Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.2 (PDK1)
2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intramitochondrial substrate metabolism was examined in cultured neuroblastoma NB41A3 cells exposed to endotoxin in order to elucidate possible causes for the changes in [ATP]/[ADP][Pi] and [NAD+]/[NADH] reported by us previously in these cells [1]. Flux through pyruvate dehydrogenase (PDH), measured with [1-14C]-pyruvate, was inhibited by 54% within 10 min in endotoxin-treated cells (0.99 nmol/min/mg dry wt vs 0.46 nmol/min/mg dry wt). In contrast, flux through 2-oxoglutarate dehydrogenase, measured with [1-14C]-glutamate was unaltered (0.79 nmol/min/mg dry wt). Dichloroacetate, an inhibitor of PDH kinase, restored flux through PDH to control levels. In endotoxin-treated cells, only 44% of the total PDH complex was in the active (nonphosphorylated) form as compared to 72% in control cells. Equilibrium uptake studies with 45Ca2+ and atomic absorption measurements showed that intracellular [Ca2+] in endotoxin-treated cells was about 20% lower than in control cells. It is postulated that binding of endotoxin to the plasma membrane triggers a sequence of events that lead to an initial decline in intracellular calcium concentration and that this latter event may be responsible for the inhibition of PDH phosphatase and consequent conversion of the complex to its inactive phosphorylated form.
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PMID:Cellular effects of endotoxin in vitro. I. Effect of endotoxin on mitochondrial substrate metabolism and intracellular calcium. 635 31

Bacitracin is a proteolytic inhibitor which interacts with the intracellular processing of insulin. Its effects on pyruvate, fatty acid and amino acid metabolism were examined in rat hepatocyte suspensions. Bacitracin (0.25-1.0 mM) increased the oxidation of [1-14C]pyruvate by 50-70% and presumably therefore increased the flux through pyruvate dehydrogenase. This was found both in the presence of extracellular Ca2+ and in its absence, but not in the presence of 2 mM-2-chloropropionate, which inhibits pyruvate dehydrogenase kinase. Insulin did not further stimulate [1-14C]pyruvate oxidation in the presence of 1 mM-bacitracin. Bacitracin decreased 14CO2 formation from [2-14C]pyruvate (20-40%) and [U-14C]palmitate (30-70%), suggesting a decreased flux through the tricarboxylic acid cycle. Fatty acid oxidation before acetyl-CoA formation was also decreased. Bacitracin decreased the incorporation of label from [3H]leucine into protein in the absence of insulin, but not in its presence. Bacitracin is commonly used in studies on insulin action. Our results suggest that in such studies the effects noted may be related not only to an interaction of bacitracin with the intracellular processing of insulin but also to direct metabolic effects of bacitracin independent of insulin.
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PMID:Metabolic effects of bacitracin in isolated rat hepatocytes. 641 32

The action of dichloroacetate (DCA) on pyruvate dehydrogenase (PDH) activity of rat brain has been studied in vitro and in vivo. In a crude brain mitochondrial fraction, DCA inhibits PDH kinase and in rat brain slices this compound increases PDH activity and stimulates glucose oxidation. In the whole animal, intraperitoneal injection of DCA causes activation of brain PDH, indicating that this inhibitor crosses the blood-brain barrier. The same treatment with DCA also produced a large increase in heart PDH activity. Further studies of the effects of DCA on the CNS should lead to results of considerable importance.
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PMID:Effects of dichloroacetate on brain pyruvate dehydrogenase. 668 96

Propionate inhibited the metabolic flux through the pyruvate dehydrogenase reaction in the perfused rat liver when the perfusate concentration of propionate was below 10 mM and the perfusate pyruvate concentration was held within the physiological range. At higher propionate concentrations (e.g., 20 mM) the inhibition of pyruvate dehydrogenase was alleviated and the activation state of the pyruvate dehydrogenase complex was nearly doubled. In livers perfused with a high pyruvate concentration (e.g., 5 mM), propionate coinfusion at all concentrations inhibited the rate of pyruvate decarboxylation. Additional studies were performed in liver mitochondria maintained in State 3 where the ATP/ADP and the NADH/NAD+ ratios were held constant. Low propionate concentrations (e.g., 0.5 mM) inactivated the mitochondrial pyruvate dehydrogenase complex, whereas propionate levels in excess of 1 mM activated the enzyme complex. CoA distribution analyses of the mitochondrial incubations indicated that the presence of either 0.5 or 10 mM propionate caused a substantial accumulation of propionyl-CoA and methylmalonyl-CoA at the expense of free CoASH. Experiments were performed in which the ratios of various acyl-CoA derivatives to CoASH were varied by sequentially increasing the L-carnitine concentrations in the incubation. An inverse relationship between the propionyl-CoA/CoASH and methylmalonyl-CoA/CoASH ratios and the activity of the pyruvate dehydrogenase complex was observed. Experiments using freeze-thawed liver mitochondrial membranes indicated that propionate protected the pyruvate dehydrogenase complex from ATP-mediated inactivation by the pyruvate dehydrogenase kinase. It is our contention that the inactivation of pyruvate dehydrogenase complex at low propionate levels may be due to an increase in the mitochondrial acyl-CoA/CoASH ratios, whereas the activation of the enzyme complex demonstrated at high propionate levels is due to the inhibition of the pyruvate dehydrogenase kinase in a manner similar to that caused by pyruvate or dichloroacetic acid.
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PMID:The effect of propionate on the regulation of the pyruvate dehydrogenase complex in the rat liver. 682 32

