J Biol Chem, 1994 Mar 18, 269(11), 8366 - 75 Intracellular analysis of in vitro modified HIV Tat protein; Koken SE et al.; Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that specifically activates transcription from the viral long terminal repeat . To characterize the properties of the Tat proteins, we have expressed them in Escherichia coli . The purified Tat protein was biochemically analyzed and tested for activity upon electroporation into human cell lines . This protein electroporation was used for the intracellular analysis of in vitro modified Tat protein . Our results indicate that the transcriptionally active form of the Tat protein is a monomer . Furthermore, we found that Tat activity is dramatically inhibited by preincubation of the protein with strongly reducing agents . In contrast, no inhibitory effect was measured upon incubation with metal-chelating reagents . These results suggest that the cysteine residues of Tat are involved in the formation of intramolecular disulfide bonds. J Biol Chem, 1994 Mar 18, 269(11), 8334 - 40 A novel 39-kDa phosphoribosylpyrophosphate synthetase-associated protein of rat liver . Cloning, high sequence similarity to the catalytic subunits, and a negative regulatory role; Kita K et al.; The rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of the 34-kDa catalytic subunits (PRS I and II) and other 39- and 41-kDa proteins (Kita, K., Otsuki, T., Ishizuka, T., and Tatibana, M . (1989) J . Biochem . (Tokyo) 105, 736-741), which are termed here PRPP synthetase-associated proteins (PAPs) . We have cloned the cDNA for the major one of 39 kDa (PAP39) from a rat liver cDNA library . Nucleotide sequencing showed that the clone encoded 356 amino acids containing sequences of all five peptides derived from PAP39 . Surprisingly, the deduced amino acid sequence is markedly similar to those of the 34-kDa catalytic subunits . Excluding two regions (about 45 residues in total), PAP39 has a 48% identity with PRS I . Northern analysis detected a major 1.9-kilobase transcript in all 16 rat tissues examined, and the relative amounts of PAP39 mRNA to PRS I mRNA varied with tissues . Covalent cross-linking experiments gave definitive evidence for molecular interaction of PAP39 with the catalytic subunits . Immunoprecipitation experiments revealed that all the catalytic subunits existed as complexes containing PAP39 . When PAPs were eliminated from the rat liver enzyme complex by gel filtration in the presence of 1 m MgCl2, a lyotrope, or by mild tryptic treatment, the enzyme activity of the remaining catalytic subunits increased . Based on these results, we propose that PAP39, the major component of PAPs, plays a negative regulatory role in PRPP synthesis and hence is an important factor controlling nucleotide syntheses in general. J Biol Chem, 1994 Mar 18, 269(11), 8280 - 6 Transcription factor (TF) IIB and TFIIA can independently increase the affinity of the TATA-binding protein for DNA; Imbalzano AN et al.; The general transcription factors (TF) IIB and TFIIA are the first factors to associate with the TATA-binding protein (TBP) during formation of a transcription initiation complex on RNA polymerase II promoters . DNase I footprint titration was used to measure the effects of TFIIB and TFIIA on binding of TBP to a consensus TATA box . Under reaction conditions optimized for TBP-DNA complex formation, the presence of TFIIB increased affinity of TBP for the TATA box by 2.5-fold, while TFIIA had no effect . When TBP binding conditions were sub-optimal, both TFIIB and TFIIA independently increased TBP affinity by approximately 10-fold . Therefore both TFIIB and TFIIA have the intrinsic ability to directly increase the affinity of TBP for the TATA box . We suggest that this property of TFIIA and TFIIB may increase the range of conditions under which high affinity TBP-DNA interactions can occur and may therefore favor the formation of the preinitiation complex. J Biol Chem, 1994 Mar 18, 269(11), 8246 - 54 Purification and biochemical characterization of Escherichia coli RecA proteins mutated in the putative DNA binding site; Cazaux C et al.; Escherichia coli RecA protein plays a central role both in DNA repair and in recombination . We report biochemical properties of three new RecA proteins mutated at positions 199 (RecA694), 207 (RecA659), and 211 (RecA611) in the putative DNA binding site . RecA694 had a wild-type phenotype, whereas RecA611 and RecA659 were deficient in promoting both the self-cleavage of LexA repressor and the DNA-strand exchange reaction . In order to determine the origin of this inhibition, we examined the capacity of wild-type and mutant proteins to bind to single-stranded DNA (with and without single-stranded binding protein, SSB), double-stranded DNA, and ATP . DNA strand exchange defects were correlated with the inability of mutant proteins to displace SSB from DNA . For the recA659 mutation this inhibition was reversed by equimolar wild-type protein . In contrast, mixtures of either wild-type/RecA659 or wild-type/RecA611 proteins remained deficient in LexA cleavage, suggesting that the dominant negative phenotype of the mutant proteins may be a consequence of the formation heterologous RecA complexes . Various mutations in the putative DNA binding site of RecA protein altered ATP binding, ATPase activity, displacement of SSB from single-stranded DNA, and protein-protein interactions . These results are consistent with the hypothesis that DNA binding to this site of RecA relays allosteric effects to several functional domains throughout the protein. J Biol Chem, 1994 Mar 18, 269(11), 8220 - 5 The sulfurtransferase activity and structure of rhodanese are affected by site-directed replacement of Arg-186 or Lys-249; Luo GX et al.; Two mutants of the enzyme rhodanese that replace Arg-186 with Leu (R186L) or Lys-249 with Ala (K249A) were prepared to test suggestions that these residues are involved in catalysis and structure . The predominant effect with R186L was functional, and Km for the sulfur donor, thiosulfate, increased from 3.7 mM to 73 mM with a modest decrease in Vmax (672 IU/mg to 576 IU/mg) . However, K249A was virtually inactive using thiosulfate, but it was active with thiosulfonates such as p-toluene-, 2-aminoethane-, or ethanethiosulfonate, and these compounds could be demonstrated to form persulfide-substituted rhodanese . Compared with wild type enzyme, K249A had (a) reduced stability, (b) comparable secondary structure, (c) more easily exposed hydrophobic surfaces, and (d) a core structure that denatured similarly to the wild type enzyme . Thus, Arg-186 and Lys-249 are important in rhodanese catalysis, and Lys-249 is particularly critical for substrate selectivity and protein stability . Finally, the results suggest that there can be active rhodanese species in vivo that will be undetected using thiosulfate as a sulfur donor. J Biol Chem, 1994 Mar 18, 269(11), 8182 - 8 The NAD(P)H:flavin oxidoreductase from Escherichia coli as a source of superoxide radicals; Gaudu P et al.; The NAD(P)H:flavin oxidoreductase (encoded by the fre gene) of Escherichia coli is a soluble enzyme which, under aerobic conditions and together with NAD(P)H and flavins, generates superoxide radicals selectively . This was demonstrated from spin trapping experiments and from the ability of the flavin reductase to achieve a superoxide dismutase (SOD)-sensitive reduction of cytochrome c . The participation of the flavin reductase to O2- . generation in E . coli cells has been studied . Superoxide production in dialyzed cytosolic fraction of SOD-deficient E . coli was stimulated by the addition of flavins . There was no stimulation in soluble extracts of flavin reductase-deficient strains . Moreover, using fusions of sodA promoter to lacZ, we showed that sodA transcription was diminished in flavin reductase-deficient E . coli and that the induction of MnSOD by flavin reductase was SoxRS-independent . These results suggest that the flavin reductase might: (i) in vivo, be an important cytosolic site of O2- . generation; (ii) in vitro, serve as a simple, efficient, and selective O2- . generator. J Biol Chem, 1994 Mar 18, 269(11), 8146 - 52 Anti-metatype antibodies stabilize the fluorescein single-chain antibody 4-4-20 complex against dissociation by hydrostatic pressure; Coelho-Sampaio T et al.; Hydrostatic pressure was used to promote dissociation of fluorescein (Fl) from single-chain antibody 4-4-20 (SCA 4-4-20) . Fl fluorescence intensity was quenched by 97% upon binding to SCA 4-4-20 . Increasing pressure to 2.4 kbar enhanced Fl fluorescence from the remaining 3% to 14-17% . The capacity of anti-metatype antibodies (anti-Met), which specifically recognize liganded anti-Fl antibodies, to protect against pressure-induced Fl dissociation was tested . Both polyclonal and monoclonal anti-Met antibodies protected against Fl dissociation, reducing the fluorescence intensity at 2.4 kbar from 14-17% to 6-8% . Additive effects of anti-Met antibodies in protection against pressure-induced Fl dissociation were suggested by the fact that a 2-fold molar excess polyclonal anti-Met reagent promoted additional protection relative to an equimolar amount . On the other hand, combination of different monoclonal anti-Met antibodies did not promote additive protection, suggesting recognition of overlapping metatopes by these monoclonals . The complex formed by SCA 4-4-20 and the Fl analog HPF was more sensitive to pressure than the Fl-SCA 4-4-20 complex . Addition of both polyclonal and monoclonal anti-Met antibodies reduced the Fl fluorescence recovery at 2.4 kbar from 75% to 40-55% . In order to directly study binding of anti-Met antibodies to mAb 4-4-20, monoclonal anti-Met antibody 3A5-1 was labeled with 2-dimethylaminonaphthalene-5-sulfonyl chloride (2,5-Dns-Cl) and Dns fluorescence anisotropy measured . Unliganded mAb 4-4-20 did not bind to 2,5-Dns-3A5-1 as indicated by the absence of measurable changes in Dns fluorescence anisotropy upon increasing mAb concentration . Addition of mAb 4-4-20 bound to Fl produced a sigmoidal increase in Dns anisotropy, compatible with association of the primary immune complex and 3A5-1 . An affinity constant, K0.5, of 1.5 x 10(-7) M and a cooperativity coefficient (n) of 3.1 were calculated for formation of the Fl-mAb 4-4-20 complex . The HPF-mAb 4-4-20 complex was also recognized by 2,5-Dns-3A5-1 but with lower affinity, indicating that the monoclonal anti-Met 3A5-1 distinguished between mAb 4-4-20 liganded to different haptens. J Biol Chem, 1994 Mar 18, 269(11), 8091 - 8 Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction; Morell MK et al.; The side chain of residue threonine 65 within the active site of ribulosebisphosphate carboxylase participates in a network of hydrogen bonds and ionic interactions involving the phosphate moiety attached to C-1 of the substrate . This residue was replaced with serine, alanine, and valine in the enzyme from Synechococcus PCC 6301 . The mutant enzymes were stable, expressed abundantly by Escherichia coli, and retained the ability to form gel-filterable complexes with the reaction-intermediate analog, 2'-carboxyarabinitol-1,5-bisphosphate . The substitutions reduced the kcat/Km(CO2) (where kcat is the substrate-saturated turnover rate) of the enzyme from 17- to 340-fold with the more radical substitutions causing more severe reductions . The CO2/O2 specificity also deteriorated progressively, the valine replacement causing a 2.3-fold reduction . In concert with these changes, a compound tentatively identified as 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate, the product of beta elimination of the 2,3-enediol(ate) intermediate of the catalytic reaction, appeared among the reaction products in progressively increasing amounts . In the case of the valine substitution, it comprised 13% of the ribulose bisphosphate consumed . The mutant enzymes also partitioned more of their reaction flux to pentulose bisphosphate isomers of ribulose bisphosphate . By contrast, the diversion of carboxylated product to pyruvate, as a result of beta elimination of the three-carbon aci-carbanion intermediate of the carboxylation reaction, was ameliorated by the replacements, the valine mutant showing a 5-fold improvement in this parameter . These observations focus attention on a geometric conflict which exists between the requirements for stabilization of the 5-carbon enediol(ate) and 3-carbon aci-carbanion intermediates . This conflict must be resolved by a change in the angle of the C-1/bridge oxygen bond during each catalytic cycle . The network of hydrogen bonds involving the side chain of threonine 65 must play a crucial role in facilitating reaction of the enediol(ate) with the gaseous substrate and in shepherding this subsequent movement. J Biol Chem, 1994 Mar 18, 269(11), 8029 - 35 Tn10/IS10 transposase