatic rings on both sides of the hydrophobic zone coincide with the boundary between non-polar and polar moieties of the detergents . CONCLUSIONS: In the tetragonal crystal form of OmpF porin, the membrane-exposed area is accessible from the aqueous solution . It is coated by a film of detergent molecules, which presumably mimics the interactions of the protein with lipids in the biological membrane . In the trigonal form, protein-protein interactions predominate in the hydrophobic zone . These may reflect the tight interactions between trimers that are observed in the biological membrane. Structure, 1995 Oct 15, 3(10), 1041 - 50 The structure of OmpF porin in a tetragonal crystal form; Cowan SW et al.; BACKGROUND: OmpF porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules, such as nutrients and waste products, across the outer membrane of Escherichia coli . Very few membrane proteins have been crystallized in three dimensions, yet this stable protein can be obtained in several crystal forms . Comparison of the structures of the same membrane protein in two different packing environments is of major interest, because it allows us to explore the integrity of the structure outside the natural membrane environment . RESULTS: The structure of OmpF porin in a tetragonal crystal form with two trimers per asymmetric unit has been determined at 3.2 A resolution and compared with that obtained previously in a trigonal crystal form . The lattice contacts involve only polar atoms, whereas extensive hydrophobic protein-protein interactions were found in the trigonal lattice . The trimer structure is virtually identical in both . CONCLUSIONS: Our comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints and, thus, presumably bears close resemblance to the in vivo structure . The tetragonal crystal structure has provided the starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article. Structure, 1995 Oct 15, 3(10), 1009 - 19 Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores; Kenney JM et al.; BACKGROUND: Hepatitis B virus is a major human pathogen which has been extensively studied, yet its structure is unknown . Cryo-electron microscopy of the viral cores expressed in Escherichia coli or isolated from infected liver provides a means for determining the structure of the hepatitis B nucleocapsid . RESULTS: Using cryo-electron microscopy and three-dimensional image reconstruction, we have determined the structures of duck and human hepatitis B virus cores and find that they have similar dimer-clustered T = 3 and T = 4 icosahedral organizations . The duck virus core protein sequence differs from the human in both length and amino acid content; however, the only significant structural differences observed are the lobes of density on the lateral edges of the projecting (distal) domain of the core protein dimer . The different cores contain varying amounts of nucleic acid, but exhibit similar contacts between the core protein and the nucleic acid . Immunoelectron microscopy of intact cores has localized two epitopes on the core surface corresponding to residues 76-84 and 129-132 . CONCLUSIONS: The bacterial expression system faithfully reproduces the native hepatitis B virus core structure even in the absence of the complete viral genome . This confirms that proper assembly of the core is independent of genome packaging . Difference imaging and antibody binding map three sequence positions in the structure: the C terminus and the regions near amino acids 80 and 130 . Finally, we suggest that the genome-core interactions and the base (proximal) domain of the core dimer are evolutionarily conserved whereas the projecting domain, which interacts with the envelope proteins, is more variable. Eur J Pharmacol, 1995 Oct 15, 291(2), 67 - 72 Thrombin triggers the de novo expression of an inducible NO synthase in porcine aortic valve endothelial cells; De Meyer E et al.; The nitric oxide (NO) production by porcine aortic valve endothelial cells was estimated in cusps incubated at 37 degrees C by measuring their cyclic GMP content and the nitrite levels of the incubation medium . After a stabilization period, incubation for 5 min with acetylcholine, bradykinin, ADP and bovine thrombin resulted in a receptor-mediated increase in cyclic GMP which could be blocked by EGTA, N-omega-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA) . Incubation