Biomedical subjects

R K Scopes

Publications and source records attributed to R K Scopes.

At least 37 records · Page 2Linked to original sources

PubMed1995

The effect of temperature on enzymes used in diagnostics.

A number of enzymes that are used in clinical analysis have been studied in relation to the effect of temperature on their activity. Both Vmax and Km were determined over a temperature range from 13 to 55 degrees C. Whereas Vmax values increased steadily until denaturation point with all enzymes, the effect of temperature on Km was more variable. With most enzymes there was a gradual increase in Km, often with a sharp rise close to the denaturation temperature. In most cases, Km did not increase as fast as Vmax, consequently the enzyme efficiency, Vmax/Km, also increased slightly with temperature. However this was not the case with rabbit muscle lactate dehydrogenase, for which the Km for pyruvate increased faster than Vmax. As a consequence, it was predicted and confirmed that enzymatic analysis of pyruvate using lactate dehydrogenase is more rapid at 20 degrees C than at 35 degrees C or 50 degrees C.

Alcohol Dehydrogenase↗

PubMed1995

Kinetic analysis of the activation of Zymomonas mobilis glucokinase by phosphate.

A detailed kinetic analysis of glucokinase EC 2.7.1.2 from Zymomonas mobilis has been carried out. This enzyme has an absolute requirement for inorganic phosphate as activator, and the kinetic behaviour can be interpreted as a steady-state ordered mechanism in which glucose is the first substrate. Values for each of the kinetic constants have been obtained for the conditions I = 0.12, 30 degrees C, and pH 7.0. Direct binding studies have confirmed that ATP does not bind to the enzyme without glucose present. Phosphate does not affect ATP binding to the enzyme-glucose complex; when saturated with both ATP and glucose, the dissociation constant for phosphate (determined kinetically) is 0.045 mM. When saturated with the other substrate and phosphate, the Km values for glucose and MgATP are 0.095 mM and 0.19 mM, respectively. The ionic form of phosphate is not important, as the apparent Km for phosphate did not change significantly over the pH range 6.4 to 7.5. Raising the temperature increased Vmax at the high rate of 10% per degree, which correlates well with the fermentation rates between 20 and 30 degrees C, giving further support to the concept that glucokinase is the rate-controlling enzyme in Z. mobilis glucose fermentation.

Adenosine Triphosphate↗

PubMed1994

Purification and characterization of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brockii.

Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2-propanol or poly(ethylene glycol) with MgCl2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 +/- 0.4 Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein-folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.

Adenosine Triphosphatases↗

PubMed1994

Polymerase chain reaction-based random mutagenesis: production and characterization of thermostable mutants of Zymomonas mobilis alcohol dehydrogenase-2.

The adhB gene encoding alcohol dehydrogenase-2 from Zymomonas mobilis has been subjected to random mutagenesis to obtain more thermostable variants of the enzyme. Random mutagenesis was accomplished using the polymerase chain reaction in mutagenic conditions. The optimum conditions involved restricting the concentration of one nucleotide to approximately one-tenth the normal amount. This introduced mutations at an average rate of 1 base in 600 in a 30-cycle PCR, sufficient to ensure that the majority of encoding DNA sequences in the mutant library have at least one base change from wild-type. Seven thermostable mutants were isolated from one library screening of 3000 colonies; two of these were selected for detailed study and purified using dye-ligand chromatography. Mutant TS-1 (F9S, V295A) was 3 degrees C more stable than the wild-type and had altered kinetic characteristics, with reduced affinity for ethanol and acetaldehyde and reduced ethanol oxidation Vmax. Mutant TS-2 (M13I, E19K, M192I) also had increased thermostability of 3 to 4 degrees C, but its kinetic characteristics were similar to that of the wild-type. Of the base changes found after sequencing a wide selection of mutants, 90% were transitions and 10% were transversions. Included were several T to C base changes which did not correspond with the nucleotide limitation used to create the mutant libraries.

Alcohol Dehydrogenase↗

PubMed1994

Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4).

Amino Acid Sequence↗

PubMed1994

Use of hydrophobic chromatography for purification of the membrane-located choline dehydrogenase from a Pseudomonas strain.

Choline dehydrogenase has been purified using hydrophobic chromatography 250-fold from a Pseudomonas strain. Although the enzyme is associated with the cell membrane and could be extracted from membrane preparations, it was best purified from a complete cell extract made with a non-ionic detergent. Only phenazine methosulfate was able to act as electron acceptor; there was no evidence of bound flavin, but there was evidence of pyrroloquinoline quinone cofactor. The purified enzyme had a specific activity of up to 67 units/mg, which is at least ten times higher than the values reported for mitochondrial choline dehydrogenases, and up to 100 times higher than previous reports for the Pseudomonas enzyme. The estimated subunit size of 66 kDa, which corresponds with the native size, is close to that deduced from the gene sequence of the Escherichia coli betA gene, and preliminary N-terminal sequencing shows homology with this deduced sequence. The next enzyme in the degradation pathway of choline, betaine aldehyde dehydrogenase, was also purified from the same extract.

