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Biomedical subjects

H F Gilbert

Publications and source records attributed to H F Gilbert.

At least 55 records · Page 3Linked to original sources
PubMed1989

The effects of mevinolin on the thiol/disulfide exchange between 3-hydroxy-3-methyglutaryl-coenzyme A reductase and glutathione.

The feeding of mevinolin plus cholestyramine to rats results in the production of a form of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR-CM) having thiol/disulfide redox properties different from those of 3-hydroxy-3-methylglutaryl-CoA reductase isolated from animals which had been given only cholestyramine (HMGR-C). The second-order rate constant for the inactivation of HMGR-CM by GSSG is 7-fold slower than for HMGR-C, while the second-order rate constant for the reactivation of oxidized enzyme by GSH is 100-fold slower. However, in the presence of saturating concentrations of both substrates, the rate constants for thiol/disulfide exchange are similar for both forms of the enzyme. HMGR-CM behaves as if a protein-glutathione mixed disulfide having a Kox of 27 +/- 4 is formed at equilibrium. In contrast, HMGR-C has previously been shown to form a protein-protein disulfide (Cappel, R. E., and Gilbert, H. F. (1988) J. Biol. Chem. 263, 12204-12212). Both forms of the enzyme are more difficult to oxidize thermodynamically in the presence of saturating levels of both substrates. For HMGR-CM, NADPH alone has no effect on the equilibrium constant for oxidation, but hydroxymethylglutaryl-CoA alone makes the enzyme approximately twice as difficult to oxidize. Under physiological conditions, HMGR-CM is thermodynamically more difficult to oxidize than HMGR-C. HMGR-C can be converted to HMGR-CM by in vitro treatment with mevinolinate. A direct or indirect interaction of mevinolin with HMGR-C results in some persistent, as yet undefined, structural alteration which inhibits the formation of a protein-SS-protein disulfide upon oxidation by glutathione disulfide.

Animals↗
PubMed1988

Inhibition of glucose phosphorylation by fatty acids in the perfused rat heart.

The flux of glucose entering the glycolytic pathway under various metabolic conditions has been indirectly monitored in the Langendorff perfused rat heart using 31P-NMR spectroscopy. By totally inhibiting (greater than 95%) glyceraldehyde-3-phosphate dehydrogenase with low concentrations of iodoacetic acid (0.2 mM) in the perfusion medium, active glycolysis results in the accumulation of sugar phosphate species (fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde 3-phosphate) which can be observed in the 31P-NMR spectrum. Using this technique, it has been shown that butyrate (10 mM) in the perfusion medium decreases the flux through the initial steps of the glycolytic pathway by at least 6-fold and that both glucose phosphorylation and glycogenolysis are inhibited. Upon total global ischemia in the presence of both glucose and butyrate, the glycolysis rate is stimulated approx. 100-fold.

Adenosine Triphosphate↗
PubMed1988

Thiol/disulfide exchange between 3-hydroxy-3-methylglutaryl-CoA reductase and glutathione. A thermodynamically facile dithiol oxidation.

In glutathione redox buffers, rat liver, microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase rapidly equilibrates between a reduced, active form and an oxidized, inactive form. At pH 7.0, 37 degrees C, the second order rate constant for inactivation of the reduced enzyme by GSSG is 1700 +/- 200 M-1 min-1, approximately 20-fold faster than the reaction of GSSG with a typical, unhindered thiol of pKa 7.7. High concentrations of GSH or lower concentrations of dithiothreitol restore the activity of the oxidized enzyme. The oxidation of the enzyme by GSSG is only 30-fold slower in the presence of saturating levels of both substrates. The incomplete inhibition of thiol/disulfide exchange by substrates can lead to significant changes in the activity of the enzyme during the assay when glutathione is present. At redox equilibrium, both in the absence and presence of substrates, the activity of the enzyme depends on the quantity [GSH]2/[GSSG], suggesting that the redox transition involves the formation of a protein-SS-protein disulfide. The equilibrium constant for the reaction HMGRred + GSSG in equilibrium HMGRox + 2 GSH is 0.55 +/- 0.07 M in the absence of substrates and 0.20 +/- 0.02 M in the presence of saturating levels of both substrates. Thus, HMG-CoA reductase is very sensitive to dithiol oxidation both kinetically and thermodynamically. Significant changes in the oxidation state and activity of this enzyme could be expected to result from normal changes in the thiol/disulfide oxidation state of the cellular glutathione redox buffer.