purification, activation, and in vitro reaction; Chalmers RM et al.; We describe a method for the purification of Tn10/IS10 transposase that relies on the aggregation of the protein after overexpression in Escherichia coli . Aggregated transposase was solubilized before the final purification step, a gel-filtration column, using a combination of salt and detergent . This procedure is the first reported for the preparation of concentrated and active transposase from any IS element . The yield is 11 mg of purified protein at a concentration of 1 mg/ml from 2.5 g of cells . The procedure can be scaled up with ease . We also describe a treatment that activates transposase in either a crude or purified state . This involves dilution into a solution of salt plus organic solvent . In transposition reactions using supercoiled substrate plasmid, the activity was directly proportional to the amount of transposase added over a wide range of transposase/DNA ratios (0.2-2.0 molecules/DNA substrate molecule) . In this range 8 transposase molecules were added per transposition event . Maximum conversion of substrate to product (40%) was with 18 transposase molecules/transposition event . At higher levels of transposase with a constant amount of substrate, activity was reduced but could be restored by addition of nonspecific DNA . Both the specific activity of transposase and the type of products generated can be altered by changing in vitro assay conditions . The effects of salts, solvents, and pH value on the reaction are described. J Biol Chem, 1994 Mar 18, 269(11), 7982 - 8 Lactate monooxygenase . I . Expression of the mycobacterial gene in Escherichia coli and site-directed mutagenesis of lysine 266; Muh U et al.; Lactate monooxygenase utilizes oxygen in the conversion of L-lactate to acetate, CO2, and water . The gene for the enzyme from Mycobacterium smegmatis had been cloned into Escherichia coli (Giegel, D . A., Williams, C . H., Jr., and Massey, V . (1990) J . Biol . Chem . 265, 6626-6632) and the derived amino acid sequence compared to glycolate oxidase and flavocytochrome b2, enzymes of known three-dimensional structure (Lindqvist, Y., and Branden, C . I . (1989) J . Biol . Chem . 264, 3624-3628; Xia, Z . X., and Mathews, S . F . (1990) J . Mol . Biol . 212, 837-863) . There is strong homology, especially around residues in the active site . The mechanism proposed for lactate monooxygenase involves an intermediate having a negative charge at the N(1)-position of the FMN . Based on the homology, lysine 266 is the residue suggested to neutralize that charge . Wild type enzyme and several forms of the enzyme altered at active site residues by site-directed mutagenesis have been expressed in E . coli and purification procedures developed . The properties determined for the recombinant wild type enzyme were, in every case, the same as those previously determined for the enzyme isolated from M . smegmatis . Mutation of lysine 266 to a methionine created K266M . The semiquinone showed spectral features different from those found in the wild type enzyme and was no longer thermodynamically stable . This indicates a redox potential for the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple . The two-electron redox potential was determined to be -180 mV at 25 degrees C, pH 7.0 . In wild type enzyme, attack of the flavin ring by sulfite creates a negative charge at the FMN N(1)-position . In K266M, the stabilization of the sulfite adduct was 17,000-fold weaker (Kd approximately 10(-3) M) than in the wild type enzyme, with a rate of association that is lowered by 10,000-fold (kon = 1.2 M-1 s-1) . The rate of reduction with L-lactate is significantly decreased in K266M . Unexpectedly, binding of substrate and inhibitors is significantly weaker in K266M than in the wild type enzyme . In all properties involving a negative charge at position N(1) of the FMN, K266M is distinctly different from wild type enzyme . This makes it quite likely that lysine 266 serves the postulated role of interacting with this negative charge. J Biol Chem, 1994 Mar 18, 269(11), 7970 - 5 A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the carboxyl-terminal domain is active, monomeric, and generates antibodies that inhibit enzymatic activity; Schenkman S et al.; trans-Sialidase isolated from trypomastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is multimeric and heterogeneous in size . We show here that limited proteolysis of tans-sialidase with papain yields a single monomeric polypeptide chain of 70 kDa that conserves full enzymatic activity on soluble and membrane-bound substrates . The papain fragment lacks most of the 12-amino acid repeats of the carboxyl-terminal domain that comprises about 50% of the native trans-sialidase . When injected into rabbits, the papain-generated fragment induces antibodies that inhibit trans-sialidase activity and trypomastigote sialylation . The repeats are also not required for the stability of the enzyme or for the correct folding during the biosynthesis in Escherichia coli, but seem essential for trans-sialidase oligomerization . We conclude that trans-sialidase is composed of two structurally and functionally independent domains. J Biol Chem, 1994 Mar 18, 269(11), 7901 - 7 Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli . Determination of sites for putrescine activation of activity and processing; Stanley BA et al.; Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity . To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis . Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B . A., and Pegg, A . E . (1991) J . Biol . Chem . 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine . Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur . Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing . The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography . Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease . Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q . It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity. J Biol Chem, 1994 Mar 18, 269(11), 7869 - 73 Differential estrogenic regulation of small M(r) heat shock protein expression in osteoblasts; Cooper LF et al.; Polymerase chain reaction amplification and sequencing of heat shock protein (HSP) 27-related transcripts present in cDNAs generated from heat-shocked osteoblast RNA revealed the expression of three related open reading frames that encode proteins of 208, 197, or 175 amino acids . Their expression as recombinant proteins in Escherichia coli demonstrated that they encode 27,000, 25,000, or 22,000 M(r) proteins . Northern blot analysis of heat shock protein 27-related transcript expression revealed that, while HSP 27 expression (850-nucleotide transcript) was induced by heat shock alone, the expression of a smaller transcript was facilitated by estrogen treatment prior to heat shock . This corresponded with the co-induction of 18,000 and 22,000 M(r) proteins by estrogen-pretreated, heat-shocked osteoblasts as revealed by fluorography . The identification of these multiple small M(r) heat shock-induced proteins demonstrated that the stress response of mammalian cells is composed of multiple, related proteins whose expression is differentially regulated . The importance of estrogen-regulation of the small M(r) heat shock protein component of the stress response may be of particular significance to osteoblast physiology. J Biol Chem, 1994 Mar 18, 269(11), 7843 - 6 The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane; Knott TG et al.; The presence of secA and secY gene homologues in the plastid genomes of red algae and cyanophytes has raised the possibility that the products of these genes are involved in protein translocation across the thylakoid membrane . Bacterial SecA proteins are effectively inhibited by azide, and we have tested the effects of this compound on the transport of lumenal proteins across the thylakoid membrane in pea chloroplasts . Recent studies have shown that lumenal proteins are transported by two different mechanisms, one dependent on the thylakoidal delta pH and the other requiring the presence of a stromal protein factor and ATP . In this report we show that azide inhibits the transport across the thylakoid membrane of the latter group of proteins, which includes plastocyanin and the lumenal 33-kDa protein of photosystem II; translocation of proteins by the delta pH-dependent pathway is unaffected . Following import into isolated chloroplasts in the presence of azide, a substantial proportion of plastocyanin and the 33-kDa protein is found as the stromal intermediate form; the proportion increases with lower ATP concentrations, suggesting that azide and ATP may compete for a single site . The presence of azide completely inhibits the import of the 33-kDa protein by isolated thylakoids, but import is restored if the azide is removed from the stromal extract or thylakoids, prior to the import incubation . The data thus indicate that azide reversibly inhibits the transport of a subset of proteins across the thylakoid membrane, consistent with the involvement of a SecA homolog . The results also indicate that azide is potentially a valuable tool for the future assignment of novel lumenal proteins to one of the thylakoidal protein transport mechanisms. Science, 1994 Mar 18, 263(5153), 1625 - 9 Mutation of a mutL homolog in hereditary colon cancer; Papadopoulos N et al.; Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene . A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene . One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds . Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease . These results suggest that defects in any of several mismatch repair genes can cause HNPCC. Nature, 1994 Mar 17, 368(6468), 261 - 5 Destabilization of the complete protein secondary structure on binding to the chaperone GroEL; Zahn R et al.; Protein folding in vivo is mediated by helper proteins, the molecular chaperones, of which Hsp60 and its Escherichia coli variant GroEL are some of the best characterized . GroEL is an oligomeric protein with 14 subunits each of M(r) 60K, which possesses weak, co-operative ATPase activity and high plasticity . GroEL seems to interact with non-native proteins, binding one or two molecules per 14-mer in a 'central cavity', but little is known about the conformational state of the bound polypeptides . Here we use nuclear magnetic resonance techniques to show that the interaction of the small protein cyclophilin with GroEL is reversible by temperature changes, and all amide protons in GroEL-bound cyclophilin are exchanged with the solvent, although this exchange does not occur in free cyclophilin . The complete secondary structure of cyclophilin must be disrupted when bound to GroEL. Biochim Biophys Acta, 1994 Mar 16, 1205(1), 146 - 50 Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase; Vertessy BG et al.; dUTP nucleotidohydrolase (dUTPase, EC 3.6.1.23) from E . coli contains a total of six tyrosine residues per trimer . About half of them were found to be susceptible to acetylation with N-acetylimidazole or to nitration with tetranitromethane with concomitant loss of activity . Deacetylation with N-hydroxylamine leads to full reactivation . Inhibitory products of dUTP hydrolysis, i.e., dUMP and inorganic pyrophosphate together with the cofactor Mg2+ protect significantly against inactivation and chemical modification . In the Cu(2+)-dUTPase complex, charge transfer from Cu2+ to the tyrosinate anion was perturbed by the presence of the substrate dUTP . These results, together with the occurrence of one tyrosine residue in a strictly conserved sequence motif suggest the critical importance of this residue for the function of the enzyme. Biochim Biophys Acta, 1994 Mar 16, 1205(1), 113 - 22 Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues; Fukuchi T et al.; We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat . To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied . cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli . Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end . The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies . Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity . They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability . Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition . The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis . There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum. FEMS Microbiol Lett, 1994 Mar 15, 117(1), 79 - 83 Phenylalanine- and tyrosine-dependent production of enterobactin in Escherichia coli; Foster MS et al.; Under low-iron conditions, Escherichia coli synthesizes the siderophore enterobactin . When compared to wild-type cells grown in iron sufficient medium, cells grown under iron limitation, in the absence of tyrosine and phenylalanine or the presence of both, increased catechol production (a measure of enterobactin and its degradation product 2,3-dihydroxybenzoic acid) 5- to 9-fold while cells supplemented with tyrosine alone produced a 10- to 20-fold increase . Mutations in fur, tyrA, pheA, or pheU generally resulted in increased enterobactin production, while a tyrR mutant was unaffected by combinations of tyrosine and phenylalanine. J Immunol, 1994 Mar 15, 152(6), 3064 - 72 Mast cell and neutrophil expression of dog mast cell protease-3 . A novel tryptase-related serine protease; Yezzi MJ et al.; In previous work the primary structure of a previously unknown protease was deduced from the sequence of a dog mastocytoma cDNA . The predicted preproprotein shares some features with mast cell tryptases but is no more than 49% identical in sequence to known trypsin-like enzymes, including dog tryptase . This study explores the expression of this protein, termed dog mast cell protease-3 (dMCP-3) . A polyclonal Ab was raised to a peptide corresponding to residues 166-181 of the deduced sequence . Anti-dMCP-3(166-181) Ig recognizes dMCP-3 expressed as a CheY fusion protein in Escherichia coli and binds to a approximately 36-kDa protein in extracts of dog mastocytomas . The Ab does not recognize dog tryptase, dMCP-3s closest known relative in mastocytoma cells . When used with fluorescein-conjugated and alkaline phosphatase-conjugated secondary Abs, anti-dMCP-3(166-181) Ig yields punctate cytoplasmic staining in mastocytoma cells, suggesting localization to intracellular granules . Staining is greatly reduced by preincubation with synthetic dMCP-3 peptide, supporting the specificity of the Ab . Immunohistochemical staining of normal dog tissues reveals scattered dMCP-3 reactive cells in skin, intestine, trachea, and lung parenchyma . Double staining with Ab and methylene blue shows that anti-dMCP-3(166-188) Ig recognizes extravascular mononuclear tissue cells with metachromatic granules . In addition, cytoplasmic staining is seen in polymorphonuclear leukocytes within vessels in tissue sections and in leukocytes harvested from blood . Hybridization of dMCP-3 cDNA to dog skin RNA provides further evidence of dMCP-3 gene transcription in normal tissue . Thus, this study provides immunochemical evidence of dMCP-3 expression in dog mast cell tumors, normal tissue mast cells, and neutrophils. Experientia, 1994 Mar 15, 50(3), 253 - 60 Some features of base pair mismatch repair and its role in the formation of genetic recombinants; Fox MS et al.; For the formation of recombinants involving closely linked markers, two distinct processes play a role . The recombinational interaction between homologous DNA molecules results in the presence of heteroduplex DNA joining the parental components of the recombinant . The presence of markers distinguishing the parents in the region of heteroduplex DNA can result in base pair mismatches . The post recombination repair of such mismatches can contribute to the separation of closely linked markers . The processes responsible for such repair also play roles in mutation avoidance . The specificities, functions and contribution to the formation of recombinants for closely linked markers of the processes in Escherichia coli are described. Experientia, 1994 Mar 15, 50(3), 216 - 22 Processing of Holliday junctions by the Escherichia coli RuvA, RuvB, RuvC and RecG proteins; Muller B et al.; Recent work has led to significant advances in our understanding of the late steps of genetic recombination and the post-replicational repair of DNA . The RuvA and RuvB proteins have been shown to interact with recombination intermediates and catalyse the branch migration of Holliday junctions . Although both proteins are required for branch migration, each plays a defined role with RuvA acting as a specificity factor that directs RuvB (an ATPase) to the junction . The RuvB ATPase provides the motor for branch migration . The next step is catalysed by RuvC protein which recognises Holliday junctions and promotes their resolution by endonucleolytic cleavage . New data indicates an alternative pathway for Holliday junction processing . This pathway involves RecG, a branch migration protein which is functionally analogous to RuvAB, and a protein (activated by a rus mutation) which works with RecG to process intermediates independently of RuvA, RuvB and RuvC. Experientia, 1994 Mar 15, 50(3), 204 - 15 In vitro reconstitution of homologous recombination reactions; Kowalczykowski SC; The proteins essential to homologous recombination in E . coli have been purified and their individual activities have been identified, permitting biochemical reconstitution of steps that comprise the cellular recombination process . This review focuses on the biochemical events responsible for the initiation and homologous pairing steps of genetic recombination . The properties of an in vitro recombination reaction that requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(chi), are described . The recBCD enzyme serves as the initiator of this reaction; its DNA helicase activity produces single-stranded DNA that is used by the recA protein to promote homologous pairing and DNA strand invasion of supercoiled (recipient) DNA . The SSB protein acts to trap the single-stranded DNA produced by recBCD enzyme and to facilitate pairing by the recA protein . The chi regulatory sequence acts in cis by attenuating the nuclease, but not the helicase, activity of recBCD enzyme . This attenuation assures the preservation of ssDNA produced by the DNA helicase activity and is responsible for the simulation in vitro and, presumably, in vivo . The attenuation of nuclease activity by chi results in the loss or functional inactivation of the recD subunit. Experientia, 1994 Mar 15, 50(3), 192 - 203 Structure and function of RecA-DNA complexes; Stasiak A et al.; While the E . coli RecA protein has been the most intensively studied enzyme of homologous recombination, the unusual RecA-DNA filament has stood alone until very recently . It now appears that this protein is part of a universal family that spans all of biology, and the filament that is formed by the protein on DNA is a universal structure . With RecA's role in recombination given new and greatly increased significance, we focus in this review on the energetics of the RecA-mediated strand exchange and the relation between the energetics and recombination spanning heterologous inserts. Eur J Biochem, 1994 Mar 15, 220(3), 739 - 44 Escherichia coli elongation-factor-Tu mutants with decreased affinity for aminoacyl-tRNA; Andersen C et al.; The two evolutionary well-conserved histidine residues, His66 and His118, of Escherichia coli elongation factor Tu have been subjected to mutational analysis . The two histidines have each been replaced by alanines, denoted H66A and H118A, respectively . His118 has also been substituted by glutamate, H118E . The three mutants have been characterized with respect to thermostability, GTPase activity and affinity for aminoacylated tRNA . Most conspicuously, the tRNA affinity is reduced or almost abolished . k-1 for dissociation of the ternary complex increases by factors of 14, 40 and 48 for H66A, H118A and H118E, respectively, when compared to the wild type . The half-lives for the non-enzymic deacylation of aminoacylated tRNA in the ternary complex are 391, 107, 69, 54 and 61 min for wild type, H66A, H118A, H118E and free aminoacylated tRNA, respectively . The Kd is about 20-times higher for H66A compared to wild type . Our results strongly suggest that His66 and His118 play major roles in stabilization of the ternary complex. Eur J Biochem, 1994 Mar 15, 220(3), 693 - 701 Isolation and expression of a gene specifying a cdc2-like protein kinase from the human malaria parasite Plasmodium falciparum; Ross-Macdonald PB et al.; A partially redundant oligonucleotide based on conserved protein sequences of cdk and cdc2-like proteins was used to isolate from genomic libraries of Plasmodium falciparum fragments of chromosome XIII carrying a 288-residue open-reading frame encoding a protein kinase sharing 57-58% identity with yeast p34cdc2 . Based on sequence data, base composition and the striking similarity with other cdk and related proteins, four intervening sequences were identified . Their removal in vitro allowed expression of the gene, designated PfPK5, in Escherichia coli, the resulting product having kinase activity against casein and histone H1 . Western blotting using a polyclonal antibody raised against the expressed protein showed that the kinase was located in the parasite's cytosol and was present in approximately constant amounts throughout the intra-erythrocytic asexual reproductive stage of the life cycle . The PSTAIRE region of the PfPK5 protein differs at three sites from that of p34cdc2, and the gene failed to complement cdc2/28 yeast mutants . However, Western blotting showed that the gene was not expressed in yeast, so this does not eliminate the possibility that it is the malarial version of cdc2. EMBO J, 1994 Mar 15, 13(6), 1310 - 7 Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway; Michl D et al.; The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin . This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase . The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids . Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates . The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism . In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II . The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH . This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1994 Mar 15, 13(6), 1263 - 9 The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity; Bode W et al.; Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling . They have been implicated in various disease processes including tumour invasion and joint destruction . These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition . Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III . Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine . The 2.0 A crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a five-stranded beta-sheet, two alpha-helices, and bridging loops . The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite . Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites . The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor . In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 748 - 54 A novel human homologue of a dead-box RNA helicase family; Kitajima Y et al.; Putative cDNA clones for a nuclear antigen that cross-reacts with anti-human aldolase A monoclonal antibody MAb1A2 were isolated from the HeLa lambda gt11 cDNA library and a candidate clone (clone 3) was analyzed . The cDNA has an open reading frame (ORF) of 1,317 bp encoding a novel RNA helicase belonging to the DEAD RNA helicase family . The ORF also contains a nuclear targeting signal and the epitope for MAb1A2 . The putative RNA helicase has sequence similarity to Escherichia coli RNA helicase DEAD, mouse translation factor eIF-4A, and human p68 and p54. Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 577 - 81 Site-directed mutagenesis of two highly conserved residues near the active site of phosphofructo-1-kinase; Zheng RL et al.; Mutations of either of two highly conserved residues near the active site of Escherichia coli phosphofructokinase, Ile-126 or Asn-128, produce no changes in the Km for ATP, relatively small changes in kcat, and a large increase in the Km for fructose 6-P, despite the fact that these residues are not directly involved in substrate binding . A computer graphics analysis of the three-dimensional structure suggests that the mutations effect the orientation of Arg-252, a residue important both for fructose 6-P binding and for catalysis. Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 1041 - 8 Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro; Engel M et al.; We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2) . The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used . The homologous NDPK's from Yeast and E . coli were also substrates for CK-2 in vitro, but not Drosophila NDPK . Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent . The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122 . The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis. Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2265 - 9 Cloning and expression of a distinctive class of self-incompatibility (S) gene from Papaver rhoeas L; Foote HC et al.; We present the identification, cloning, and characterization of a self-incompatibility (S) gene from Papaver rhoeas that has no significant homology to any previously reported gene sequences, including S genes from other species . This result suggests that a different self-incompatibility mechanism may be operating in this species and has important implications for the evolutionary relationships between the S genes . The S1 cDNA was cloned by using an oligonucleotide based upon N-terminal amino acid sequence data from stigmatic proteins that show complete linkage with the S1 gene . The single-copy gene has been expressed in Escherichia coli to test biological activity . Although the recombinant S1 protein (S1e) is not processed in the same way as the protein produced in the plant, it exhibits, in vitro, the specific pollen inhibitory activity expected of an S gene product; pollen carrying the S1 allele is inhibited, whereas pollen not carrying S1 is not inhibited . These results provide definitive demonstration that the product of a cloned S gene has S-specific pollen inhibitory activity. Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2240 - 4 Cloning of a 3-methyladenine-DNA glycosylase from Arabidopsis thaliana; Santerre A et al.; We have isolated an Arabidopsis thaliana cDNA that complements the methyl methanesulfonate-sensitive phenotype of an Escherichia coli double mutant deficient in 3-methyladenine glycosylases (DNA-3-methyladenine glycosidases I and II, EC 3.2.2.20 and 3.2.2.21, respectively, encoded by tag and alkA) . Expression of the Arabidopsis cDNA enhances the methyl methanesulfonate resistance of the E . coli double mutant by nearly four orders of magnitude . The cDNA corresponds to a single-copy, nuclearly encoded sequence which specifies a predicted 28.1-kDa protein with a charge of +8 at pH 7.0 . Enzymatic analysis of extracts prepared from the transformed mutants indicates that the cDNA encodes a 3-methyladenine glycosylase . The predicted amino acid sequence of the Arabidopsis glycosylase has significant homology to other eukaryotic 3-methyladenine glycosylases. Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2191 - 5 Role of an Escherichia coli stress-response operon in stationary-phase survival; Weiner L et al.; The phage shock protein operon (pspABCE) of Escherichia coli is strongly expressed in response to stressful environmental conditions, such as heat shock, ethanol treatment, osmotic shock, and filamentous phage infection . We show that bacteria lacking the pspABC genes exhibit a substantial decrease in the ability to survive prolonged incubation in stationary phase under alkaline conditions (pH 9) . The psp mutant bacteria grow approximately as well as wild-type strains in the alkaline medium, and stationary-phase survival of the psp mutants improves substantially at pH values closer to the optimal growth range (pH 6-8) . In late stationary-phase (1- to 2-day-old) cells, the operon can be strongly induced under certain conditions, and PspA can become one of the most highly expressed bacterial proteins . The combination of stationary-phase starvation and alkaline pH is likely to place a severe strain on the maintenance of endogenous energy sources, and, consistent with these effects, we find that psp expression is also induced by uncouplers of oxidative phosphorylation and other agents that interfere with energy production . The death rate of psp mutants in stationary phase is accelerated by the presence of wild-type bacteria in the same culture, suggesting that the psp operon may play a significant role in enabling E . coli to compete for survival under nutrient- or energy-limited conditions. Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2146 - 50 Folded proteins occur frequently in libraries of random amino acid sequences; Davidson AR et al.; A library of synthetic genes encoding 80- to 100-residue proteins composed mainly of random combinations of glutamine (Q), leucine (L), and arginine (R) has been expressed in Escherichia coli . These genes also encode an epitope tag and six carboxyl-terminal histidines . Screening of this library by immunoblotting showed that 5% of these QLR proteins are expressed at readily detectable levels . Three well-expressed QLR proteins were purified and characterized . Each of these proteins has significant alpha-helical content, is largely resistant to degradation by Pronase, and has a distinct oligomeric structure . In addition, one protein unfolds in a highly cooperative manner . These properties of the QLR proteins demonstrate that they possess folded structures with some native-like properties . The QLR proteins differ from most natural proteins, however, in being remarkably resistant to denaturant-induced and thermal-induced unfolding and in being relatively insoluble in the absence of denaturants. Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2120 - 4 RNA polymerase binding using a strongly acidic hydrophobic-repeat region of sigma 54; Tintut Y et al.; sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties . It contains a strongly acidic region of previously unknown function . Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter . Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding . Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase . This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases. Biochemistry, 1994 Mar 15, 33(10), 3063 - 70 Arrangement of messenger RNA on Escherichia coli ribosomes with respect to 10 16S rRNA cross-linking sites; Bhangu R et al.; The arrangement of the mRNA on the Escherichia coli ribosome with respect to ribosomal RNA sites has been investigated by photochemical cross-linking experiments . mRNA analogues 51-54 nucleotides in length contained a Shine-Dalgarno sequence, a single codon for tRNA(Gly), and 4-thiouridine (s4U) in the 5' third of the mRNA (-20 to -12), in the middle third of the mRNA (-3 to +6), or in the 3' third of the mRNA (+20 to +26), where the position numbers are counted from the first nucleotide of the codon . Complexes were formed with these mRNAs and 70S ribosomes in the absence or presence of tRNA(Gly) and were irradiated . The extent of cross-linking and the identity of cross-linked rRNA sites were determined on agarose gels and by primer extension . 16S rRNA nucleotides A412, A532, G693 (weakly), U723, and U1381 (weakly) cross-linked with s4U in the 3' third; A532, G693, U723, A1167 (weakly), U1381, G818 (weakly), and A845 cross-linked with s4U in the middle; A532, G693, U723, A1167, G818 (weakly), and A845 cross-linked with s4U in the 5' third . All of these cross-links occur with tRNA independence . Cross-links at C1395 and A1196 occur for all three mRNAs with tRNA dependence . The pattern of these sites provides information about the order of the rRNA sites along the mRNA track, and they also point out the apparent overlapping neighborhoods for the mRNA track . Models for the track of the mRNA on the 30S subunit are considered to explain this pattern of interactions. Biochemistry, 1994 Mar 15, 33(10), 2951 - 60 Unfolding studies of the protease domain of urokinase-type plasminogen activator: the existence of partly folded states and stable subdomains; Nowak UK et al.; The domain structure and the stability against thermal and chemical denaturation of urokinase-type plasminogen activator (u-PA) have been investigated by NMR spectroscopy and differential scanning calorimetry (DSC) . At least five structurally autonomous regions of this three-domain protein have been found to exist . Two of these are the EGF-like and the kringle domains; the others are all within the third domain, which is a serine protease . The latter undergoes three unfolding transitions in its enzymatically active form . Reaction with a specific affinity label (L-Glu-L-Gly-L-Arg-chloromethyl ketone) to produce an inactivated protein results in a stabilization of the structure involved in two of these transitions, and an increase in cooperativity to give a domain which unfolds in two, not three, distinct steps . These are attributed to the denaturation of the two major subdomains of the protease structure . One of the subdomains has exceptional stability, being unfolded only under extreme conditions such as 75 degrees C at pH 2.5 or 4 M GuDCl at pH 4.5 and 29 degrees C . This region has been identified by isolation and characterization of a fragment (residues Ile-159 to Thr-277) obtained by limited proteolysis with thermolysin under conditions where the protease domain was partly unfolded . The NMR data are consistent with this stable region being at the N-terminus of the protein and indicate that its structure and stability are similar to those of the corresponding region of the native protein . These results support the idea that the u-PA protease domain has structural resemblance to the digestive serine proteases, but that stabilizing interactions within the structure can differ significantly between a group of homologous proteins. Biochemistry, 1994 Mar 15, 33(10), 2945 - 50 A molecular wedge for triggering the amidotransferase activity of carbamoyl phosphate synthetase; Mareya SM et al.; The reactive cysteine residue within the small subunit of Escherichia coli carbamoyl phosphate synthetase has been identified using the technique of site-directed mutagenesis . Three cysteine residues have previously been found to react with N-ethylmaleimide (NEM) under controlled reaction conditions . Two of these cysteine residues are found on the large subunit, while the third cysteine is located on the small subunit . In the present investigation, Cys-248 of the small subunit has been identified as the residue that reacts with NEM in the presence of MgATP and bicarbonate . Three cysteine residues of the small subunit at positions 131, 214, and 248 were individually mutated to serine residues . These site-specific changes, in addition to N-ethylmaleimide-labeling studies, demonstrated that Cys-248 is the amino acid that reacts with N-ethylmaleimide . Substitution of Cys-248 of the small subunit with larger residues (Asp, Phe, Arg, and Trp) was conducted in order to more closely mimic the observed properties of the NEM-labeled enzyme . The partial glutaminase activity of the C248D mutant increased 40-fold relative to the wild-type enzyme, while the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished . Similar, but less dramatic, effects were observed for the other mutants, C248S, C248R, C248F, and C248W . There was good correlation between the extent of enhancement of the partial glutaminase activity and an uncoupling of the phosphorylation reactions that occur on the large subunit.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Mar 15, 33(10), 2873 - 9 Cellular retinoid-binding proteins: limited proteolysis reveals a conformational change upon ligand binding; Jamison RS et al.; Intracellular retinoid-binding proteins are small, tightly folded, compact proteins, which appear to be involved in the delivery of retinoids to microsomal metabolic enzymes, among other potential roles . Recently, it has been demonstrated that two of these binding proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein type II {CRBP(II)}, interact with the same microsomal enzyme but in different manners, depending on the absence or presence of ligand {Herr, F.M., & Ong, D.E . (1992) Biochemistry 31, 6748-6755} . The structural components of the binding proteins responsible for these differential interactions are presently unknown . In addition, it is not clear how the ligand is able to gain entry into the solvent-inaccessible interior binding cavity . Limited proteolysis of the apo and holo forms of CRBP and CRBP(II) was used to probe the conformational differences between the different states of these two proteins in solution . It was found that the apo forms of both proteins were significantly more susceptible to proteolysis, and probably adopted a more open conformation, than the holo forms . The initial cleavage site of endoproteinase Arg-C in the apo forms occurred at a conserved arginine residue near a possible site of ligand entry . Similar results were obtained by limited proteolysis of cellular retinoic acid-binding protein and heart fatty acid-binding protein, indicating that a common ligand-induced conformational change may occur for other members of this family of intracellular binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Mar 15, 33(10), 2852 - 8 Determination of the secondary structure and folding topology of an RNA binding domain of mammalian hnRNP A1 protein using three-dimensional heteronuclear magnetic resonance spectroscopy; Garrett DS et al.; The secondary structure and folding topology of the first RNA binding domain of the human hnRNP A1 protein was determined by multidimensional heteronuclear NMR spectroscopy . The 92 amino acid long domain exhibits a beta alpha beta beta alpha beta folding pattern, arranged in a four-stranded antiparallel beta-sheet flanked by two alpha-helices, which is very similar to that found for other members of this family . Regions of marked variation between the structurally characterized RNA binding proteins of this class to