Alcohol Oxidoreductases↗

PubMed1993

Cloning, sequencing and expression of the gene encoding NADH oxidase from the extreme anaerobic thermophile Thermoanaerobium brockii.

The gene encoding the enzyme NADH oxidase from the extreme thermophile Thermoanaerobium brockii has been isolated from a recombinant library of genomic DNA and sequenced. An open reading frame corresponds to the 651 amino acids of the enzyme's subunit, which include characteristic FAD- and NADH-binding sequences, as well as cysteines which are involved in the FeS cluster present in the enzyme. The enzyme is expressed either from its own promoter or from vector promoters in Escherichia coli. After heat-treating the recombinant extracts at 70 degrees C, most of the host proteins are denatured, leaving the NADH oxidase 5- to 10-fold enriched.

Amino Acid Sequence↗

PubMed1993

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.

2,3-Diphosphoglycerate↗

PubMed1992

A thermostable NADH oxidase from anaerobic extreme thermophiles.

A high-abundance NADH-oxidizing enzyme (NADH: acceptor oxidoreductase, EC 1.6.99.3) has been identified and isolated from a range of anaerobic extreme thermophiles, including strains of Clostridium thermohydrosulfuricum and Thermoanaerobium brockii. By use of a pseudo-affinity salt-promoted adsorbent, a nearly pure sample was obtained in one step; remaining impurities were separated by ion-exchange. The fully active purified enzyme contains FAD (two molecules per subunit of 75-78 kDa) and iron-sulphur, and is hexameric in its most active form. The reaction with oxygen is a one- or two-electron transfer to produce superoxide radical and H2O2; other acceptors include tetrazolium salts, dichlorophenol-indophenol, menadione and ferricyanide. The role of the enzyme is not clear; it was found not to be NAD:ferredoxin oxidoreductase, which is a major NADH-utilizing enzyme in these organisms.

Chromatography, Gel↗

PubMed1992

Changes in the fluorescence of bound nucleotide during the reaction catalysed by glucose-fructose oxidoreductase from Zymomonas mobilis.

The reduction of gluconolactone by glucose-fructose oxidoreductase containing tightly bound NADPH (enzyme-NADPH) is biphasic in nucleotide fluorescence. The initial rapid decrease, which represents quenching of the fluorescence by bound lactone, is followed by a slower decrease which corresponds to the change in absorbance. At low glucose concentrations, the oxidation of glucose by enzyme-NADP+ involves a single first-order process with similar rate constants in fluorescence and absorbance. At higher glucose concentrations, the apparent first-order rate constants for the fluorescence change are less than those for the absorbance change. This is consistent with a mechanism in which the fluorescence change occurs during the lactone dissociation step, which is slower than the hydrogen transfer step during which the absorbance change occurs. The rate constant for gluconolactone dissociation is 360 +/- 10 s-1 and this step is therefore rate-determining for the overall reaction. Reduction of fructose by enzyme-NADPH is first order with a limiting rate constant of at least 2000 s-1.

Carbohydrate Dehydrogenases↗

PubMed1992

Immunocytochemical localization of glycolytic and fermentative enzymes in Zymomonas mobilis.

Gold-labeled antibodies were used to examine the subcellular locations of 11 glycolytic and fermentative enzymes in Zymomonas mobilis. Glucose-fructose oxidoreductase was clearly localized in the periplasmic region. Phosphogluconate lactonase and alcohol dehydrogenase I were concentrated in the cytoplasm near the plasma membrane. The eight remaining enzymes were more evenly distributed within the cytoplasmic matrix. Selected enzyme pairs were labeled on opposite sides of the same thin section to examine the frequency of colocalization. Results from these experiments provide evidence that glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and alcohol dehydrogenase I form an enzyme complex.

Alcohol Dehydrogenase↗

PubMed1992

Cloning, sequence analysis, and expression of the structural gene encoding glucose-fructose oxidoreductase from Zymomonas mobilis.

The gene encoding glucose-fructose oxidoreductase (gfo) from Zymomonas mobilis was cloned in Escherichia coli and sequenced. An open reading frame of 439 amino acids encoded a protein of 49 kDa. A leader sequence of 52 amino acids preceded the N-terminal sequence of the enzyme, indicating cleavage of the precursor protein at an Ala-Ala site to give rise to an active form of the enzyme of 43 kDa. Processing of the glucose-fructose oxidoreductase leader sequence, although not complete, was demonstrated in an in vitro translation system. The two Z. mobilis promoters of the gfo gene show considerable homology to other highly expressed Z. mobilis genes (pdc, adhB, gap, and pgk) as well as to the E. coli consensus sequence. Although translation of the gfo gene was demonstrated in vitro in an E. coli S30 coupled transcription-translation system, a functional stable protein was not produced in the E. coli clone. However, the gfo gene cloned into a shuttle vector was shown to overexpress glucose-fructose oxidoreductase to levels of up to 6% of the soluble protein in Z. mobilis.