with lipopolysaccharide (endotoxin) from E . coli O111:B4 or bovine for 5 h, dose-dependently increased nitrite production as well as cyclic GMP content . The elevated nitrite production was completely abolished in the presence of the protein synthesis inhibitor cycloheximide, was reduced by more than 50% by dexamethasone but was not affected by EGTA . L-NMMA dose-dependently reduced the increased nitrite production and cyclic GMP content . These results suggest that besides the presence of a constitutive NO synthase in porcine aortic valve endothelial cells thrombin, like lipopolysaccharide, triggers the de novo expression of an inducible Ca(2+)-independent NO synthase. Genes Dev, 1995 Oct 15, 9(20), 2470 - 81 The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast; Lafontaine D et al.; The m6(2)A1779m6(2)A1780 dimethylation at the 3' end of the small subunit rRNA has been conserved in evolution from bacteria to eukaryotes . The yeast 18S rRNA dimethylase gene DIM1 was cloned previously by complementation in Escherichia coli and shown to be essential for viability in yeast . A conditional GAL10::dim1 strain was constructed to allow the depletion of Dim1p from the cell . During depletion, dimethylation of the pre-rRNA is progressively inhibited and pre-rRNA processing at cleavage sites A1 and A2 is concomitantly lost . In consequence, the mature 18S rRNA and its 20S precursor drastically underaccumulate . This has the effect of preventing the synthesis of nonmethylated rRNA . To test whether the processing defect is a consequence of the absence of the dimethylated nucleotides or of the Dim1p dimethylase itself, a cis-acting mutation was created in which both dimethylated adenosines are replaced by guanosine residues . Methylation cannot occur on this mutant pre-rRNA, but no clear pre-rRNA processing defect is seen . Moreover, methylation of the wild-type pre-rRNA predominantly occurs after cleavage at sites A1 and A2 . This shows that formation of the m6(2)A1779m6(2)A1780 dimethylation is not required for pre-rRNA processing . We propose that the binding of Dim1p to the pre-ribosomal particle is monitored to ensure that only dimethylated pre-rRNA molecules are processed to 18S rRNA. Eur J Biochem, 1995 Oct 15, 233(2), 498 - 505 Involvement of the C-terminal tail in the activity of Drosophila alcohol dehydrogenase . Evaluation of truncated proteins constructed by site-directed mutagenesis; Albalat R et al.; Drosophila alcohol dehydrogenase belongs to the heterogeneous family of short-chain dehydrogenases/reductases, which does not include the well characterized mammalian alcohol dehydrogenases . Although it is clear that the main biological role of this enzyme is in alcohol oxidation, in the absence of the three-dimensional conformation only partial information on the protein regions involved in the active site, and the coenzyme and substrate interacting cavities is available . Two segments have already been identified, a coenzyme-binding segment at the N-terminus, and the reactive Tyr152 and Lys156 residues . Limited proteolytic assays had suggested the involvement of the 13 C-terminal amino acids in the function of the enzyme . By site-directed mutagenesis, we have constructed eight different truncated mutant enzymes and expressed them in Escherichia coli . The purified mutant enzymes have been recovered and characterized using monoclonal antibodies . Kinetic analysis and stability assays have been performed, and clearly demonstrate the contribution of the last 13 amino acids to the activity . We hypothesize that the C-terminal tail constitutes an essential region for maintaining the hydrophobicity of the catalytic pocket needed for binding of the substrate. Eur J Biochem, 1995 Oct 15, 233(2), 478 - 83 Reconstitution of the Fo complex of Escherichia coli ATP synthase from isolated subunits . Varying the number of essential carboxylates by co-incorporation of wild-type and mutant subunit c after purification in organic solvent; Dmitriev OY et al.; Subunit c of the Escherichia coli F1F0-ATPase, purified in chloroform/methanol (2:1), was reconstituted with detergent-solubilized F0 subunits a and b to form a functionally active H+ channel . The rates of H+ uptake by the proteoliposomes containing the reconstituted F0 complex were comparable to those observed with native F0 reconstituted without subunit dissociation . The F0 reconstituted from purified subunits was also shown to form an active ATP-driven H+ pump upon binding of the F1-ATPase sector of the complex . Reconstitution of D61N and D61G mutant c subunits with wild-type subunits a and b produced an inactive F0 . Hybrid F0 complexes, formed with mixtures of wild-type and D61N or D61G mutant c subunits, were also prepared . Formation of an active F0 was prevented by addition of relatively small proportions of D61N or D61G mutant c subunits, i.e . active F0 formation was gradually disrupted as the mutant/wild-type ratio was increased from 0.05 to 0.2 . The hybrid reconstitution studies support a model where inactivation of one of the 9-12 c subunits found in F0 is sufficient to abolish activity. Eur J Biochem, 1995 Oct 15, 233(2), 473 - 7 Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O44:H18; Staaf M et al.; The O-antigen polysaccharide of the lipopolysaccharide from the enteroaggregative Escherichia coli O44:H18 has been investigated . Sugar and methylation analysis, 1H- and 13C-NMR spectroscopy revealed that the polysaccharide is composed of pentasaccharide repeating units . The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond correlation experiments . The structure of the repeating unit of the O-antigen from Escherichia coli O44:H18 is as follows . {formula: see text} Eur J Biochem, 1995 Oct 15, 233(2), 419 - 25 The identification of the single-stranded DNA-binding domain of the Escherichia coli RecA protein; Gardner RV et al.; To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure . Synthetic peptides derived from amino acid residues 185-219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E . coli RecA in this region did not . The binding of RecA to ssDNA examined using a gel-shift assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301-329 of the C-terminus or from N-terminal residues 6-39 . A peptide corresponding to amino acid positions 152-169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 microM . We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193-212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA . Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185-224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303-353) . In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays . These findings suggest that a portion of the region extending from amino acid residues 193-212 is either part of or the whole ssDNA-binding domain of the RecA protein. Eur J Biochem, 1995 Oct 15, 233(2), 406 - 13 Molecular cloning, expression, and characterization of a human intestinal 15-kDa protein; Fujita M et al.; We have isolated a cDNA encoding a human intestinal 15-kDa protein (I-15P) from a human ileal lambda gt 11 cDNA library, using a full-length rat I-15P cDNA . One clone encompassed 571 nucleotides and encoded a 128-amino-acid protein with a calculated molecular mass of 14355 Da . The deduced amino acid sequence of human I-15P showed high similarity to the rat counterpart (78%), mouse ileal lipid-binding protein (80%) and porcine gastrotropin (75%) . It also exhibited 36% similarity to human liver fatty-acid-binding protein (L-FABP) . Northern blot analysis of human I-15P revealed a single transcript only in ileum, however, the reverse-transcription/PCR demonstrated expression in ovary and placenta, but it was much lower than in ileum . Transformation of Escherichia coli with the I-15P cDNA resulted in the efficient expression of a protein that was identical to the ileal cytosolic I-15P . In vitro binding studies revealed that the bacterially expressed recombinant I-15P showed much lower affinities for palmitate and oleate than L-FABP . However, it showed similar affinity for taurocholate, compared with a control, BSA . Comparison of the structural features of human I-15P and human L-FABP suggested that loss of a long alpha-helix region and hydrophobic profile of I-15P may be attributable to a unique ligand-binding specificity of I-15P. J Immunol, 1995 Oct 15, 155(8), 4090 - 4 Paradoxical effects of IL-10 in endotoxin-induced uveitis; Rosenbaum JT et al.; Uveitis, or intraocular inflammation, can be provoked in laboratory rodents by the local or systemic injection of bacterial endotoxin . Many of the inflammatory effects of endotoxin are potentially due to the induction of cytokine synthesis . IL-10 is a cytokine that potently inhibits