Regulation of pyruvate dehydrogenase complex in ischemic rat heart

1984 ◽  
Vol 246 (6) ◽  
pp. H858-H864 ◽  
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.

Effects of aging on the activities of pyruvate dehydrogenase complex and its kinase in rat heart

Life Sciences ◽  
1997 ◽  
Vol 60 (25) ◽  
pp. 2309-2314 ◽  
Author(s):  
Naoya Nakai ◽  
Yuzo Sato ◽  
Yoshiharu Oshida ◽  
Atsushi Yoshimura ◽  
Noriaki Fujitsuka ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Effects Of Aging ◽  
Dehydrogenase Complex

scholarly journals Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat

1978 ◽  
Vol 173 (2) ◽  
pp. 669-680 ◽  
Author(s):  
N J Hutson ◽  
A L Kerbey ◽  
P J Randle ◽  
P H Sugden
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Diabetic Rats ◽  
Atp Depletion ◽  
Heart Mitochondria ◽  
Active Complex ◽  
Phosphate Complex ◽  
Rat Hearts ◽  
Dehydrogenase 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.

On The Unique Structural Organization of the Saccharomyces Cerevisiae Pyruvate Dehydrogenase Complex

1998 ◽  
Vol 4 (S2) ◽  
pp. 954-955
Author(s):  
James K. Stoops ◽  
Z. Hong Zhou ◽  
John P. Schroeter ◽  
Steven J. Kolodziej ◽  
R. Holland Cheng ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Structural Organization ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Oxidative Decarboxylation ◽  
Multienzyme Complex ◽  
The Core ◽  
Dehydrogenase Complex

Dihydrohpoamide acetyl transferase (E2), a catalytic and structural component of a multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate, forms the central core to which the other components are bound. We have utilized protein engineering and 3-D electron microscopy to study the structural organization of the largest multienzyme complex known (Mr ∼ 107). The structures of the truncated 60-mer core (tE2) and complexes of the tE2 associated with a binding protein (BP), and the BP associated with its dihydrohpoamide dehydrogenase (BP'E3) and the intact E2 associated with BP and the pyruvate dehydrogenase (E1) were determined (Figs. 1 and 2). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges.Previous studies have given rise to the generally accepted belief that BP and BP'E3 components are bound on the outside of the E2 scaffold and that E1 is similarly bound to the core in variable positions by flexible tethers.

Computer simulation of metabolism in pyruvate-perfused rat heart. III. Pyruvate dehydrogenase

1979 ◽  
Vol 237 (3) ◽  
pp. R167-R173 ◽  
Author(s):  
M. C. Kohn ◽  
M. J. Achs ◽  
D. Garfinkel
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Active Form ◽  
Realistic Model ◽  
Specific Protein ◽  
Active Species ◽  
Perfused Rat Heart ◽  
Synergistic Activation ◽  
The Difference

A physiologically and biochemically realistic model of the regulation of pyruvate dehydrogenase complex (PDH) was constructed for the perfused rat heart. It includes conversion between inactive (phospho) and active (dephospho) forms by a specific protein kinase (PDHK) and phosphoprotein phosphatase (PDHP). The activity of the tightly bound PDHK is influenced by synergistic activation/inhibition by acetyl CoA/CoASH and NADH/NAD. PDHK in this simulation was more sensitive to the fraction of ADP that was Mg2+-chelated than to the ATP-to-ADP ratio. Ca2+ stimulates binding of Mg2+-dependent PDHP to the complex; the bound enzyme was considered to be the active species. The fraction of PDH in the active form, rather than substrate and inhibitor levels, determines PDH activity under these conditions. This fraction depends on the present value and recent history of the difference between PDHK and PDHP activities. Both of these are active continuously and continuously control PDH.

scholarly journals Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

1982 ◽  
Vol 205 (2) ◽  
pp. 389-396 ◽  
Author(s):  
Leonard C. Packman ◽  
Richard N. Perham ◽  
Gordon C. K. Roberts
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Random Coil ◽  
Cross Linking ◽  
Enzyme Complex ◽  
Cross Links ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the 1H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T2, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the 1H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by 1H n.m.r. spectroscopy.

scholarly journals Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide

1976 ◽  
Vol 154 (2) ◽  
pp. 327-348 ◽  
Author(s):  
A L. Kerbey ◽  
P J. Randle ◽  
R H. Cooper ◽  
S Whitehouse ◽  
H T. Pask ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Alloxan Diabetes ◽  
Total Activity ◽  
Diabetic Rats ◽  
Pyruvate Dehydrogenase Kinase ◽  
Perfused Rat Heart ◽  
Kinase Reaction ◽  
The Matrix ◽  
Rat Heart Mitochondria

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

scholarly journals Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides

1980 ◽  
Vol 189 (1) ◽  
pp. 161-172 ◽  
Author(s):  
C E Henderson ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Icosahedral Symmetry ◽  
Enzyme Complex ◽  
Molecular Masses ◽  
Dehydrogenase Complex

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.

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