date are mainly localized in the loops connecting the secondary structure elements. Biochemistry, 1994 Mar 15, 33(10), 2838 - 42 1H NMR studies of mouse ribonucleotide reductase: the R2 protein carboxyl-terminal tail, essential for subunit interaction, is highly flexible but becomes rigid in the presence of protein R1; Lycksell PO et al.; Mouse ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone . It has earlier been shown that the carboxyl-terminal part of the R2 protein is essential for subunit association to form the active enzyme complex . We now demonstrate that protein R2 gives rise to a number of sharp 1H NMR resonances, significantly narrower than the major part of the resonances . This line narrowing of certain resonances indicates segmental mobility in the molecule . In two-dimensional 1H TOCSY spectra of protein R2, cross-peak patterns from about 25 amino acid residues are visible . Most of these were assigned to the carboxyl-terminal part of the protein by comparisons with cross-peak patterns of oligopeptides corresponding to the carboxyl terminus of mouse R2 and to the patterns of a seven amino acid residue carboxyl-terminal truncated form of protein R2 . These results and the magnitude of the chemical shifts of the assigned residues demonstrate that the carboxyl-terminal part of mouse R2 protein is highly mobile compared to the rest of the protein and essentially unstructured . When protein R1 is added to a solution of protein R2, the sharp resonances are broadened, suggesting that the mobility of the carboxyl-terminal tail of protein R2 is reduced . The possibility of making direct observations of subunit interaction in native and mutagenized R1/R2 proteins should allow discrimination between effects of amino acid replacements on the catalytic mechanism and effects on subunit interaction. Biochemistry, 1994 Mar 15, 33(10), 2773 - 81 Solubilizing buried domains of proteins: a self-assembling interface domain from glutathione reductase; Leistler B et al.; In the dimeric glutathione reductase (GR) from Escherichia coli, the interface domain is largely surrounded by the other three domains in each subunit of the protein . Subgenes encoding three forms of the interface domain have been expressed in E . coli and the products purified from inclusion bodies: INT is the excised interface domain, as it is found in native GR; INTN and INTFN are variants carrying exchanges of surface residues in what would have been hydrophobic contact regions with other neighboring domains . The isolated INT domain was found to be a soluble and folded protein, but it was isolated as a mixture of the dimer and at least two species of higher molecular weight . The latter were believed to arise by further association of the dimer via the newly exposed and unsatisfied hydrophobic contact regions . In the variant INTN, three hydrophobic residues normally involved in the contact with the NADPH-binding domain in GR were replaced . This partly suppressed the further aggregation of the dimers . However, continued aggregation at high protein concentrations suggested that at least one further site of unwanted aggregation was still present . After four additional amino acid replacements in the region normally in contact with the FAD-binding domain, the resulting variant INTFN exhibited no unspecific aggregation, even at concentrations as high as 3.2 mg/mL.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Mar 15, 33(10), 2761 - 72 Three-dimensional solution structure of Escherichia coli periplasmic cyclophilin; Clubb RT et al.; The solution structure of the periplasmic cyclophilin type cis-trans peptidyl-prolyl isomerase from Escherichia coli (167 residues, MW > 18.200) has been determined using multidimensional heteronuclear NMR spectroscopy and distance geometry calculations . The structure determination is based on a total of 1720 NMR-derived restraints (1566 distance and 101 phi and 53 chi 1 torsion angle restraints) . Twelve distance geometry structures were calculated, and the average root-mean-square (rms) deviation about the mean backbone coordinate positions is 0.84 +/- 0.18 A for the backbone atoms of residues 5-165 of the ensemble . The three-dimensional structure of E . coli cyclophilin consists of an eight-stranded antiparallel beta-sheet barrel capped by alpha-helices . The average coordinates of the backbone atoms of the core residues of E . coli cyclophilin have an rms deviation of 1.44 A, with conserved regions in the crystal structure of unligated human T cell cyclophilin {Ke, H . (1992) J . Mol . Biol . 228, 539-550} . Four regions proximal to the active site differ substantially and may determine protein substrate specificity, sensitivity to cyclosporin A, and the composite drug:protein surface required to inhibit calcineurin . A residue essential for isomerase activity in human T cell cyclophilin (His126) is replaced by Tyr122 in E . coli cyclophilin without affecting enzymatic activity. Biochemistry, 1994 Mar 15, 33(10), 2757 - 60 Shift in pH-rate profile and enhanced discrimination between dicarboxylic and aromatic substrates in mitochondrial aspartate aminotransferase Y70H; Pan P et al.; Tyr70 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by oligonucleotide-directed mutagenesis . Aspartate aminotransferase Y70H retained at pH 7.5 13% of the activity toward dicarboxylic amino acids, whereas the activity toward aromatic amino acids was only 0.6% of that of the wild-type enzyme, corresponding to a 22-fold increase in the ratio of the activities toward these two types of substrates . In comparison to that of the wild-type enzyme, the low-pH limb of the pH-activity profile of the mutant enzyme was shifted to higher pH values, very likely reflecting the titration curve of the newly introduced histidine residue with a pKa' of 6.3 . Apparently, a positively charged residue at position 70 abolishes enzymic activity . The spectrophotometrically determined pKa' value of the internal aldimine formed between pyridoxal 5'-phosphate and Lys258 in the mutant enzyme was 6.0, similar to that in the wild-type enzyme . The rate constant of the dissociation of pyridoxamine 5'-phosphate from the mutant enzyme was increased only 3 times over that of the wild-type enzyme, in contrast to the 80-fold increase in Escherichia coli aspartate aminotransferase Y70F {Toney, M . D., & Kirsch, J . F . (1987) J . Biol . Chem . 262, 12403-12405}, suggesting that His70 can replace Tyr70 in forming a hydrogen bond to the coenzyme. Structure, 1994 Mar 15, 2(3), 151 - 8 AB5 ADP-ribosylating toxins: comparative anatomy and physiology; Burnette WN; The crystal structures recently determined for pertussis toxin, cholera toxin, and E . coli heat-labile toxins promise advances in rational vaccine design and improved understanding of G protein-mediated signal transduction. J Physiol, 1994 Mar 15, 475(3), 531 - 7 Enterotoxin Escherichia coli STa activates a nitric oxide-dependent myenteric plexus secretory reflex in the rat ileum; Rolfe V et al.; 1 . Mucosally added enterotoxin Escherichia coli STa increased the electrogenic Cl- secretion measured as the short-circuit current (Isc) across isolated muscle-stripped and muscle-unstripped rat mid-ilea incubated in vitro . 2 . Pretreatment with serosal L-NAME (N omega-nitro-L-arginine methyl ester) or tetrodotoxin (TTX) significantly reduced the maximum Isc and the duration of action of STa in the unstripped but not stripped ilea . D-NAME (serosal), indomethacin or 5-hydroxy-tryptamine-desensitization was ineffective on STa-induced Isc in either stripped or unstripped ilea . 3 . Serosal capsaicin reduced the maximum Isc of STa and its duration of action in unstripped ilea . 4 . L-Arginine induced a significantly larger increase in the Isc across unstripped ilea than across stripped ilea; this could be significantly reduced by serosal L-NAME or TTX, although these were ineffective in stripped ilea . 5 . Pretreatment of anaesthetized rats with I.P . L-NAME suppressed the fluid secretion induced by luminal STa in ilea in vivo but had no effect on that induced by luminal carbachol . 6 . Mucosal STa increased electrogenic Cl- secretion across intact rat ileum in vitro by activating a capsaicin-sensitive, nitric oxide-dependent myenteric plexus-mediated secretory reflex . The suppression by L-NAME of STa induced ileal fluid secretion in vivo probably involves the inhibition of this reflex. J Biotechnol, 1994 Mar 15, 33(1), 43 - 53 Application of an alkaline phosphatase fusion protein system suitable for efficient screening and production of Fab-enzyme conjugates in Escherichia coli; Weiss E et al.; We report a novel vector system suitable for the efficient preparation of alkaline phosphatase (PhoA)-labelled antibody Fab fragments in Escherichia coli . The previously described pGE20 vector used for the functional expression of truncated heavy (Fd) and light (L) chains of Fab into the bacterial culture medium was modified by inserting the E . coli PhoA coding region 3' to the Fd cloning sites . The secreted Fd-PhoA fusions and L proteins were found to be disulfide linked and Fab-PhoA complexes, prepared with IgG1 antibodies recognizing specifically human tumor necrosis factor alpha, were shown to be useful for the rapid detection of antigen . When an additional short peptide was interposed between the Fd and PhoA domains, nearly all of the recombinant Fab-PhoA conjugates present in the culture supernatant retained both antigen binding and enzymatic activity . A method for the detection and selection of bacterial colonies expressing bifunctional hybrid molecules of desired antigen specificity was also developed . Taken together, the systems described permit the generation and production of Fab-PhoA conjugates in E . coli, which can replace conventionally prepared PhoA-labelled antibodies in appropriate immunoassays. Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 969 - 76 Sequence analysis and expression of phospholipase A2 from Taiwan cobra; Pan FM et al.; Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of venom glands in Taiwan cobras to facilitate the cloning and sequencing of phospholipase A2 (PLA2) gene . The PCR product was then subcloned into pUC18 vector and transformed in E . coli strain JM109 . Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method . Sequencing several clones containing about 0.5 kb DNA inserts constructed a complete and unambiguous full-length reading frame of 468 base pairs covering a precursor for phospholipase A2 with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid segment of signal peptide . The sequenced major PLA2 with pI 4.991 shows a high degree of sequence homology to those PLA2 of the same or closely-related genus . The deduced protein sequence allows us to correct and resolve some discrepancy between the sequences determined by conventional protein sequencing (Toxicon, 19, 141(1981)) and X-ray crystallography (Science, 250, 1560(1990)) . Expression of PLA2 in E . coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified PLA2 from the same cobra venom albeit with a much lower activity. Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 504 - 10 Nitric oxide production depends on preceding tetrahydrobiopterin synthesis by endothelial cells: selective suppression of induced nitric oxide production by sepiapterin reductase inhibitors; Schoedon G et al.; Using murine vascular endothelial cells expressing both constitutive and inducible nitric oxide synthases (cNOS and iNOS), we explored the feasibility of suppressing cytokine-induced nitric oxide (NO) production without affecting constitutive NO production by inhibition of the tetrahydrobiopterin (BH4) biosynthesis . We show in this study that in endothelial cells cytokine/endotoxin-activated BH4 synthesis precedes the induction of NO generation . Using the sepiapterin reductase inhibitors phenprocoumon or dicumarol as BH4 synthesis inhibitors, we achieved a pronounced and selective suppression of induced NO production in cytokine-activated endothelial cells . Addition of exogenous BH4, but not sepiapterin, restored NO production in the presence of the inhibitors . Despite profound inhibition of the BH4 biosynthesis, constitutive NO synthesis was not affected, thereby demonstrating the selectivity and specificity of the inhibitors . Suppression of enhanced NO production by sepiapterin reductase inhibitors such as cumaroles could provide pharmacologic means for therapeutic interventions in NO-mediated pathophysiologic events. Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2125 - 9 Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon"; Dao V et al.; The efficiency of translation depends on correct tRNA-ribosome interactions . The ability of chemically synthesized yeast tRNA(Phe) anticodon domains to effectively inhibit the binding of native yeast tRNA(Phe) to poly(U)-programmed Escherichia coli 30S ribosomal subunits was dependent on a Mg(2+)-stabilized stem and an open anticodon loop, both facilitated by base modifications . Analysis of tRNA sequences has revealed that base modifications which negate canonical hydrogen bonding are found in 95% of those tRNA anticodon loop sequences with the potential to form two Watson-Crick base pairs across the loop . Therefore, we postulated that a stable anticodon stem and an open loop are prerequisites for ribosome binding . To test this hypothesis, DNA analogs of the yeast tRNA(Phe) anticodon domain were designed to have modification-induced, Mg(2+)-stabilized stems and open loops . The unmodified DNA analog neither bound to poly(U)-programmed 30S ribosomal subunits nor inhibited the binding of native tRNA(Phe) . However, specifically modified DNA analogs did bind to ribosomal subunits and effectively inhibited tRNA(Phe) from binding . Thus, modification-dependent Mg(2+)-stabilized anticodon domain structures with open loops have evolved as the preferred anticodon conformations for ribosome binding. FEBS Lett, 1994 Mar 14, 341(1), 54 - 8 The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a five-stranded alpha-helical bundle between residues 20 and 83; Efimov VP et al.; The N-terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20-83, was over-expressed in E . coli and purified under non-denaturing conditions . The fragment forms pentamers similar to the assembly domain of the native protein . Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71 . The fragment adopts a predominantly alpha-helical structure as judged by circular dichroism spectroscopy . On the basis of these findings we propose the model of a five-stranded alpha-helical bundle for the assembly domain of COMP . The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers. FEBS Lett, 1994 Mar 14, 341(1), 131 - 4 Expression and stability of recombinant RQ-mRNAs in cell-free translation systems; Ugarov VI et al.; Expression of dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CAT) mRNAs in cell-free Escherichia coli translation systems is greatly enhanced as a result of their insertion into RQ135 RNA, a naturally occurring satellite of phage Q beta . The enhancement is due to protection of the recombinant mRNAs against endogenous ribonucleases and to an increased initial rate of translation in the case of the RQ-CAT mRNA. FEBS Lett, 1994 Mar 14, 341(1), 79 - 85 The crystal structure of human CskSH3: structural diversity near the RT-Src and n-Src loop; Borchert TV et al.; SH3 domains are modules occurring in diverse proteins, ranging from cytoskeletal proteins to signaling proteins, such as tyrosine kinases . The crystal structure of the SH3 domain of Csk (c-Src specific tyrosine kinase) has been refined at a resolution of 2.5 A, with an R-factor of 22.4% . The structure is very similar to the FynSH3 crystal structure . When comparing CskSH3 and FynSH3 it is seen that the structural and charge differences of the RT-Src loop and the n-Src loop, near the conserved Trp47, correlate with different binding properties of these SH3 domains . The structure comparison suggests that those glycines and acid residues which are very well conserved in the SH3 sequences are important for the stability of the SH3 fold. Nucleic Acids Res, 1994 Mar 11, 22(5), 767 - 72 Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication; Tanaka K et al.; The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV) . If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins . Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above . These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site) . Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation . We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication. Nucleic Acids Res, 1994 Mar 11, 22(5), 711 - 21 The DNA binding domains of the varicella-zoster virus gene 62 and herpes simplex virus type 1 ICP4 transactivator proteins heterodimerize and bind to DNA; Tyler JK et al.; The product of varicella-zoster virus gene 62 (VZV 140k) is the functional counterpart of the major transcriptional regulatory protein of herpes simplex virus type 1 (HSV-1), ICP4 . We have found that the purified bacterially expressed DNA binding domain of VZV 140k (residues 417-647) is a stable dimer in solution . As demonstrated by the appearance of a novel protein--DNA complex of intermediate mobility in gel retardation assays, following in vitro co-translation of a pair of differently sized VZV 140k DNA binding domain peptides, the 140k DNA binding domain peptide binds to DNA as a dimer . In addition, the DNA binding domain peptide of HSV-1 ICP4 readily heterodimerizes with the VZV 140k peptide on co-translation, indicating that HSV-1 ICP4 and VZV 140k possess very similar dimerization interfaces . It appears that only one fully wild type subunit of the dimer is sufficient to mediate sequence specific DNA recognition in certain circumstances . Co-immunoprecipitation analysis of mutant DNA binding domain peptides, co-translated with an epitope-tagged ICP4 DNA binding domain, shows that the sequence requirements for dimerization are lower than those necessary for DNA binding. Science, 1994 Mar 11, 263(5152), 1440 - 3 Coding of hemolysins within the ribosomal RNA repeat on a plasmid in Entamoeba histolytica; Jansson A et al.; The pathogenesis of amoebic dysentery is a result of cytolysis of the colonic mucosa by the parasitic protozoan Entamoeba histolytica . The cytolysis results in extensive local ulceration and allows the amoeba to penetrate and metastasize to distant sites . Factors involved in this process were defined with three clones that express hemolytic activities in Escherichia coli . These potential amoebic virulence determinants were also toxic to human colonic epithelial cells, the primary cellular targets in amoebal invasion of the large intestine . The coding sequences for the hemolysins were close to each other on a 2.6-kilobase segment of a 25-kilobase extrachromosomal DNA element . The structural genes for the hemolysins were within inverted repeats that encode ribosomal RNAs. J Mol Biol, 1994 Mar 11, 236(5), 1299 - 309 Partitioning of plasmid R1 . The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element; Jensen RB et al.; The parA partitioning system of plasmid R1 mediates efficient stabilization of R1 and F-derived replicons . The parA system is encoded by a continuous DNA segment of approximately 1600 base-pairs and consists of three components . Two adjacent genes, parM and parR, coding for the trans-acting proteins ParM and ParR, and the cis-acting parC site . The centromere-like parC site is located upstream of parM and parR and contains the parA promoter . The parM and parR genes are co-transcribed as an operon from the parA promoter . The 5' end of the parA encoded transcript was mapped to the center of the parC region at +115 . The -10 and -35 core promoter sequences are flanked by the two sets of five direct repeats in parC (the ParR boxes) . The parA promoter was found to be negatively regulated by the parR gene product, whereas the parM gene product seemingly was not involved in the regulation . Surprisingly, a region downstream of the parA promoter enhanced transcription from the promoter many-fold (30 to 50-fold) . The parC site titrated the ParR protein, suggesting that the ParR protein interacts directly with the parC site . Using an engineered parA system we found that the parC site could be complemented in cis by the parM and parR genes . Furthermore, the proper function of the parC site was highly dependent on the expression level of ParM and ParR . The incompatibility associated with the parC site could not be suppressed by overexpression of the ParM and ParR proteins . Based on these results we suggest a novel partition model involving pairing of newly replicated plasmid molecules. J Mol Biol, 1994 Mar 11, 236(5), 1289 - 98 Partitioning of plasmid R1 . Ten direct repeats flanking the parA promoter constitute a centromere-like partition site parC, that expresses incompatibility; Dam M et al.; The parA partitioning system of plasmid R1 consists of three different components: the cis-acting centromere-like parC site, and the two trans-acting proteins ParM and ParR . These three components are contained within a region of 1.6 kb . The parC site is located upstream of the two genes, parM and parR, which are expressed as an operon from the parA promoter . The parC site contains an array of ten 11 base-pair direct repeats, organized in two sets of five repeats flanking the parA core promoter sequences . Deletions and point mutations were introduced in the parA locus, resulting in partially stable and unstable plasmids . An analysis of these parA- plasmids showed that ParM and ParR are transacting . The 160 bp minimal parC region contained sufficient in cis information for efficient trans-complementation . Both proteins were required for maximal stabilization of a parC+ mini-R1 plasmid, although ParR alone, donated either in cis or in trans, yielded partial stabilization . Plasmids that overexpressed ParR caused destabilization of a co-resident parA+ plasmid, whereas overexpression of ParM had no such effect . The parC site exerted incompatibility (incA) at high but not at low copy number . Likewise, the entire parA system exerted incompatibility in a copy number-dependent fashion, and stronger than the incompatibility expressed by parC alone. J Mol Biol, 1994 Mar 11, 236(5), 1283 - 8 Asymmetric arrangement of two alpha subunits within Escherichia coli RNA polymerase . Involvement of one alpha subunit in contact with cAMP receptor protein; Zou C et al.; Class I transcription factors of Escherichia coli have been proposed to make contact with contact site I on the alpha subunit, C-terminal region of RNA polymerase with the subunit composition of alpha 2 beta beta ' sigma . Both a reconstituted mutant holoenzyme containing two C-terminally truncated alpha-235 subunits and a hybrid enzyme containing one wild-type alpha (alpha-329) and one C-terminal truncated alpha (alpha-235) subunit were found to be as active in transcription from factor-independent simple promoters as the wild-type holoenzyme . The mutant enzyme was, however, inactive in cAMP receptor protein (CRP)-dependent transcription from lacP1 promoter, but the hybrid enzyme was about 50% as active in lacP1 transcription as the wild-type enzyme . The results indicate that only one specific alpha subunit makes contact with CRP. J Biol Chem, 1994 Mar 11, 269(10), 7689 - 95 Identification of temperature-sensitive mutants of the human immunodeficiency virus type 1 protease through saturation mutagenesis . Amino acid side chain requirements for temperature sensitivity; Manchester M et al.; Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus . An Escherichia coli expression and processing assay system was used to screen 285 protease mutants for temperature-sensitive activity . Fourteen protease mutants had a temperature-sensitive phenotype, and approximately half resulted from conservative amino acid substitutions . Of the 14 substitutions that conferred a temperature-sensitive phenotype, 11 substitutions occurred at 6 positions that represent 3 pairs of residues in the protease that contact each other in the three-dimensional structure . These mutants assist in pinpointing regions of the protease that are important for enzyme activity and stability. J Biol Chem, 1994 Mar 11, 269(10), 7674 - 81 The sequence of a second member of the lima bean lectin gene family and the expression and characterization of recombinant lectin in Escherichia coli; Jordan ET et al.; The lima bean lectin recognizes terminal alpha-D-GalNAc groups and agglutinates human type A erythrocytes . We have cloned a portion of the gene encoding the alpha subunit of the lima bean lectin . The clone was obtained using the polymerase chain reaction and verified from a genomic clone encoding the mature protein of 253 amino acids . The deduced amino acid sequence has significant overall homology with other leguminous plant lectins and contains all of the known peptide sequences isolated from lima bean lectin (LBL) . Southern blot analysis reveals the presence of several genes which hybridize to the cloned gene and which we propose are genes included in the lima bean lectin gene family . We report here the sequence, expression, and characterization of LBL 2, the second member of this gene family . Milligram quantities of soluble active recombinant lima bean lectin (rLBL) were obtained from Escherichia coli, using the T7 RNA polymerase expression system . SDS-polyacrylamide gel electrophoresis and Western blot analysis indicate expression of one protein band of about 27 kDa in induced E . coli cells . This protein cross-reacts with polyclonal antibodies raised against seed lectin (sLBL) and gave a