scholarly journals Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria

1980 ◽  
Vol 188 (2) ◽  
pp. 409-421 ◽  
Author(s):  
G J Sale ◽  
P J Randle
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Diabetic Rats ◽  
Heart Mitochondria ◽  
Kinase Reaction ◽  
Rat Heart Mitochondria ◽  
Dehydrogenase Complex

1. Evidence is given for three sites of phosphorylation in the alpha-chains of the decarboxylase component of purified rat heart pyruvate dehydrogenase complex, analogous to those established for procine and bovine complexes. Inactivation of rat heart complex was correlated with phosphorylation of site 1. Relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. 2. Methods are described for measurement of incorporation of 32Pi into the complex in rat heart mitochondria oxidizing 2-oxoglutarate + L-malate (total, sites 1, 2 and 3). Inactivation of the complex was related linearly to phosphorylation of site 1 in mitochondria of normal or diabetic rats. The relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. Rates of site-2 and site-3 phosphorylation may have been closer to that of site 1 in mitochondria of diabetic rats than in mitochondria of normal rats. 3. The concentration of inactive (phosphorylated) complex was varied in mitochondria from normal rats by inhibiting the kinase reaction with pyruvate at concentrations ranging from 0.15 to 0.4 mM. The results showed that the concentration of inactive complex is related linearly to incorporation of 32Pi into site 1. Inhibition of 32Pi incorporations with pyruvate at all concentrations over this range was site 3 greater than site 2 greater than site 1. 4. With mitochondria from diabetic rats, pyruvate (0.15-0.4 mM) inhibited incorporation of 32Pi into site 3, but it had no effect on the concentration of inactive complex or on incorporations of 32Pi into site 1 or site 2. It is concluded that site-3 phosphorylation is not required for inactivation of the complex in rat heart mitochondria. 5. Evidence is given that phosphorylation of sites 2 and 3 may inhibit reactivation of the complex by dephosphorylation in rat heart mitochondria.

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.

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 Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart

1998 ◽  
Vol 329 (1) ◽  
pp. 197-201 ◽  
Author(s):  
Pengfei WU ◽  
Juichi SATO ◽  
Yu ZHAO ◽  
Jerzy JASKIEWICZ ◽  
M. Kirill POPOV ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Insulin Treatment ◽  
Pyruvate Dehydrogenase Complex ◽  
Immunoblot Analysis ◽  
Diabetic Rats ◽  
Pyruvate Dehydrogenase Kinase ◽  
Specific Effects ◽  
Dehydrogenase Complex

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.

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 Proportion of active dephosphorylated pyruvate dehydrogenase complex in heart and isolated heart mitochondria is decreased in obese hyperinsulinaemic mice

1984 ◽  
Vol 217 (1) ◽  
pp. 117-121 ◽  
Author(s):  
A L Kerbey ◽  
I D Caterson ◽  
P F Williams ◽  
J R Turtle
Keyword(s):  
Insulin Resistance ◽  
Enzyme Activity ◽  
Pyruvate Dehydrogenase ◽  
Glucose Oxidation ◽  
Isolated Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Heart Mitochondria ◽  
Mouse Heart ◽  
Inhibitory Effects ◽  
Dehydrogenase Complex

The proportion of active, dephosphorylated, pyruvate dehydrogenase complex was decreased in the mouse heart by obesity (by 56%), and this decrease in enzyme activity persisted during preparation and extraction of heart mitochondria. Phosphorylation and inactivation of pyruvate dehydrogenase may be a major factor in mediating the inhibitory effects of obesity on glucose oxidation in muscle, and this may represent an important mechanism in the development and/or expression of cellular insulin-resistance.

scholarly journals Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria

1980 ◽  
Vol 190 (1) ◽  
pp. 107-117 ◽  
Author(s):  
R M Denton ◽  
J G McCormack ◽  
N J Edgell
Keyword(s):  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Ruthenium Red ◽  
Heart Mitochondria ◽  
Heart Cells ◽  
Maximal Effect ◽  
Rat Heart Mitochondria ◽  
20 Nm

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2–3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.

scholarly journals Pyruvate dehydrogenase kinase/activator in rat heart mitochondria, Assay, effect of starvation, and effect of protein-synthesis inhibitors of starvation

1982 ◽  
Vol 206 (1) ◽  
pp. 103-111 ◽  
Author(s):  
A L Kerbey ◽  
P J Randle
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
High Speed ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Active Form ◽  
Pyruvate Dehydrogenase Kinase ◽  
Heart Mitochondria ◽  
Supernatant Fraction ◽  
Kidney Mitochondria

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.

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