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F T Robb

Publications and source records attributed to F T Robb.

At least 19 recordsLinked to original sources
PubMed1992

Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.

Archaea↗
PubMed1992

Characterization of an extremely thermostable glutamate dehydrogenase: a key enzyme in the primary metabolism of the hyperthermophilic archaebacterium, Pyrococcus furiosus.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3) from the hyperthermophilic Archeon Pyrococcus furiosus was purified to homogeneity by chromatography on anion-exchange, molecular-exclusion and hydrophobic-interaction media. The purified native enzyme had an M(r) of 270,000 +/- 15,000 and was shown to be a hexamer with identical subunits of M(r) 46,000. The enzyme was exceptionally thermostable, having a half-life of 3.5 to more than 10 h at 100 degrees C, depending on the concentration of enzyme. The Km of the enzyme for ammonia was high (9.5 mM), indicating that the enzyme is probably active in the deaminating, catabolic direction. The coenzyme utilization of the enzyme resembled the equivalent enzymes from eukaryotes rather than eubacteria, since both NADH and NADPH were recognized with high affinity. The enzyme displayed a preference for NADP+ over NAD+ that was more pronounced at low assay temperatures (50-70 degrees C) compared with the optimal temperature for enzyme activity, 95 degrees C.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrase gene (scrB).

The nucleotide sequence of a 2.119-kb DNA fragment containing the Vibrio alginolyticus sucrase gene (scrB) was determined. The complete sequence (484 aa residues) of the sucrase was deduced and homology was detected between the sucrase enzymes from V. alginolyticus and the Gram-positive bacteria Bacillus subtilis and Streptococcus mutans. In Escherichia coli cells the cloned V. alginolyticus sucrase is translocated to the periplasm. Transposon phoA mutagenesis experiments strongly suggested that V. alginolyticus sucrase in E. coli is not exported across the cytoplasmic membrane by means of a typical signal sequence.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the Vibrio alginolyticus glnA region.

The nucleotide sequence of a 4 kb fragment containing the Vibrio alginolyticus glnA, ntrB and ntrC genes was determined. The upstream region of the glnA gene contained tandem promoters. The upstream promoter resembled the consensus sequence for Escherichia coli sigma 70 promoters whereas the presumptive downstream promoter showed homology with nitrogen regulated promoters. Four putative NRI binding sites were located between the tandem promoters. The ntrB gene was preceded by a single presumptive NRI binding site. The ntrC gene was located 45 base pairs downstream from the ntrB gene. The V. alginolyticus ntrB and ntrC genes were able to complement ntrB, ntrC deletions in E. coli.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the Vibrio alginolyticus calcium-dependent, detergent-resistant alkaline serine exoprotease A.

The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA.

Alkaline Phosphatase↗
PubMed1988

The structure of the regulatory region of the rat L1 (L1Rn, long interspersed repeated) DNA family of transposable elements.

Here we report the DNA structure of the left 1.5 kb of two newly isolated full length members of the rat L1 DNA family (L1Rn, long interspersed repeated DNA). In contrast to earlier isolated rat L1 members, both of these contain promoter-like regions that are most likely full length. In addition, the promoter-like region of both members has undergone a partial tandem duplication. A second internal region of the left end of one of the reported members is also tandemly duplicated. The propensity of the left end of rat L1 elements to undergo this form of genetic rearrangement, as well as other structural features revealed by the present work, is discussed in light of the fact that during evolution the otherwise conserved mammalian L1 DNA families have each acquired completely different promoter-like regions. In an accompanying paper [Nur, I., Pascale, E., and Furano, A. V. (1988) Nucleic Acids Res. 16, submitted], we report that one of the rat promoter-like regions can function as a promoter in rat cells when fused to the Escherichia coli chloramphenicol acyltransferase gene.

Amino Acid Sequence↗
PubMed1987

Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli.

A halotolerant collagenolytic Vibrio alginolyticus strain isolated from salted hides had intracellular sucrase activity and did not secret sucrase into the medium. The strain actively transported sucrose by a sucrose-inducible, Na+-independent process. A 10.4-kilobase DNA fragment of V. alginolyticus DNA was cloned into Escherichia coli. The recombinant E. coli(pVS100) could utilize sucrose as a sole carbon source. In contrast to V. alginolyticus, the recombinant E. coli produced both intra- and extracellular sucrase activities. Up to 20% of the total sucrase activity was in the supernatant. Sucrase synthesis in E. coli(pVS100) was inducible and was subject to glucose repression, which was relieved by cyclic AMP. Sucrose was actively transported by a sucrose-inducible, Na+-independent system in E. coli(pVS100). Sucrose uptake was inhibited by the addition of a proton conductor. The maximum velocity and apparent Km values of sucrose uptake for the V. alginolyticus strain and E. coli(pVS100) were 130 nmol/mg of protein per min and 50 microM and 6 nmol/mg of protein per min and 275 microM, respectively.

Biological Transport, Active↗
PubMed1986

Temperature and oxygen regulated expression of a glutamine synthetase gene from Vibrio alginolyticus cloned in Escherichia coli.

Glutamine synthetase (GS) synthesis in Vibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene, glnA from V. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabled Escherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. The V. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment. V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in an E. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels in E. coli.

Aerobiosis↗
PubMed1985

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain.

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by adenylylation and deadenylylation. The pH profiles assayed by the gamma-glutamyltransferase method were similar for NH4-shocked and unshocked cell extracts and isoactivity point was not obtained from these curves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH4)2SO4 and glutamine, but cells grown in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.

