scholarly journals Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria

1981 ◽  
Vol 194 (3) ◽  
pp. 721-732 ◽  
Author(s):  
R G Hansford
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Inhibitory Effect ◽  
O2 Uptake ◽  
Mitochondrial Content ◽  
Bicarbonate Ions ◽  
Direct Inhibitory Effect ◽  
Na Ions ◽  
Rat Heart Mitochondria ◽  
Stimulation Of

1. The mitochondrial content of active (dephospho) pyruvate dehydrogenase (PDHA) was found to be severalfold higher at an extramitochondrial Ca2+ concentration of 2 microM (pCa6) than at pCa7. The nature of the respiratory substrate did not affect this finding. 2. This Ca2+-dependence was shown in state-4 and 50%-state-3 conditions [see Chance & Williams (1956) Adv. Enzymol. 17, 65-134], but was absent in the presence of excess ADP (state 3). 3. Na+ and Mg2+ ions shifted the pCa value required for a maximal PDHA content to lower values. This was attributed to a stimulation of mitochondrial Ca2+ egress and an inhibition of uptake, respectively. Na+ ions diminished pyruvate dehydrogenase phosphate phosphatase activity in mitochondria which had been extensively depleted of Ca2+ ions by incubation with EGTA, raising the possibility of a direct inhibitory effect of Na+ ions, unrelated to Ca2+ movements. 4. Mg2+ ions lowered the mitochondrial PDHA content at pCa 6.24 and 6.48, but had only minimal effects in the presence of EGTA. 5. The effects of P1 and bicarbonate ions on PDHA content were also studied, as possible effectors of mitochondrial Ca2+ transport. Bicarbonate ions abolished the response to Ca2+ ions, by generating maximal values of PDHA content, but such a response was still observed when physiological concentrations of both P1 and bicarbonate were used. 6. The pCa of the medium in the range 6.33 to over 7 affected PDHA content, with only very minor changes in state-4 rates of O2 uptake and no change in [ATP]/[ADP] ratio or in mitochondrial [NADH]/[NAD+] ratio, provided that Mg2+ ions were present. Thus the effect of Ca2+ ions on PDHA content is unlikely to be mediated by changes in [ATP]/[ADP] and [NADH]/[NAD+] ratio and is more likely to be direct. Equally, changes in the [acetyl-CoA]/[CoA] ratio in response to Ca2+ ions when the substrate was pyruvate were the converse of those required to mediate changes in interconversion, and are probably secondary to changes in PDHA content.

scholarly journals Regulation of pyruvate dehydrogenase by insulin and polyamines within electropermeabilized fat-cells and isolated mitochondria

1992 ◽  
Vol 285 (2) ◽  
pp. 435-439 ◽  
Author(s):  
G A Rutter ◽  
T A Diggle ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Fat Cells ◽  
Permeabilized Cells ◽  
Isolated Mitochondria ◽  
Mitochondrial Fractions ◽  
Rat Heart Mitochondria ◽  
Rapid Stimulation ◽  
Stimulation Of

1. Regulation of the mammalian pyruvate dehydrogenase (PDH) complex by insulin and polyamines has been examined by using electropermeabilized rat epididymal fat-cells and isolated mitochondria. The complex could be regulated within the permeabilized cells not only by insulin, but also by certain low-M(r) species, including Ca2+ and the polyamine spermidine. 2. Both spermine and spermidine increased the level of active dephosphorylated PDH (PDHa) in isolated adipose-tissue mitochondria 2-3-fold, with half-maximal effects at 0.9 mM and 1.7 mM respectively. By contrast, PDH activity in rat heart mitochondria was essentially insensitive to the effects of these polyamines. 3. The effects on PDH activity of incubation of adipose-tissue mitochondria with spermine persisted through re-isolation and re-incubation of the mitochondria in the absence of the polyamine. 4. No evidence was found of any increase in the concentration of spermine associated with purified mitochondrial fractions prepared from insulin-treated tissue. 5. Overall, the data provide further evidence against a role for polyamines in the rapid stimulation of PDH by insulin, but suggest that polyamines may be important in mediating longer-term changes in the activity of the complex.