Amino Acid Sequence↗

PubMed1991

Membrane-associated ATPase from Zymomonas mobilis; purification and characterization.

The major ATPase (adenosinetriphosphatase, EC 3.6.1.3) activity present in the membrane of Zymomonas mobilis has been isolated, using a novel combination of multifunctional hydrophobic adsorbents. On subjecting the preparation to gel filtration, activity was lost, but could be restored by reconstituting fractions from the column. Subunit composition of the fractions indicated that the Zymomonas mobilis ATPase is of the F0F1 type, and so is probably involved in proton pumping. The contribution of this ATPase to the overall ATP turnover in the cells has been calculated to be approximately 20% and so it may be partly responsible for the phenomenon of 'uncoupled growth' observed with Zymomonas mobilis.

Adenosine Triphosphatases↗

PubMed1991

Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein.

The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One- and two-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignments were made by comigration with purified proteins, comparison with overexpressed genes in recombinant strains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced both by exposure to ethanol and by elevated temperature (45 degrees C). This enzyme, encoded by the adhB gene, is expressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus sequence for heat shock proteins.

Alcohol Dehydrogenase↗

PubMed1991

The use of multifunctional adsorbents to purify membrane-bound phosphatases from Zymomonas mobilis. Purification of acid phosphatase, alkaline phosphatase and ATPase.

The purification of detergent-solubilized membrane-bound phosphatases from Zymomonas mobilis using novel adsorbents is described. The prepared adsorbents have a hydrophobic core with functional groups attached. These functional groups may either increase or decrease the hydrophobicity of the adsorbent, or participate in other forms of interactions. Adsorption of acid phosphatase (ACP), alkaline phosphatase (ALP) and ATPase to these adsorbents was salt-promoted. Desorption was achieved by decreasing the salt concentration or by displacement with increasing concentration of Triton X-100. The results indicate that chromatography on multifunctional adsorbents that are predominantly hydrophobic in character is a procedure that can have a general applicability in purification of membrane proteins.

Acid Phosphatase↗

PubMed1990

Differential salt-promoted chromatography for protein purification.

A range of hydrophobic-type adsorbents for protein chromatography has been screened for the binding, at high salt concentrations, of 10 enzymes from a bacterial extract. Adsorbents were chosen for tandem chromatography, in which the first adsorbent removed much of the protein, and the second and subsequent columns bound the desired enzymes. Simple schemes for isolating Zymomonas mobilis and yeast alcohol dehydrogenases are described, in which the enzymes are affinity eluted by NAD+.

Alcohol Dehydrogenase↗

PubMed1988

The kinetics of glucose-fructose oxidoreductase from Zymomonas mobilis.

Glucose-fructose oxidoreductase operates by a classic ping-pong mechanism with a single site for all substrates: glucose, fructose, gluconolactone and sorbitol. The Km values for these substrates were determined. The values of kcat are 200 s-1 and 0.8 s-1 for the forward and reverse directions respectively. The overall catalytic process consists of two half-reactions with alternate reduction of NADP+ and oxidation of NADPH tightly bound to the enzyme. Reduction of enzyme-NADP+ by glucose and oxidation of enzyme-NADPH by gluconolactone involve single first-order processes. The values of the rate constants at saturating substrate are 2100 s-1 and 8 s-1 respectively; deuterium isotope effects indicate that these are for the hydrogen transfer step. Oxidation of enzyme-NADPH by fructose is first order with a limiting rate constant of at least 430 s-1. The reaction of enzyme-NADP+ with sorbitol is biphasic, with rate constants for both phases less than 1 s-1. This behaviour is explained by a mechanism in which the slow cyclisation of the acyclic form of fructose follows its dissociation from the enzyme. The rate-determining steps for the overall reaction are probably dissociation of gluconolactone in the forward direction and hydrogen transfer from sorbitol to enzyme-bound NADP+ in the reverse direction.

Carbohydrate Dehydrogenases↗

PubMed1987

Nucleotide sequence of the pyruvate decarboxylase gene from Zymomonas mobilis.

Pyruvate decarboxylase (EC 4.1.1.1), the penultimate enzyme in the alcoholic fermentation pathway of Zymomonas mobilis, converts pyruvate to acetaldehyde and carbon dioxide. The complete nucleotide sequence of the structural gene encoding pyruvate decarboxylase from Zymomonas mobilis has been determined. The coding region is 1704 nucleotides long and encodes a polypeptide of 567 amino acids with a calculated subunit mass of 60,790 daltons. The amino acid sequence was confirmed by comparison with the amino acid sequence of a selection of tryptic fragments of the enzyme. The amino acid composition obtained from the nucleotide sequence is in good agreement with that obtained experimentally.

Amino Acid Sequence↗