the synthesis of many cytokines, including IL-1 and TNF-alpha . We have assessed the ability of IL-10 to inhibit endotoxin-induced uveitis in rabbits and mice . The intravitreal injection of 1 micrograms of human recombinant IL-10 was extremely effective in rabbits in reducing the inflammation produced by the intravitreal injection of 250 ng of Escherichia coli endotoxin, as judged by the reduced accumulation of cells and protein in the aqueous humor . Locally injected IL-10 was similarly effective in blocking the ocular inflammatory effects of intravitreally injected endotoxin in a mouse model . If the injection of IL-10 was delayed subsequent to the endotoxin injection, the reduced inflammatory effects in the rabbit model were diminished . In contrast to its ability to inhibit the local inflammatory effect of endotoxin in the eye, IL-10 did not reduce the inflammation induced by a local ocular injection of 400 U of human recombinant IL-alpha . Paradoxically, in a mouse model of uveitis subsequent to intraperitoneally injected endotoxin, the simultaneous injection of 1 micrograms of IL-10 and endotoxin potentiated the ocular inflammation, as judged by the number of leukocytes seen in histologic sections . This effect was dose dependent, since eye inflammation was markedly inhibited by 100 micrograms of IL-10 injected i.p . These observations are compatible with the hypothesis that locally injected IL-10 acts by reducing cytokine synthesis in these uveitis models . Intraperitoneally injected IL-10 can either inhibit or suppress endotoxin-induced eye inflammation in a dose-dependent manner. Mech Ageing Dev, 1995 Oct 13, 84(2), 113 - 26 Cytokine production and lymphocyte subpopulations in aged humans . An assessment during nocturnal sleep; Born J et al.; The view of a general impairment of immune functions associated with aging has been challenged by recent studies including a more detailed evaluation of various cytokines and lymphocyte subsets . In the present human study, effects of age on the production of cytokines by T cells and monocytes were assessed, together with age-dependent changes in subset populations of mononuclear cells (MNC) . Blood was collected every 30 min during nocturnal sleep in 16 aged (mean: 79.6 +/- 7.5 years) and in 16 young controls (mean: 24.6 +/- 3.1 years) . Nocturnal sleep was chosen as a well-defined period within the 24-h cycle with minimal exogenous influences . The in vitro production of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) was measured after mitogen stimulation with lipopolisaccharide from E . coli (LPS) . Production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was measured after stimulation with phytohemagglutinin (PHA) . Regarding MNC subsets, monocytes, lymphocytes, CD3+, CD4+, CD8+, HLA-DR, CD16+, CD25+, and CD19+ were determined . Advanced age was associated with a decreased number of T cells (CD3+) and decreases in the major T cell subsets (CD4+, CD8+, P < 0.001) . Production of IL-2 was not affected . However, production of IFN-gamma tended to be enhanced, and numbers of activated T cells (HLA-DR/CD3+), natural killer cells (CD16+), and T cells expressing IL-2 receptors (CD25+/CD3+) were markedly increased in the aged . While monocyte counts were unchanged in the elderly production of IL-1 beta and TNF-alpha mainly derived from these cells, was enhanced (p < 0.05) . Results indicate a state of enhanced responsiveness of the T cell compartment and of monocytes in aged which may compensate for the substantial decrease in T cells. J Biol Chem, 1995 Oct 13, 270(41), 24609 - 14 Subunits coupling H+ transport and ATP synthesis in the Escherichia coli ATP synthase . Cys-Cys cross-linking of F1 subunit epsilon to the polar loop of F0 subunit c; Zhang Y et al.; Second site suppressor mutations at position 31 of F1 subunit epsilon recouple ATP-driven H+ translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F1F0 ATP synthase (Zhang, Y., Oldenburg, M., and Fillingame, R . H . (1994) J . Biol . Chem . 