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.

STUDIES ON THE INHIBITION OF THE MITOCHONDRIAL ATP-ase BY REDUCTION OF THE RESPIRATORY CHAIN

1960 ◽  
Vol 38 (1) ◽  
pp. 1195-1214 ◽  
Author(s):  
W. Chefurka
Keyword(s):  
Electron Transport ◽  
Oxidative Phosphorylation ◽  
Respiratory Chain ◽  
Transport System ◽  
Heart Muscle ◽  
Rat Heart ◽  
Complete Inhibition ◽  
Muscle Preparation ◽  
Rat Heart Mitochondria ◽  
Stimulation Of

The effects of inhibition of the electron transport system on the stimulation of the ATP-ase reaction in liver and rat-heart mitochondria by dinitrophenol were studied. Cyanide at concentrations 10−3M and higher effectively reduced the stimulation of ATP-ase by dinitrophenol. A similar but less striking inhibition was observed for rat-heart sarcosomes. This ATP-ase reaction was also inhibited in the presence of 10−4M cyanide with glutamate, β-hydroxybutyrate, DPNH, and succinate as reductants of the respiratory chain. Anaerobiosis also caused a substantial decrease in the ATP-ase reaction. In all instances, complete inhibition of the ATP-ase reaction could not be achieved when the respiratory chain was reduced. The magnesium-stimulated ATP-ase of rat-heart sarcosomes, of aged mitochondria, and of the Keilin–Hartree heart-muscle preparation was insensitive to reduction of the carriers suggesting that this reaction may constitute only part of the total ATP-ase reaction. The compatability of the various mechanisms of oxidative phosphorylation with these results is discussed.

The Inhibitory Effect of Tannins on Lipid Peroxidation of Rat Heart Mitochondria

1995 ◽  
Vol 47 (2) ◽  
pp. 138-142 ◽  
Author(s):  
CHUANG-YE HONG ◽  
CHEIN-PING WANG ◽  
SHIANG-SUO HUANG ◽  
FENG-LIN HSU
Keyword(s):  
Lipid Peroxidation ◽  
Rat Heart ◽  
Inhibitory Effect ◽  
Heart Mitochondria ◽  
Rat Heart Mitochondria

scholarly journals Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria

1996 ◽  
Vol 320 (1) ◽  
pp. 329-334 ◽  
Author(s):  
Vida MILDAZIENE ◽  
Rasa BANIENE ◽  
Zita NAUCIENE ◽  
Ausra MARCINKEVICIUTE ◽  
Ramune MORKUNIENE ◽  
...  
Keyword(s):  
Rat Heart ◽  
Mitochondrial Membrane ◽  
Isolated Rat Heart ◽  
Heart Mitochondria ◽  
Proton Leak ◽  
Succinate Oxidation ◽  
Kinetic Dependence ◽  
Intermediate States ◽  
Rat Heart Mitochondria ◽  
Stimulation Of

Stimulation of mitochondrial respiration by physiological concentrations of Ca2+ was studied to determine which components of oxidative phosphorylation are affected by Ca2+. The kinetic dependence of the respiratory chain, phosphorylation subsystem and proton leak on the mitochondrial membrane potential in isolated rat heart mitochondria respiring on 2-oxoglutarate or succinate was measured at two different concentrations of external free Ca2+. The results show that proton leak is not directly affected by Ca2+, but that both the respiratory and phosphorylation systems can be directly stimulated by Ca2+ depending on conditions. Although Ca2+ directly stimulates the phosphorylation system, this has relatively little effect on respiration rate with 2-oxoglutarate in States 3 and 4 because the subsystem has little control over respiration. However, in intermediate states, the phosphorylation system has greater control and Ca2+ stimulation of this system contributes substantially to the stimulation of respiration and phosphorylation. In the case of succinate oxidation neither the respiratory subsystem nor the phosphorylation system is stimulated by Ca2+.

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.

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