Succinyl-CoA synthetase and the alpha-subunit of pyruvate dehydrogenase are phosphorylated after incubation of mitochondria from brain, heart, and liver with [gamma-32P]ATP. Dichloroacetate, a known specific inhibitor for pyruvate dehydrogenase kinase, inhibits not only the phosphate incorporation into the alpha-subunit of pyruvate dehydrogenase but also the autophosphorylation of succinyl-CoA synthetase. AMP also inhibits the phosphorylation of both proteins. Phosphorylation of the alpha-subunit of pyruvate dehydrogenase in liver mitochondria is significantly lower than in mitochondria from other tissues.
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PMID:The effect of dichloroacetate on the phosphorylation of mitochondria proteins. 683 84

The effects of myocardial ischemia and reperfusion on pyruvate dehydrogenase (PDH) activity were studied in isolated rat hearts. PDH remained largely (80%) in the active form during 10 min of whole heart ischemia in hearts receiving 11 mM glucose as substrate. With reperfusion, PDH was converted to the inactive form (45% by 2 min) and then returned slowly to control levels. Addition of pyruvate (10 mM) to the glucose containing perfusate during reperfusion prevent the reperfusion inactivation of PDH (96% active). The maintenance of a high percent of PDH in the active form during ischemia occurred in spite of high mitochondrial ratios of NADH/NAD and acetyl CoA/CoA and was related to a very low mitochondrial ATP/ADP ratio. The low ATP and high ADP would restrict PDH kinase phosphorylation and inactivation of PDH during ischemia. Reperfusion resulted in a rapid increase in mitochondrial ATP/ADP ratio and the increased availability of ATP as substrate for the kinase coupled with continued high levels of NADH and acetyl CoA which stimulate kinase activity may have accounted for the early inactivation of PDH with reperfusion. Addition of pyruvate to the perfusate probably inhibited the PDH kinase and prevent the reperfusion inactivation of PDH.
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PMID:Effects of ischemia and reperfusion on pyruvate dehydrogenase activity in isolated rat hearts. 687 85

The effects of increased cardiac work, pyruvate and insulin on the state of pyruvate dehydrogenase (PDH) activation and rate of pyruvate decarboxylation was studied in the isolated perfused rat heart. At low levels of cardiac work, 61% of PDH was present in the active form when glucose was the only substrate provided. The actual rate of pyruvate decarboxylation was only 5% of the available capacity calculated from the percent of active PDH. Under this condition, the rate of pyruvate decarboxylation was restricted by the slow rate of pyruvate production from glycolysis. Increasing cardiac work accelerated glycolysis, but production of pyruvate remained rate limiting for pyruvate oxidation and only 40% of the maximal active PDH capacity was used. Addition of insulin along with glucose reduced the percent of active PDH to 16% of the total at low cardiac work. This effect of insulin was associated with increased mitochondria NADH/NAD and acetyl CoA/CoA ratios. With both glucose and insulin the calculated maximum capacity of active PDH was about the same as measured rates of pyruvate oxidation indicating that pyruvate oxidation was limited by the activation state of PDH. In this case, raising the level of cardiac work increased the active PDH to 85% and although pyruvate oxidation was accelerated, measured flux through PDH was only 73% of the maximal activity of active PDH. With pyruvate as added exogenous substrate, PDH was 82% of active at low cardiac work probably due to pyruvate inhibition of PDH kinase. In this case, the measured rate of pyruvate oxidation was 64% of the capacity of active PDH. However, increased cardiac work still caused further activation of PDH to 96% active. Thus, actual rates of pyruvate oxidation in the intact tissue were determined by (1) the supply of pyruvate in hearts receiving glucose alone, (2) by the percent of active PDH in hearts receiving both glucose and insulin at low work and (3) by end-product inhibition in hearts receiving glucose and insulin at high work or at all levels of work with pyruvate as substrate. The increase in active PDH with higher levels of cardia work was associated most closely with reduced mitochondrial NADH/NAD ratios and with decreased acetyl CoA/CoA ratios when insulin or pyruvate were present.
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PMID:Mechanism of pyruvate dehydrogenase activation by increased cardiac work. 687 86

Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffective. Hydrogen peroxide and t-butyl hydroperoxide were inactive. The data indicate pyruvate dehydrogenase kinase (EC 2.7.1.99) contains a thiol group (or groups) that is involved in maintaining a conformation of the enzyme that facilitates phosphorylation and inactivation of its protein substrate, pyruvate dehydrogenase (EC 1.2.4.1). These findings suggest that modulation of pyruvate dehydrogenase kinase activity by thiol-disulfide exchange may be an important physiological mechanism for regulation of kinase activity and, hence, activity of the pyruvate dehydrogenase complex.
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PMID:Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange. 695 81

Rat liver mitoplasts (inner mitochondrial membrane and matrix) contain protein kinase activity. This activity increases twofold on addition of Triton X-100. The activity observed in absence of Triton X-100 is probably exposed on the outer surface of mitoplasts, since it is sensitive to trypsin treatment. Most of the remaining protein kinase is bound to the membrane fraction, presumably on the inside of (or else hidden in) the inner mitochondrial membrane. Only a small part of the kinase activity is found in the mitochondrial matrix. A phosphoprotein band, partly resolved into a doublet, was observed on electrophoresis in SDS-polyacrylamide gels after endogeneous phosphorylation of mitoplasts, inner mitochondrial membrane or matrix. When isolated fractions are phosphorylated approximately 75% of the phosphoprotein is found in the matrix, and the remainder in the inner membrane. The phosphorylation of the doublet is inhibited by inhibitors to pyruvate dehydrogenase kinase, suggesting that it represents the phosphorylated subunit of pyruvate dehydrogenase.
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PMID:Localization of protein kinase activity and phosphoproteins in mitoplasts from rat liver. 733 41

Evidence for a reversible process resulting in stable activated and inactivated states of the mitochondrial branched chain alpha-keto acid dehydrogenase complex in isolated perfused rat heart is presented. The inactivation process is mediated by pyruvate infusion, while activation (up to 18-fold) is facilitated by branched chain alpha-keto acid substrates. The low activity state of the branched chain complex characteristic of freshly excised rat hearts could be maintained by infusion of either pyruvate or glucose. Activation of the complex in the perfused rat heart was achieved slowly by substrate-free perfusion, while rapid activation was accomplished by infusion of branched chain alpha-keto acids. The fully activated enzyme complex resulting from branched chain alpha-keto acid infusion subsequently could be inactivated maximally by infusion of pyruvate alone or intermediate degrees of inactivation could be produced by certain ratios of co-infused pyruvate and branched chain alpha-keto acid. alpha-Ketoisocaproate was an order of magnitude more effective than alpha-keto isovalerate either in preventing inactivation or in stimulating the opposing activation process when co-infused with pyruvate. The mitochondrial pyruvate transport inhibitor, alpha-cyanocinnamate, effectively prevented inactivation of the complex by infused pyruvate. Differential changes in the activation states of the branched chain alpha-keto acid dehydrogenase and pyruvate dehydrogenase complexes were evident when the two complexes were compared in apparently similar flux-inhibited (via octanoate infusion) and flux-stimulated (via dichloroacetate infusion) metabolic conditions. The differential effect of pyruvate concentration on the activity states of the two complexes was also well-defined. The results of the present study suggest distinct systems for the regulation of the activity of the two multienzyme complexes of interest. While our results argue neither for nor against an inactivation of the branched chain alpha-keto acid dehydrogenase complex by a protein kinase, the regulatory properties of such an intramitochondrial protein kinase may not be similar to the pyruvate dehydrogenase kinase. The mechanistic nature of the suggested novel regulatory system concerned with the pyruvate-mediated inactivation of the branched chain alpha-keto acid activation cannot be inferred at the present time.
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PMID:Studies on the activation and inactivation of the branched chain alpha-keto acid dehydrogenase in the perfused rat heart. 743 Jan 1


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