Animals↗
PubMed1988

Determination of buffering capacity of rat myocardium during ischemia.

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.

Adenosine Triphosphate↗
PubMed1987

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics.

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.

Bacterial Proteins↗
PubMed1986

Thiol/disulfide exchange between rabbit muscle phosphofructokinase and glutathione. Kinetics and thermodynamics of enzyme oxidation.

Reversible thiol/disulfide exchange equilibria between rabbit muscle phosphofructokinase and glutathione redox buffers results in a dependence of the activity of the enzyme on the thiol to disulfide ratio of the redox buffer (Gilbert, H. F. (1982) J. Biol. Chem. 257, 12086-12091). The transition between fully reduced (active) and fully oxidized (inactive) enzyme is half complete at a [GSH]/[GSSG] ratio of 6.5 +/- 1 at pH 8.0 and 5.6 +/- 0.9 at pH 7.2. In the presence of excess GSSG approximately 40-50% of the activity is lost in a rapid process (k = 110 M-1 min-1), while the remaining activity is lost more slowly (k = 1.9 M-1 min-1). Two equivalents of radiolabeled glutathione are incorporated covalently, one coincident with each phase of inactivation. The most rapidly oxidized sulfhydryl group is also the most rapidly reduced by GSH in the reverse reaction (k = 150 M-1 min-1). Reduction of a more slowly reacting protein-glutathione mixed disulfide is required to regenerate the original activity (k = 0.33 M-1 min-1). The thiol/disulfide oxidation equilibrium constant (Kox) for the most rapidly oxidized sulfhydryl group is estimated to be 0.7 while that for the more slowly oxidized group is 6.1. The sulfhydryl group which is more easily oxidized kinetically is the more thermodynamically resistant to oxidation. The magnitude of the equilibrium constants for these reversible oxidations would suggest that the oxidation state (and activity) of phosphofructokinase would not be significantly affected by typical metabolic changes in the glutathione oxidation state in vivo.

Animals↗
PubMed1986

Thiol/disulfide redox equilibrium between glutathione and glycogen debranching enzyme (amylo-1,6-glucosidase/4-alpha-glucanotransferase) from rabbit muscle.

Rabbit skeletal muscle glycogen debranching enzyme is inactivated in a kinetically biphasic manner by GSSG at pH 8.0. The rapid phase results in the loss of 30% activity, while the slower phase leads to total enzyme inactivation. Both the glucosidase and the transferase activities of the enzyme are inhibited by GSSG. The inactivation by disulfides is fully and rapidly reversed in a biphasic manner by reduction with excess reduced dithiothreitol or GSH. After a fast initial recovery of 70% of the initial activity, the remaining 30% of the activity is recovered more slowly. Equilibration of the enzyme with a redox buffer of GSH and GSSG shows a monophasic equilibration of the activity. The ratio of GSH/GSSG where the enzyme is 50% active (R0.5) is 0.06 +/- 0.03. The R0.5 does not vary significantly with the total concentration of glutathione species suggesting formation of protein-SSG mixed disulfides. The ratios of the observed second-order rate constants for GSSG inactivation and GSH reactivation do not lead to a correct value of the observed thiol/disulfide oxidation equilibrium constant. Although the enzyme has sulfhydryl groups, the oxidation of which leads to activity changes, the kinetic and thermodynamic resistance to oxidation suggests that the enzyme is not likely to be subject to regulation by thiol/disulfide exchange in vivo.

Animals↗
PubMed1986

Cooperative behavior in the thiol oxidation of rabbit muscle glycogen phosphorylase in cysteamine/cystamine redox buffers.

