Ethanol and oleate inhibition of alpha-ketoisovalerate and 3-hydroxyisobutyrate metabolism by isolated hepatocytes.

Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.     

Effect of Streptomycin on Some Enzyme Systems of Bacillus subtilis

Streptomycin slightly inhibited lactic and malic dehydrogenases of Bacillus subtilis, and inhibited isocitric dehydrogenase to about 60%. The formation of lactic dehydrogenase, glutamic dehydrogenase, alpha-alanine dehydrogenase, and succinic dehydrogenase was stimulated by the antibiotic at a concentration causing 50% inhibition of bacterial growth. Streptomycin had practically no influence on the formation of malic dehydrogenase, but the antibiotic produced 48% inhibition of the synthesis of isocitric dehydrogenase.     

Inhibition of pyruvate dehydrogenase complex by moniliformin.

The mechanism for the inhibition of pyruvate dehydrogenase complex from bovine heart by moniliformin was investigated. Thiamin pyrophosphate proved to be necessary for the inhibitory action of moniliformin. The inhibition reaction was shown to be time-dependent and to follow first-order and saturation kinetics. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin-inactivated complex only partially reversed inactivation. Moniliformin seems to act by inhibition of the pyruvate dehydrogenase component of the enzyme complex and not by acting on the dihydrolipoamide transacetylase or dehydrogenase components, as shown by monitoring the effect of moniliformin on each component individually. On the basis of these results, a suicide inactivator mechanism for moniliformin on pyruvate dehydrogenase is proposed.     

Changes in enzymatic activities involved in glucose metabolism by acyl-CoAs in Trypanosoma cruzi

This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+ afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.     

Nickel-specific, slow-binding inhibition of carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide

The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 microM KCN. Dissociation constants of 8.46 microM (KCN) and 4.70 microM (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.     

Selective action of mycobacillin on the uptake of releasable cell materials by Aspergillus niger.

The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant.     

Purification and kinetic properties of 3 beta-hydroxysteroid dehydrogenase from bovine adrenocortical microsomes

3 beta-Hydroxysteroid dehydrogenase was purified from bovine adrenocortical microsomes and its properties were studied. The purified dehydrogenase gave a single homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed no steroid delta 5-delta-4 isomerase activity. The molecular weight of the dehydrogenase was estimated to be 41,000 for the monomer and the isoelectric point was determined to be at pH 6.3. The Km values of the dehydrogenase were 6.2 microM for NAD+, 4.9 mM for NADP+, 2.0 microM for pregnenolone, and 5.3 microM for 17 alpha-hydroxypregnenolone. The mechanism of inhibition by trilostane of the dehydrogenase was also examined kinetically. The inhibition was found to be competitive, with Ki values of 0.14 microM for 17 alpha-hydroxypregnenolone and 0.38 microM for pregnenolone.     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.     

Regulation of succinate oxidation by NAD+ in mitochondria purified from potato tubers

NAD⁺ supplied to purified Solanum tuberosum mitochondria caused progressive inhibition of succinate oxidation in State 3. This inhibition was especially pronounced at alkaline pH and at low succinate concentrations. Glutamate counteracted the inhibition. NAD⁺ promoted oxaloacetate accumulation in State 3; supplied oxaloacetate inhibited O₂ uptake in the presence of succinate much more severely in State 3 than in State 4. NAD reduction linked to succinate oxidation by ATP-dependent reverse electron transport was likewise inhibited by oxaloacetate. We conclude that NAD⁺-induced inhibition of succinate oxidation is due to an inhibition of succinate dehydrogenase resulting from increased accumulation of oxaloacetate generated from malate oxidation via malate dehydrogenase. The results are discussed in the context of the known regulatory characteristics of plant succinate dehydrogenase.     

The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase in situ PMR studies

The kinetics of inhibition of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by iodoacetate were studied in the intact cell and in vitro. The kinetics were determined using ¹H-NMR to follow solvent exchange of ¹H and ²H at the C-2 position of lactate. The exchange occurs via a series of enzyme-catalysed reactions, including that catalysed by glyceraldehyde-3-phosphate dehydrogenase. A direct assay with quenching of the inhibition was also used to check the results. Iodoacetate was shown to act as an active site-directed inhibitor of the dehydrogenase. The enzyme inhibition patterns, which are characterised by a binding step and a kinetic step, are similar in situ and in vitro. Membrane binding, however, was found to alter the inhibition pattern for the enzyme in vitro.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases.

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.     

Time-dependence of the effect of X-irradiation on the formation of glycerol phosphate dehydrogenase and other dehydrogenases in the developing rat brain

X-irradiation (100-1500 r) administered to the heads of rats 8-30 days of age inhibited the development of glycerol phosphate dehydrogenase (l-glycerol 3-phosphate-NAD oxidoreductase, EC 1.1.1.8) in the brain stem and cerebral hemispheres. At 40 days of age and older no effect was observed. This inhibition was a delayed phenomenon, dose-dependent and with no recovery. It is proposed that the inhibition of enzyme formation is related to radiation damage caused to DNA. Actinomycin D inhibited the development of glycerol phosphate dehydrogenase in a manner similar to ionizing radiation. Four other dehydrogenases also showed age-dependent radiosensitivities. ;Malic enzyme' (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27) and malate dehydrogenase (EC 1.1.1.37) ceased to be radiosensitive at about 8 days of age and isocitrate dehydrogenase (NADP) (EC 1.1.1.42) at 16 days. The correlation between developmental increase in enzyme activity and radiosensitivity held closely for glycerol phosphate dehydrogenase and isocitrate dehydrogenase and to a smaller extent for the others.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10(-6)M for aconitase, 10(-4)M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10(-3)M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10(-3)M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The Ki values for the purified enzyme in the presence of 5 microM alloxan were 0.22 microM with citrate, 4.0 microM with cis-aconitate and 0.62 microM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.     

Substrate inhibition of soluble and bound lactate dehydrogenase (isoenzyme 5)

Substrate inhibition of chicken lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5, was studied with the enzyme in the soluble phase and bound to muscle subcellular particulate structures. Inhibition studies were performed by incubating bound or soluble enzyme with NAD⁺ prior to measuring the reaction with a stopped-flow technique at 40 °C and a concentration of enzyme of 10^?7m. The value of V for soluble lactate dehydrogenase was 610 nmoles per sec, and for the bound enzyme it was 262. k_m (pyruvate) values were similar for both enzymes. Under our experimental conditions, up to 73% inhibition of the soluble enzyme was observed. On the other hand, there was no detectable inhibition of bound lactate dehydrogenase. It is suggested that the resistance to substrate inhibition of bound lactate dehydrogenase may possibly be due to the prevention of dissociation of the enzyme into monomeric or other subunits because of attachment to the particulate structures.     

Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.