scholarly journals Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios

1988 ◽  
Vol 252 (1) ◽  
pp. 181-189 ◽  
Author(s):  
G A Rutter ◽  
R M Denton
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Rat Heart ◽  
Adenine Nucleotides ◽  
Heart Mitochondria ◽  
Maximal Effect ◽  
Dehydrogenase System ◽  
Rat Heart Mitochondria ◽  
Oxoglutarate Dehydrogenase ◽  
Intact Heart

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.

scholarly journals Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart

1994 ◽  
Vol 303 (2) ◽  
pp. 461-465 ◽  
Author(s):  
B J Nichols ◽  
M Rigoulet ◽  
R M Denton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Isocitrate Dehydrogenase ◽  
Rat Heart ◽  
Adenine Nucleotides ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rat Heart Mitochondria ◽  
Oxoglutarate Dehydrogenase ◽  
Regulatory Properties

The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions. Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+. In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP. Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+. All the enzymes studied were activated by decreases in pH, but to differing extents. The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate. These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available.

scholarly journals The regulation of the oxidation of fatty acids and other substrates in rat heart mitochondria by changes in the matrix volume induced by osmotic strength, valinomycin and Ca2+

1987 ◽  
Vol 244 (1) ◽  
pp. 159-164 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Rat Heart ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Acid Oxidation ◽  
Maximal Effect ◽  
Physiological Conditions ◽  
Matrix Volume ◽  
The Matrix ◽  
Rat Heart Mitochondria

1. The rate of ADP-stimulated respiration with various substrates and the matrix volume of rat heart mitochondria were measured over a range of osmolarities of the medium. 2. The rate of oxidation of palmitoylcarnitine (in the presence of malate) was stimulated 7-fold by increasing the matrix volume from 0.6 to 1.0 microliter/mg of protein. Oxidation of octanoate showed a similar sensitivity to the matrix volume, whereas oxidation of other substrates showed little sensitivity until the volume fell below 0.7 microliter/mg of protein. 3. The matrix volume of heart mitochondria incubated under physiological conditions was about 0.8 microliter/mg of protein. 4. Low concentrations of valinomycin added to mitochondria incubated under such physiological conditions could activate the rate of ADP-stimulated palmitoylcarnitine oxidation by at least 100%. 5. Decreasing the matrix volume increased the reduction of the electron-transferring flavoprotein (ETF), suggesting an effect on electron flow between ETF and ubiquinone, as has been observed for liver mitochondria [Halestrap & Dunlop (1986) Biochem. J. 239, 559-565]. 6. A rapid decrease in light-scattering by heart mitochondria incubated in State 4 was induced by addition of Ca2+, reaching 50% of the maximal effect after about 30 s at 30 degrees C and with K0.5 for Ca2+ of 0.3 microM. This was not associated with a change in matrix volume, and is discussed in terms of a conformational change whose identity remains to be determined. 7. However, incubation of heart mitochondria at 37 degrees C in the presence of 0.65 microM-Ca2+ for 4 min did increase the matrix volume significantly, by 0.181 +/- 0.029 microliter/mg of protein (n = 7, P less than 0.001), similar to the Ca2+-induced changes observed with liver mitochondria [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. 8. The possible significance of these results in the co-ordinate regulation of fatty acid oxidation and the citric acid cycle in the heart responding to increased work load or hormonal stimulation is discussed.

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.

Substrate specific effects of calcium on metabolism of rat heart mitochondria

1996 ◽  
Vol 270 (4) ◽  
pp. H1398-H1406 ◽  
Author(s):  
A. V. Panov ◽  
R. C. Scaduto
Keyword(s):  
Rat Heart ◽  
Redox State ◽  
Adenine Nucleotides ◽  
Enzyme Reaction ◽  
Isolated Rat Heart ◽  
Heart Mitochondria ◽  
Specific Substrate ◽  
Metabolic States ◽  
Tightly Coupled ◽  
Rat Heart Mitochondria

Oxidative metabolism in the heart is tightly coupled to mechanical work. Because this coupling process is believed to involve Ca2+, the roles of mitochondrial Ca2+ in the regulation of oxidative phosphorylation was studied in isolated rat heart mitochondria. The electrical component of the mitochondrial membrane potential (delta psi) and the redox state of the pyridine nucleotides were determined during the oxidation of various substrates under different metabolic states. In the absence of added adenine nucleotides, the NADP+ redox couple was almost completely reduced, regardless of the specific substrate and the presence of Ca2+, whereas NAD+ couple redox state was highly dependent on the substrate type and the presence of Ca2+. Titration of respiration with ADP, in the presence of excess hexokinase and glucose, showed that both respiration and NAD(P)+ reduction were very sensitive to ADP. The maximal enzyme reaction rate of ADP-stimulated respiration Michaelis constants (Km) for ADP were dependent on the particular substrate employed. delta psi was much less sensitive to ADP. With either alpha-ketoglutarate or glutamate as substrate, Ca2+ significantly increased reduction of NAD(P)+.Ca2+ did not influence NAD(P)+ reduction with either acetylcarnitine or pyruvate as substrate. In the presence of ADP, delta psi was increased by Ca2+ at all metabolic states with glutamate plus malate, 0.5 mM alpha-ketoglutarate plus malate, or pyruvate plus malate as substrates. The data presented support the hypothesis that cardiac respiration is controlled by the availability of both Ca2+ and ADP to mitochondria. The data indicate that an increase in substrate supply to mitochondria can increase mitochondrial respiration at given level of ADP. This effect can be produced by Ca2+ with substrates such as glutamate, which utilize alpha-ketoglutarate dehydrogenase activity for oxidation. Increases in respiration by Ca2+ may mitigate an increase in ADP during periods of increased cardiac work.

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