Glycogen phosphorylase a and b are irreversibly inactivated by oxidation with the disulfide cystamine. The mechanism is complex and involves oxidation of at least two classes of sulfhydryl groups. The oxidation of one or more of the first class of 4 +/- 1 sulfhydryl groups is reversible, but the equilibrium constant for the oxidation is so unfavorable (1 X 10(-4)) that the micromolar concentrations of cysteamine released stoichiometrically with enzyme oxidation are sufficient to prevent complete oxidation even in the presence of 100 mM cystamine. The rapid phase of inactivation of phosphorylase b, which is first order in cystamine (k = 2.9 +/- 0.3 M-1 min-1), is followed by the oxidation of 5 +/- 1 groups in an irreversible process that is second order in cystamine concentration (k = 3.9 +/- M-2 min-1). Similar behavior is observed for phosphorylase a, although the behavior is complicated by association/dissociation equilibrium. The second-order dependence of the rate of irreversible inactivation on cystamine concentration is interpreted in terms of a "cooperative" model in which a rapidly reversible thermodynamically unfavorable equilibrium oxidation of one or more sulfhydryl groups must precede the irreversible oxidation of one or more additional sulfhydryl groups. The thiol/disulfide oxidation equilibrium constant for the initial reversible reaction is estimated to be at least 10(4) less favorable than that for the reversible oxidation of phosphofructokinase.

Adenosine Monophosphate↗
PubMed1986

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase.

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.

Acetyl Coenzyme A↗
PubMed1985

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction.

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.

Animals↗
PubMed1985

Interaction between citrate synthase and thiolase.

Thiolase, a mitochondrial matrix enzyme which produces CoASAc from fatty acids, is shown to interact with citrate synthase, the mitochondrial matrix enzyme responsible for CoASAc utilization. The interaction is demonstrated in three ways: the two enzymes co-precipitate in polyethylene glycol; thiolase causes a change in the fluorescence anisotropy of labeled citrate synthase; and the two enzymes co-elute in gel permeation chromatography. The interactions are shown to be specific by the use of enzymes not metabolically related to citrate synthase.

Acetyl-CoA C-Acyltransferase↗
PubMed1984

Fluorescence energy transfer measurements of spatial relationships between sulfhydryl groups of thiolase I from porcine heart.

Mitochondrial thiolase I from pig heart has been found to have at least two and possibly three reactive sulfhydryl residues at or near the active site [Izbicka-Dimitrijević, E., & Gilbert, H. F. (1982) Biochemistry 21, 6112-6118; Izbicka-Dimitrijević, E., & Gilbert, H. F. (1984) Biochemistry 23, 4318-4324]. In the native enzyme, fluorescein mercuric acetate reacts with two of the sulfhydryl groups and inactivates the enzyme with a rate constant of 1.6 X 10(-4) M-1 s-1 in 0.1 M Tris-acetate, pH 7.0. The presence of saturating (250 microM) concentrations of acetoacetyl coenzyme A protects against both modification and inactivation. The acetyl enzyme, a normal intermediate in the reaction catalyzed by thiolase, is not inactivated by fluorescein mercuric acetate although one sulfhydryl group out of five per mole of thiolase subunit is still available for reaction with the reagent. Fluorescein mercuric acetate and S-mercurio-N-dansyl-L-cysteine (Dns-Cys-SHg+) have been used to differentially label two of the sulfhydryl groups in thiolase. The distance between Dns-Cys-SHg+ (donor) and fluorescein mercuric acetate (acceptor) determined by the fluorescence energy transfer is less than 14 A. The fluorescent analogue of coenzyme A, 1,N6-etheno coenzyme A, is recognized by thiolase as a substrate (Km = 21 microM); however, substrate inhibition and equilibrium dialysis show that the affinity of the free enzyme for CoA is quite low (Ki = 100 microM). The quantum yield of the fluorescence of the three thiolase tryptophan residues is low (0.024), corresponding to about 12% of the fluorescence expected from equivalent concentrations of tryptophan.

Acetyl-CoA C-Acetyltransferase↗
PubMed1984

Multiple oxidation products of sulfhydryl groups near the active site of thiolase I from porcine heart.