269, 10221-10224) . This finding suggests a functional interaction between subunit c and subunit epsilon during the coupling of H+ transport through F0 to ATP synthesis of F1 . However, the physical proximity of the two subunits remained to be defined . In this study, Cys residues were introduced into residues in the polar loop region of subunit c surrounding Gln42 and at position 31 of subunit epsilon to see whether the subunits could be cross-linked . Disulfide bridge formation between subunit c and subunit epsilon was observed in membranes of three double mutants, i.e . cA40C/epsilon E31C, cQ42C/epsilon E31C, and cP43C/epsilon E31C, but not in wild type membranes or in membranes of the cA39C/epsilon E31C double mutant . These results indicate that the polar loop of subunit c and the region around residue 31 of subunit epsilon are physically close to each other in the F1F0 complex and support the hypothesis that these two subunits interact directly in the coupling of H+ transport to ATP synthesis . Disulfide cross-linking of the Q42C subunit c and E31C subunit epsilon leads to inhibition of ATPase coupled H+ transport, as might be expected in a model where the catalytic sites of the F1 ATPase alternate during H+ transport-coupled ATP hydrolysis/synthesis . However, a quantitative relationship between the extent of inhibition of transport and the extent of cross-linking could not be established by the methods used here, and the possibility remains that the epsilon-c cross-linked F1F0 complex retains residual H+ transporting activity. J Biol Chem, 1995 Oct 13, 270(41), 24604 - 8 Binding of Neu differentiation factor with the extracellular domain of Her2 and Her3; Horan T et al.; The interaction of neu differentiation factor (NDF) with the extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been studied using native gels, light scattering, and sedimentation equilibrium . The full-length NDF beta 2 was shown to bind sHer3 with a dissociation constant of 26 +/- 9 nM, while it showed a 1000-fold weaker binding to sHer2 . Taken together, these results demonstrate that NDF is a high affinity ligand for Her3, but not for Her2 . No increase in affinity of the NDF beta 2 for sHer3 was observed upon addition of sHer2 to the NDF beta 2-sHer3 mixture . Binding of NDF beta 2 to sHer3 did not induce receptor dimerization or oligomerization, the stoichiometry being one sHer3 per one NDF molecule . This finding suggests that transmembrane and/or intracellular domains of receptor family members or perhaps additional unidentified components may be involved in NDF induced dimerization and autophosphorylation, or alternatively, that dimerization is not the mechanism for Her3 autophosphorylation and signal transduction. J Biol Chem, 1995 Oct 13, 270(41), 24459 - 67 Strand specificity of nicking of DNA at Chi sites by RecBCD enzyme . Modulation by ATP and magnesium levels; Taylor AF et al.; RecBCD enzyme is essential for the major pathway of homologous recombination of linear DNA in Escherichia coli . It is a potent nuclease and helicase and, during its unwinding of double-stranded DNA, makes single-strand scissions in the vicinity of Chi recombination hot spots . We report here that both the strand that is cut and the position of the cuts relative to Chi depended on the ATP to Mg2+ ratio . With ATP in excess, Chi-dependent nicks occurred, as we have previously reported, four to six nucleotides to the 3'-side of the Chi octamer (5'-GCTGGTGG-3') and were detected only on the strand bearing that sequence . Three differences were seen with Mg2+ in excess . 1) Chi-dependent 3'-ends were produced on the GCTGGTGG-containing strand closer to and within the Chi octamer . 2) Chi-dependent cuts occurred on the complementary DNA strand . 3) RecBCD enzyme destroyed the 3'-terminated strand of DNA from its entry point up to the vicinity of the Chi site, as others have previously reported . We show here that, with Mg2+ in excess, the enzyme continued to travel along DNA, after encountering a Chi site, releasing both strands of the DNA distal to Chi as single strands . We discuss potential biological consequences of these two modes of RecBCD enzyme-Chi interaction. J Biol Chem, 1995 Oct 13, 270(41), 24451 - 8 Monomeric RecBCD enzyme binds and unwinds DNA; Taylor AF et al.; RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli . It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA . The functional form of RecBCD enzyme has been unclear, since M(r) of 250,000-655,000 have been previously reported . We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide . We show here that the monomeric form of the enzyme (M(r) approximately 330,000) can form a stable initiation complex on the end of ds DNA . Depending on the nature of