reaction of identity with seed lectin by Ouchterlony double diffusion, specifically agglutinates type A blood cells, and is specifically inhibited by D-GalNAc . The isoelectric point of rLBL is 5.86, whereas those of the seed lectin subunits were determined to be 5.86, 5.58, and 5.20 (previously designated alpha, beta, alpha', respectively) . rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL . However, despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including mobility on gel filtration, aggregation in solution, and its CD spectrum . These differences may be due to a number of factors, which will be discussed. J Biol Chem, 1994 Mar 11, 269(10), 7532 - 7 Arginine 41 of subunit c of Escherichia coli H(+)-ATP synthase is essential in binding and coupling of F1 to F0; Fraga D et al.; Two substitutions were made for Arg41 in the polar loop of subunit c of the Escherichia coli F1F0 H(+)-transporting ATP synthase . The R41K and R41H mutants were initially studied by use of a plasmid carrying the complete c R41K or c R41H unc (F1F0) operon in a chromosomal strain deleted for the unc operon . The extent of F0 incorporation into membranes of these cells was quite variable, and the system was concluded to be unsuitable for biochemical characterization . Ultimately, the mutant genes were recombined into the chromosome using a novel method for the unc system . The biochemical phenotype of the chromosomally expressed mutants proved to be reproducible . The c R41H mutation causes a specific defect in assembly of F0, i.e . subunit a was not incorporated into the membrane despite near normal incorporation of subunits b and c . On the other hand, c R41K mutant F0 assembled normally in one of two background strains studied . (In the second genetic background, subunit a was inefficiently incorporated into the c R41K membrane.) In membranes prepared from a c R41K strain assembling a complete F0, R41K F0 was found to bind F1 with near normal affinity and to transport H+ at near normal rates . Although R41K F0 binds F1, F1-ATPase activity and H+ transport remained uncoupled . The uncoupling was indicated by a lack of ATP-driven H+ translocation and by the high proton permeability of membranes with F1 bound to F0 . The uncoupled phenotype of the R41K mutant closely resembles that previously reported for the c Q42E mutant. J Biol Chem, 1994 Mar 11, 269(10), 7494 - 500 Analysis of the translational initiation region on the Euglena gracilis chloroplast ribulose-bisphosphate carboxylase/oxygenase (rbcL) messenger RNA; Koo JS et al.; The chloroplast mRNAs from Euglena gracilis fall into two classes . One group of mRNs from this organelle contains a Shine-Dalgarno sequence 5' to the start codon, while the other group of mRNAs does not have a conserved sequence signal in the 5'-untranslated region . To investigate the start signals for E . gracilis chloroplast mRNAs that do not carry a Shine-Dalgarno sequence, 30 S initiation complex formation has been studied using a series of transcripts carrying the wild-type translational start site of ribulose-bisphosphate carboxylase/oxygenase (rbcL) or mutated derivatives of this site . Mutation of the start codon of the rbcL gene indicates that the chloroplast 30 S subunit is recognizing only the correct AUG codon . The analysis of the messages from a series of deletion mutants shows that a minimum of delta 20 residues 5' to the AUG codon is required for activity . Maximal activity requires the full 55-base leader sequence . Surprisingly, a transcript carrying the inverse complement of 48 bases in the leader is delta 60% as active as the wild-type message in promoting initiation complex formation . Introduction of a Shine-Dalgarno sequence in the 5'-leader increases the activity of the mRNA only delta 1.4-2-fold . The presence of an oligodeoxynucleotide containing a strong Shine-Dalgarno sequence does not significantly inhibit the formation of initiation complexes at the rbcL start site . Similar results are obtained when initiation complexes are formed with initiation factors from either E . gracilis chloroplasts or Escherichia coli. J Biol Chem, 1994 Mar 11, 269(10), 7483 - 8 Site-specific biotinylation of colicin Ia . A probe for protein conformation in the membrane; Qiu XQ et al.; Channel-forming colicins are Escherichia coli proteins that form voltage-dependent channels in lipid bilayer membranes and are lethal to sensitive strains of E . coli . Experiments with colicin E1 have led to a model of voltage dependence based on the insertion of alpha-helical segments of the protein into the membrane in response to cis-positive voltages . This model was tested on the partly homologous colicin Ia protein, which offers certain advantages over colicin E1 as a model channel, it is active at neutral pH and exhibits comparatively well-defined single channel conductance . We describe here the creation of a specific probe for locating a particular amino acid residue on one side or the other of a planar lipid bilayer membrane, by using the biotin-streptavidin system . Site-directed mutagenesis was used to change lysine 544 of colicin Ia to cysteine . This placed a unique cysteine at a site expected, by homology to colicin E1, to cross the membrane from the cis to the trans side in association with the opening of the channel . This unique cysteine was biotinylated chemically, so that it could serve as a target for streptavidin . Incubation of the biotinylated mutant colicin with streptavidin blocked its killing activity, in vivo; incubation of wild-type colicin, which lacks cysteine, with streptavidin, did not affect its activity . Channels formed by the biotinylated mutant protein in planar lipid bilayers were abolished by streptavidin added to the cis side of the membrane, if the channels were closed, but not if they were open . Trans streptavidin had no effect on either open or closed channels . Thus, when the channel is closed, residue 544 of colicin Ia is accessible to cis streptavidin in the closed state, but the opening of the channel eliminates this accessibility. J Biol Chem, 1994 Mar 11, 269(10), 7297 - 303 Recombinant human retinoic acid receptor beta . Binding of synthetic retinoids and transcriptional activation; Lombardo A et al.; All-trans-retinoic acid mediates cell growth and differentiation by binding to and then activating nuclear retinoid receptor proteins that regulate gene transcription . Recombinant human retinoic acid receptor beta was cloned and expressed in Escherichia coli as a fusion protein rMBP-RAR beta with maltose-binding protein to facilitate purification . After isolation from bacterial lysates, rMBP-RAR beta was used for binding with selected retinoids . Scatchard analysis with {11,12-3H2}all-trans-retinoic acid gave a Kd of 0.34 nM . Competitive binding studies with a series of conformationally restricted aromatic retinoids indicated that the Ki values for binding to rMBP-RAR beta correlated with the logs of the EC50 values for gene transcriptional activation (p < or = 0.05) and with those for the relative activation compared to that of all-trans-retinoic acid (p < or = 0.01) . Inspection of binding-activation correlation diagrams indicates candidate structures for improved retinoid agonists or antagonists. J Biol Chem, 1994 Mar 11, 269(10), 7192 - 200 Characterization of the mitochondrial binding and import properties of purified yeast F1-ATPase beta subunit precursor . Import requires external ATP; Hajek P et al.; To better understand the early events of the mitochondrial protein import process, we purified the precursor of the F1-ATPase beta subunit (pre-F1 beta) and examined its import into isolated mitochondria . Import of purified urea-denatured pre-F1 beta did not require cytosolic factors . However, the period of productive import was prolonged by the addition of reticulocyte lysate, suggesting that cytosolic factors such as molecular chaperones were acting to extend the period of import competence of pre-F1 beta . Purified pre-F1 beta bound extensively to both cardiolipin-containing liposomes and to intact mitochondria, indicating that a direct interaction between mitochondrial precursors and the mitochondrial outer membrane surface can occur . The ability to chase this surface-bound pre-F1 beta into mitochondria suggests that precursors bound to the mitochondrial surface can be maintained in an import competent conformation . Finally, our defined mitochondrial import system was used to characterize the ATP requirements of pre-F1 beta import in the absence of cytosol . We found a strong requirement for ATP on both sides of the mitochondrial inner membrane, suggesting that one or more previously undetected mitochondrial proteins outside the inner membrane utilize ATP to promote efficient pre-F1 beta import. J Biol Chem, 1994 Mar 11, 269(10), 7066 - 9 Effect of DNA cytosine methylation upon deamination-induced mutagenesis in a natural target sequence in duplex DNA; Zhang X et al.; Are 5-methylcytosine residues in DNA hot spots for transition mutagenesis? Numerous studies identify 1) structural changes induced by DNA methylation, 2) high percentages of human mutations that result from GC to AT transition pathways, and 3) differences between G.C and G.mC base pairs in susceptibility to nonenzymatic deamination . However, investigations of chemical stability necessarily involve non-physiological conditions for chemical analysis of deamination . Here we describe an experiment that compares rates of deamination-induced mutagenesis between a G.C and G.mC base pair, when both are present in duplex DNA, incubated at 37 degrees C and pH 7.4, within identical sequence contexts, in a natural mutational target (the Escherichia coli lacZ alpha gene) that selects for mutagenesis at the specific site under investigation . Under these conditions the rate of spontaneous deamination at G.mC exceeds that at G.C by more than 21-fold . Our data implicate differences in chemical stability toward deamination as a major causal factor releasing DNA cytosine methylation to spontaneous mutagenesis. J Biol Chem, 1994 Mar 11, 269(10), 7062 - 5 Mammalian ferrochelatase, a new addition to the metalloenzyme family; Ferreira GC et al.; A {2Fe-2S} cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway . Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy . In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of {2Fe-2S}+ cluster . Mossbauer spectra of the recombinant ferrochelatase, purified from a strain of E . coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in 57Fe-enriched medium, demonstrated unambiguously that the cluster is a {2Fe-2S} cluster . No change in the cluster oxidation state was observed during catalysis . The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the {2Fe-2S} center in regulation of mammalian ferrochelatases. Gene, 1994 Mar 11, 140(1), 73 - 7 Newly identified genes involved in the signal transduction of Escherichia coli K-12; Utsumi R et al.; We cloned and sequenced two Escherichia coli genes which are members of a family of an environmentally responsive two-component system . The nucleotide (nt) and deduced amino-acid sequences of these two genes were found to be homologous to those of the Bordetella pertussis bvgA and bvgS genes . They were mapped at 51 min (clones 6B9 to 7G9 of the Kohara miniset library of the E . coli chromosome) . Both proteins, deduced from their nt sequences, were identified in the coupled in vitro transcription-translation system; their molecular masses were consistent with BvgA and BvgS (23 and 135 kDa, respectively) . Furthermore, when these genes were expressed on a multicopy plasmid in an envZ deletion strain, ompC expression was induced . This expression was found to be regulated by low temperature, MgSO4 and nicotinic acid, factors known to control the virulence of B . pertussis via BvgA and BvgS . These results indicate that the newly cloned genes were structurally and functionally similar to bvgA and bvgS, and we designated these genes evgA and evgS. Gene, 1994 Mar 11, 140(1), 59 - 62 A reliable amplification technique with single-sided specificity for the isolation of 5' gene-regulating regions; Luo M et al.; A simple and efficient method is described for the isolation of extension fragments of known DNA sequences by polymerase chain reaction (PCR) using a single specific primer . With this method, size-selected genomic DNA fragments are ligated to a plasmid vector (pGEM-4Z) which contains sequencing primers and the population of chimeric plasmids is used for transforming Escherichia coli . DNA is extracted from an aliquot of the resulting mini-library and PCR performed using a sequence-specific primer and either of the standard sequencing primers of the plasmid vector . This method appears to be more versatile than inverse PCR (IPCR), since: (i) the DNA sequence needed as the specific primer can be