Ammonium Sulfate↗
PubMed1983

The amino acid sequence of D-ribose-binding protein from Escherichia coli K12.

The amino acid sequence of the D-ribose-binding protein from Escherichia coli K12 was determined from the DNA sequence of the gene. Protein sequence analyses covering 80% of the protein were consistent with the sequence deduced from the DNA. The mature binding protein has 271 amino acid residues and shows substantial homology to D-galactose-binding protein. A signal peptide sequence of 23 or 25 residues was also deduced from the DNA sequence. It shows the characteristic features of prokaryotic signal peptides.

Amino Acid Sequence↗
PubMed1982

Regulation of hut enzymes and intracellular protease activities in Vibrio alginolyticus hut mutants.

The production of alkaline protease, collagenase and histidine utilization (Hut) enzymes by Vibrio alginolyticus wild-type, hutH1 and hutU1 strains was investigated. Alkaline protease synthesis was stimulated by histidine and urocanic acid in the wild-type and hutU1 strains. The hutH1 mutant alkaline protease production was stimulated by urocanic acid and not by histidine. The Hut enzymes in the wild-type strain were coordinately induced by histidine. Urocanase and formimino-hydrolase were induced by histidine in the hutH1 mutant which lacked histidase and was not able to convert histidine to urocanic acid. Collagenase production in peptone medium was inhibited in the hut mutants. It is concluded that in V. alginolyticus urocanic acid regulates alkaline protease synthesis but that the Hut enzymes are induced by histidine. The involvement of the Hut genetic system in the regulation of alkaline protease and collagenase synthesis is discussed.

Bacterial Proteins↗
PubMed1981

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus.

The production of an extracellular collagenase and alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37 degrees C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37 degrees C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5 degrees C and there was no difference in the growth rate at 30 and 37 degrees C. Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30 degrees C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.

Cerulenin↗
PubMed1981

Regulation of extracellular alkaline protease activity by histidine in a collagenolytic Vibrio alginolyticus strain.

Vibrio alginolyticus synthesized an inducible extracellular collagenase in a peptone medium during the stationary growth phase. These cultures also possessed extracellular alkaline serine protease activity. The alkaline protease activity did not require a specific inducer and it was produced in tryptone or minimal media. The collagenase was not produced in either the tryptone or minimal media. The alkaline protease activity was sensitive to catabolite repression by a number of carbon sources, including glucose, and by amino acids and ammonium ions. Cyclic AMP, dibutyryl cyclic AMP and cyclic GMP did not relieve catabolite repression. Histidine and urocanic acid stimulated the production of alkaline protease activity in tryptone and minimal media. Other compounds associated with the histidine utilization (hut) pathway did not increase alkaline protease activity. Histidine reversed the repression of alkaline protease activity by glucose of (NH4)2SO4 in minimal medium. Histidine and the compounds associated with the hut pathway inhibited collagenase production.

Amino Acids↗
PubMed1981

Inhibition of ribonucleic acid polymerase by a bacteriocin from Bacteroides fragilis.

The Bacteroides fragilis bacteriocin which inhibits ribonucleic acid (RNA) polymerase activity had a narrow activity spectrum in vivo and only inhibited the growth of certain B. fragilis strains. In vitro the bacteriocin was not specific and inhibited RNA polymerases from widely diverse bacterial genera. RNA polymerases from rifampin-resistant strains of Bacteroides thetaiotaomicron and Clostridium acetobutylicum were resistant to the bacteriocin in vitro. Purified bacteriocin bound to partially purified RNA polymerase, and both proteins were cosedimented in a glycerol gradient. In the RNA polymerase reaction, the bacteriocin acted as a competitive inhibitor for adenosine, cytidine, and uridine 5'-triphosphates and as a noncompetitive inhibitor for guanosine 5'-triphosphate. The bacteriocin did not inhibit RNA polymerase from chicken embryos.

Bacteriocins↗
PubMed1980

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative Escherichia coli K12 mutant and from Salmonella typhimurium.

Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.

Bacterial Proteins↗
PubMed1980

Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth.

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.

Adenosine Triphosphate↗
PubMed1980

Rifampin and bacteriocin resistance in Bacteroides fragilis.

A low-molecular-weight bacteriocin produced by a Bacteroides fragilis strain inhibited ribonucleic acid polymerase activity in crude extracts of a susceptible B. fragilis indicator strain. A total of 10 rifampin-resistant mutants of the indicator strain were isolated. Nine of the rifampin-resistant mutants were resistant to the bacteriocin, and the other mutant was hypersusceptible. The rifampin- and bacteriocin-resistant mutants all adsorbed approximately the same amount of the bacteriocin as the indicator strain. Two of these rifampin- and bacteriocin-resistant mutants were investigated further, and the polymerase activity in crude extracts of the two mutants was not affected by either rifampin or the bacteriocin. The in vitro ribonucleic acid polymerase activity of the hypersusceptible strain was more susceptible to the bacteriocin than the parent indicator strain was. The bacteriocin-producing strain was susceptible to rifampin but was resistant to its own bacteriocin in vivo. The in vitro ribonucleic acid polymerase activity of the producer strain was only slightly affected by 64 arbitrary units of the bacteriocin. Increasing concentrations of the bacteriocin inhibited ribonucleic acid polymerase extracts of the producer strain.

Bacteriocins↗