the ds end, KD estimates ranged from approximately 0.1 nM to approximately 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding . We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA . A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme . Our results make such a model unlikely for RecBCD enzyme. J Biol Chem, 1995 Oct 13, 270(41), 24420 - 7 Deficient geranylgeranylation of Ram/Rab27 in choroideremia; Seabra MC et al.; Choroideremia, an X-linked form of retinal degeneration, results from defects in the Rab escort protein-1 (REP-1) gene . REP-1 and REP-2 assist in the attachment of geranylgeranyl groups to Rab GTPases, a modification essential for their action as molecular switches regulating intracellular vesicular transport . If Rabs that depend preferentially on REP-1 for prenylation exist, they will accumulate unprenylated in choroideremia cells . Using recombinant Rab geranylgeranyl transferase and REPs to label unprenylated cytosolic proteins, we identified one unprenylated protein in choroideremia lymphoblasts that was prenylated in vitro more efficiently by REP-1 than by REP-2 . This protein was purified and identified as Ram (renamed Rab27), a previously cloned Rab of unknown function . Immunohistochemistry of rat retina showed that Ram/Rab27 is expressed in the pigment epithelium and choriocapillaris, the two retinal cell layers that degenerate earliest in choroideremia . These results raise the possibility that the retinal degeneration in choroideremia results from the deficient geranylgeranylation of Ram/Rab27 or a closely related protein. J Biol Chem, 1995 Oct 13, 270(41), 24414 - 9 Mutations leading to altered CheA binding cluster on a face of CheY; Shukla D et al.; CheY is the response regulator of Escherichia coli chemotaxis and is one of the best studied response regulators of the two-component signaling system . CheY can receive phosphate from the histidine kinase, CheA . Phospho-CheY interacts with the motor-switch complex to induce clockwise flagellar rotation, thus causing the cell to tumble . We used an enzyme-linked immunosorbent assay to study the direct interaction between the kinase, CheA, and the regulator, CheY . The products of random, suppressor, and site-specific cheY mutants were assayed for their ability to bind CheA . Nine mutants showed altered binding . We sequenced and mapped these point mutations on the crystal structure of CheY, and a high degree of spatial clustering was revealed, indicating that this region of CheY is involved in CheA binding . Interestingly, five of these altered binding mutants were previously defined as being involved in motor-switch binding interactions . This suggested a possible overlap between the motor-switch binding and CheA binding surfaces of CheY . Using CheY (Trp-58) fluorescence quenching, we determined the equilibrium dissociation constants of CheA (124-257) binding for these CheY mutants . The results from the fluorescence quenching are in close agreement with our initial enzyme-linked immunosorbent assay results . Therefore, we propose that the CheA and the motor binding surfaces on CheY partially overlap and that this overlap allows CheY to interact with either the CheA or the flagellar motor, depending on its signaling (phosphorylation) state. J Biol Chem, 1995 Oct 13, 270(41), 24392 - 8 Abortive cycling and the release of polymerase for elongation at the sigma 54-dependent glnAp2 promoter; Tintut Y et al.; Transcription initiation at the sigma 54-dependent glnAp2 promoter was studied to follow the state of polymerase as RNA synthesis begins . Sigma 54 polymerase begins transcription in abortive cycling mode, i.e . after the first bond is made, approximately 75% of the time the short RNA is aborted and synthesis must be restarted . Polymerase is capable of abortive initiation until it reaches a position beyond +3 and before +7, at which stage polymerase is released from its promoter contacts and an elongation complex is formed . INitial elongation is accompanied by two transcription bubbles, one moving with the polymerase and the other remaining at the transcription start site . The sigma 54-associated polymerase shows an earlier and more efficient transition out of abortive initiation mode than prior studies of sigma 70-associated polymerase. J Biol Chem, 1995 Oct 13, 270(41), 24361 - 9 The human synapsin II gene promoter . Possible role f