Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.

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Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1991.tb21044.x ◽

1991 ◽

Vol 200 (1) ◽

pp. 29-34 ◽

Cited By ~ 16

Author(s):

Egbert SCHULZE ◽

Jacques A. E. BENEN ◽

Adrie H. WESTPHAL ◽

Arie KOK

Keyword(s):

Pyruvate Dehydrogenase ◽

Azotobacter Vinelandii ◽

Pyruvate Dehydrogenase Complex ◽

Lipoamide Dehydrogenase ◽

Dehydrogenase Complex

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Activation of pyruvate dehydrogenase complex (PDC) of rat heart mitochondria by glyburide

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(84)90399-1 ◽

1984 ◽

Vol 123 (1) ◽

pp. 202-209 ◽

Cited By ~ 6

Author(s):

Cynthia K. Buffington ◽

Abbas E. Kitabchi

Keyword(s):

Pyruvate Dehydrogenase ◽

Rat Heart ◽

Pyruvate Dehydrogenase Complex ◽

Heart Mitochondria ◽

Rat Heart Mitochondria ◽

Dehydrogenase Complex

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Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation

Biochemical Journal ◽

10.1042/bj1900705 ◽

1980 ◽

Vol 190 (3) ◽

pp. 705-710 ◽

Cited By ~ 22

Author(s):

P A Lazo ◽

A Sols

Keyword(s):

Pyruvate Dehydrogenase ◽

Metabolic Regulation ◽

Pyruvate Dehydrogenase Complex ◽

Acetyl Coa ◽

Substrate Complex ◽

Ascites Tumour ◽

Rate Limiting Step ◽

Lipoamide Dehydrogenase ◽

Dehydrogenase Complex

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich—Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17—36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme—substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.

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The Interaction Between Lipoamide Dehydrogenase and the Peripheral-Component-Binding Domain from the Azotobacter Vinelandii Pyruvate Dehydrogenase Complex

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.861_a.x ◽

1995 ◽

Vol 234 (3) ◽

pp. 861-870 ◽

Cited By ~ 10

Author(s):

Adrie H. Westphal ◽

Anna Fabisz-Kijowska ◽

Henri Kester ◽

Peter P. Obels ◽

Aart Kok

Keyword(s):

Pyruvate Dehydrogenase ◽

Azotobacter Vinelandii ◽

Pyruvate Dehydrogenase Complex ◽

Binding Domain ◽

Lipoamide Dehydrogenase ◽

Dehydrogenase Complex

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Regulation of pyruvate dehydrogenase complex in ischemic rat heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1984.246.6.h858 ◽

1984 ◽

Vol 246 (6) ◽

pp. H858-H864 ◽

Cited By ~ 3

Author(s):

T. B. Patel ◽

M. S. Olson

Keyword(s):

Pyruvate Dehydrogenase ◽

Rat Heart ◽

Pyruvate Dehydrogenase Complex ◽

Total Activity ◽

Control Flow ◽

Enzyme Complex ◽

Multienzyme Complex ◽

Flow Rates ◽

Perfused Rat Heart ◽

Dehydrogenase Complex

The effect of flow-induced ischemia on the rate of pyruvate decarboxylation and the activation state of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated, perfused rat heart. Pyruvate dehydrogenase activity in the heart decreased significantly during flow-induced ischemia and was a function of changes in the activation state (i.e., active/total activity) of the enzyme complex. In the absence of pyruvate, the activation state of pyruvate dehydrogenase decreased from nearly 100% active at the normal flow rate (10 ml/min) to 20% active as the flow was reduced to 0.5 ml/min. At high pyruvate levels (5 mM), the activation state increased from nearly 70% active at control flow rates to 100% active during ischemia. At an intermediate pyruvate concentration (0.5 mM), the enzyme complex was maintained at a relatively low activation state (30–35% active) throughout the range of flow rates tested. Ischemia caused elevated perfusate lactate concentrations only when the flow rates were less than 5.0 ml/min. The activation state of the pyruvate dehydrogenase complex in hearts perfused with glucose was also decreased during ischemia.

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