as short as about 20 nucleotides (nt); (ii) the DNA templates to be used in PCR are available in high amount, thus facilitating all manipulations; and (iii) if relinearization of the DNA by restriction enzyme digestion is desired before the PCR reaction, many restriction sites can be chosen from the vector polylinker . Using this method, we have isolated the genomic 5' region of the carrot bifunctional dihydrofolate reductase-thymidylate synthase-encoding gene dhfr-ts using a 21-nt sequence of the 5' region of the dhfr-ts cDNA clone as the specific primer. Gene, 1994 Mar 11, 140(1), 137 - 8 Sequences of the rplJL operon containing the L10 and L7/L12 genes from Brucella abortus; Oliveira SC et al.; The rplJL operon encodes the L10 and L7/L12 proteins, essential for ribosomal function and protein synthesis . In this study, we report the nucleotide sequence of the rplJ and rplL genes from Brucella abortus . The deduced amino-acid sequences show 37 and 67% identity to Escherichia coli L10 and L7/L12, respectively. Cell, 1994 Mar 11, 76(5), 925 - 32 Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport; Noueiry AO et al.; Plant viruses establish a systemic infection by moving through plasmodesmata, but little is known of the mechanism(s) involved . The roles of two movement-associated proteins of a single-stranded DNA virus were investigated in vivo, using functional proteins expressed in E . coli and microinjection into plant cells . We report here that the BL1 protein of bean dwarf mosaic geminivirus moves extensively from cell to cell, increases mesophyll plasmodesmal size exclusion limit, and potentiates the movement of double-stranded DNA from cell to cell . Movement of single- and double-stranded DNA out of the nucleus is mediated by the BR1 protein . These results provide direct experimental evidence for intercellular macro-molecular transport in plants, and suggest that the BR1 and BL1 proteins coordinate the movement of viral DNA across both nuclear and plasmodesmal boundaries. J Biol Chem, 1994 Mar 11, 269(10), 7450 - 7 Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B . Searching for the catalytic triad; Boehlein SK et al.; The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established . We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli asparagine synthetase B (AS-B) and a series of mutants . In common with other members of the purF family of amidotransferases, the recombinant enzyme possesses an NH2-terminal cysteine residue . Replacement of Cys-1 by either alanine or serine results in a loss of glutaminase and glutamine-dependent activity, without out any significant effect upon ammonia-dependent asparagine synthesis . As previously observed for human AS (Sheng, S., Moraga-Amador, D., Van Heeke, G., Allison, R . D., Richards, N . G . J., and Schuster, S . M . (1993) J . Biol . Chem . 268, 16771-16780), glutamine is an inhibitor of the ammonia-dependent reaction catalyzed by both the Cys-1-->Ala (C1A) and Cys-1-->Ser (C1S) mutants of AS-B . In the case of C1A, the inhibition pattern suggests that an abortive complex is formed . This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N . G . J., and Schuster, S . M . (1992) FEBS Lett . 313, 98-102) . In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S . Cys-1 does not appear to be required for glutamine binding . Replacement of Asp-33 by either asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B . Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H . (1989) J . Biol . Chem . 264, 16613-16619) . In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer . In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1994 Mar 11, 269(10), 7379 - 86 Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli; Franco PJ et al.; Acidic residues which are found on transmembrane segments within the lactose permease may play an important role in H+ and/or sugar recognition . To examine the functional roles of Glu-269 and Glu-325, we have constructed a variety of amino acid substitutions (e.g . aspartate, glycine, alanine, serine, or glutamine) via site-directed mutagenesis . At position 269, all mutations appear to have a detrimental effect on sugar affinity, downhill transport, and counterflow . The Asp-269 mutant was able to accumulate lactose against a concentration gradient, whereas all of the nonionizable substitutions at position 269 were completely defective . Nevertheless, in spite of their inability to actively accumulate sugars, Gly-269, Ala-269, and Gln-269 mutants were observed to transport H+ upon the addition of galactosides . Mutations at position 325 had a markedly different phenotype . For example, the Asp-325, Gly-325, and Gln-325 mutants exhibited an apparent Km for lactose transport (e.g . 0.21, 0.47, and 0.50 mM, respectively), which was actually lower than that of the wild-type strain (1.44 mM) . In counterflow assays, all position 325 mutants also appear to catalyze lactose exchange . Similar to the results obtained at position 269, the Asp-325 mutant exhibited moderate levels of accumulation, whereas none of the nonionizable mutations at position 325 were able to accumulate galactosides against a concentration gradient . However, unlike the position 269 mutants, no H+ transport was observed in the Gly-325, Ala-325, Ser-325, or Gln-325 strains upon the addition of lactose, S-beta-D-galactopyranosyl-(1,1)-beta-thiogalactopyranoside, 1-O-methyl-beta-D-galactopyranoside, or melibiose . Furthermore, in these mutants, the efflux of lactose during counterflow assays became insensitive to delta pH . Overall, these results are consistent with the notion that an acidic residue at position 325 is required for H+ transport via the lactose permease . Alternative hypotheses are also discussed. Biochim Biophys Acta, 1994 Mar 8, 1184(2-3), 284 - 90 Mutagenesis of the b'-subunit of Synechocystis sp . PCC 6803 ATP-synthase; Lill H et al.; We investigated the F0F1 ATP synthase of the cyanobacterium, Synechocystis sp . PCC 6803 . The gene for the F0-subunit b', a peptide probably located at the interface between F0 and F1, has been partially or completely evicted from the bacterial genome . We found that the complete deletion of the subunit was lethal to the cells . However, the subunit could be truncated down to its hydrophobic N-terminal stretch without much harm . Since the gene for b' probably shares a common ancestor with the gene for subunit b and emerged by gene duplication, we propose that b' gathered a new role during evolution, perhaps in the regulation of photophosphorylation. Biochemistry, 1994 Mar 8, 33(9), 2688 - 95 Evidence for participation of aspartate-84 as a catalytic group at the active site of porphobilinogen deaminase obtained by site-directed mutagenesis of the hemC gene from Escherichia coli; Woodcock SC et al.; The role of aspartate-84, an invariant residue in the active site cleft of Escherichia coli porphobilinogen deaminase, has been investigated by site-directed mutagenesis . Substitution of aspartate-84 by glutamate results in an enzyme that retains less than 1% of its activity and which can form highly stable enzyme-intermediate complexes . Substitution of aspartate-84 by either alanine or asparagine, however, results in proteins unable to catalyze the formation of preuroporphyrinogen but which, nevertheless, appear able to assemble the dipyrromethane cofactor . The mechanisms of the tetramerization reaction and cofactor assembly are discussed. Biochemistry, 1994 Mar 8, 33(9), 2644 - 50 Influence of substrates and MgADP on the time-resolved intrinsic fluorescence of phosphofructokinase from Escherichia coli . Correlation of tryptophan dynamics to coupling entropy; Johnson JL et al.; The influence of that MgADP and the substrate ligands MgATP and fructose 6-phosphate (Fru-6-P) have on the structure of E . coli phosphofructokinase (PFK) in the vicinity of the single tryptophan that exists in each subunit has been examined by employing both steady-state and time-resolved measurements of the tryptophan fluorescence . The accessibility of the tryptophan to iodide quenching is over 1 order of magnitude less than experienced by N-acetyltryptophanamide in solution but varies nonetheless with the state of ligation . Most, but not all, of these changes correlate with changes in the degree of local motion available to the tryptophan side chain as determined by steady-state and time-resolved polarization measurements . When the data obtained from differential polarization experiments are fit to a model in which the motion of the tryptophan side chain is able to move with high frequency within a cone of limited amplitude as part of an otherwise slowly tumbling spherical protein, it was found that ligands primarily affect the amplitude of the available local motion . By interpreting these effects with reference to the disproportionation equilibria which define the negative coupling free energy between MgADP and Fru-6-P and the positive coupling free energy between MgADP and MgATP, it is apparent that changes in the local motion amplitudes correlate with the sign of the component coupling entropy previously determined from van't Hoff analyses (Johnson & Reinhart, 1994).(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Mar 8, 33(9), 2635 - 43 Influence of MgADP on phosphofructokinase from Escherichia coli . Elucidation of coupling interactions with both substrates; Johnson JL et al.; A comprehensive assessment is presented of the mutual influence that MgADP, MgATP, and fructose 6-phosphate (Fru-6-P) have on each other's binding to phosphofructokinase (PFK) from E . coli . When virtually any combination of these ligands binds to PFK it produces a significant perturbation in the intrinsic tryptophan fluorescence intensity and/or polarization which not only provides a means to follow binding in titration experiments but which also underscores the fact that more than two different enzyme conformations result from the binding of these ligands . When MgATP is saturating, the binding of MgADP to the allosteric site increases the affinity the enzyme subsequently displays for Fru-6-P . However, in the absence of MgATP, MgADP can bind to both the allosteric site and the nucleotide portion of the active site, with the latter antagonizing the binding of Fru-6-P to an extent that leads to an overall inhibition of Fru-6-P binding by MgADP . MgADP binding at the allosteric site also inhibits the binding of MgATP, indicating that under many circumstances MgADP should be more properly viewed as an inhibitor rather than an activator of E . coli PFK . After quantifying all of the 20 dissociation constants and 11 coupling parameters between ligand pairs pertinent to this three-ligand system, the more significant coupling parameters have been further characterized by examining their variation with temperature to establish the apparent enthalpy and entropy contributions to the corresponding coupling free energies . For both activating and inhibitory couplings, the enthalpy and entropy terms have the same sign as the coupling free energy.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Mar 8, 33(9), 2628 - 34 Nativelike secondary structure in interleukin-1 beta inclusion bodies by attenuated total reflectance FTIR; Oberg K et al.; Attenuated total reflectance FTIR has been used to study the structure of human interleukin-1 beta in inclusion bodies (IBs) and other aggregated forms . The secondary structure composition of native wild-type IL-1 beta determined by FTIR is in excellent agreement with that previously determined by crystallography and NMR: 52% beta-sheet, 25% loop/irregular structure, and 23% turn . Remarkably, IL-1 beta inclusion bodies exhibit secondary structural composition very similar to that of the native protein . The results indicate that the IBs form from a folding intermediate that has nativelike secondary structure . The secondary structure content of aggregated IL-1 beta, formed either in refolding or by thermal denaturation, was identical within experimental error to that of the IB, indicating that these aggregates were formed from intermediates with structures similar to that of the inclusion body. FEBS Lett, 1994 Mar 7, 340(3), 281 - 6 Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli; Lloyd E et al.; Expression of recombinant horse heart myoglobin in Escherichia coli has been found to result in the production of both native and variable amounts (approximately 16-17% total) of two sulphmyoglobin isomers . The recombinant sulphmyoglobin produced consists primarily of the A and B isomers as identified by 1H NMR spectroscopy with no evidence for production of the C isomer . Conversion of recombinant sulphmyoglobin to the native protein can be achieved by reconstitution with protohaem IX . The possible relationship of this observation to recombinant expression of other heme proteins is discussed.