The inactivation of porcine heart thiolase I with the disulfide reagents 5,5'-dithiobis(2-nitrobenzoate) (DTNB) and 2,2- and 4,4-dithiopyridine in 0.2 M phosphate buffer, pH 7.5, follows second-order kinetics with rate constants of 2.2 X 10(2), 25 X 10(2), and 5.8 X 10(2) M-1 min-1, respectively. Stoichiometric concentrations of the thiol-oxidizing reagent diethyl azodicarboxylate inactivate thiolase in less than 1 min at pH 7.5. The presence of saturating concentrations of the substrate acetoacetyl coenzyme A or the formation of the acetyl enzyme (a normal catalytic intermediate) results in a significant protection against the inactivation of thiolase by DTNB, 2,2-dithiopyridine, and diethyl azodicarboxylate. All five sulfhydryl residues of native thiolase react with either of the dipyridyl disulfides, but only the equivalent of 3.2 residues react with DTNB even at high concentrations and prolonged incubation times. The reaction of thiolase with DTNB leads to the formation of 1.0-1.4 mol of intrachain disulfide and 0.65 mol of mixed disulfides. After inactivation of thiolase with an equimolar concentration of diethyl azodicarboxylate, 1.2 mol of intrachain disulfide per subunit is found. No cross-linking between the subunits occurs as a result of the reaction of thiolase with DTNB or diethyl azodicarboxylate. The DTNB-inactivated enzyme can be reactivated with excess dithiothreitol while the diethyl azodicarboxylate inactivated enzyme is totally resistant to reactivation by dithiothreitol. There appear to be at least two different ways of forming inactive, oxidized enzyme products depending on the oxidant used, suggesting the possibility of multiple sulfhydryl groups at or near the active site.

2,2'-Dipyridyl↗
PubMed1983

Self-association of rabbit muscle phosphofructokinase: role of subunit interaction in regulation of enzymatic activity.

Phosphofructokinase (PFK) has been reversibly inactivated by oxidized glutathione. The physical properties of this inactivated form were characterized by sedimentation studies. In the presence of saturating amounts of fructose 6-phosphate and the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate, the inactivated PFK sediments as a 13.5S component. Sedimentation equilibrium study identifies it to be a tetramer with a molecular weight of 320 000. Sedimentation velocity studies in buffer alone at both pH 7.00 and pH 8.55 reveal that this inactivated enzyme can still undergo self-association with the same stoichiometry as that of the native enzyme, although the equilibrium constants are in favor of the formation of lower aggregates. The presence of allosteric activators, either ADP or cAMP, enhances the association of the inactivated PFK without changing its stoichiometry, an observation similar to that of the native enzyme. However, quantitatively the enhancement of association is not to the same extent as that of the native enzyme. Conversely, citrate favors the formation of smaller aggregates quantitatively similar to that of the native enzyme. Hence, this study shows that the oxidation state of the protein sulfhydryl groups influences the physical properties of PFK and the modulation of PFK activity is linked to subunit association-dissociation and quaternary structural changes.

Adenosine Triphosphate↗
PubMed1982

Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange.

Rabbit muscle phosphofructokinase is rapidly inactivated at pH 8.0 by incubation with low concentrations of oxidized glutathione, Coenzyme A glutathione mixed disulfide, and oxidized Coenzyme A. The inactivation is first order in disulfide concentration over the concentration ranges examined (50-200 microM), and is approximately 8-fold slower at pH 7.0 than at pH 8.0. The substrates ATP and fructose 6-phosphate protect against inactivation while effector molecules such as AMP, cAMP, and citrate do not. The oxidation of the enzyme by disulfides is fully reversible. The equilibrium constant for the reaction Ered + GSSG in equilibrium Eox + GSH at pH 8.0 is 7.1 in the absence of substrates and 2.5 in the presence of 0.1 mM ATP. For comparison, the equilibrium constant for the reaction CoASH + GSSG in equilibrium CoASSG + GSH was found to be 3.1 at pH 8.0. These equilibrium constants for thiol/disulfide exchange are such that modulation of phosphofructokinase activity by thiol/disulfide exchange in vivo is feasible. The ability of the thiol/disulfide ratio in vivo to modulate the activity of the fructose 6-phosphate/fructose 1,6-diphosphate futile cycle is discussed. The possibility is considered that modulation of the thiol/disulfide ratio in vivo may serve as a "third messenger" in response to cAMP levels, and that the activity of key enzymes of glycolysis/gluconeogenesis may be regulated in response to changing thiol/disulfide ratios.

Animals↗