Comparative study of respiration kinetics and protein composition of skinned fibers from various types of rat muscle.

The respiration parameters of mitochondria from rat heart muscle and from fast-twitch and slow-twitch skeletal muscle skinned fibers were comparatively analyzed. Electrophoretic patterns of fiber protein composition were also compared. It was found that fibers with low affinity of mitochondria for ADP (i.e., heart and slow-twitch skeletal muscle soleus) contain a 27.5-kD protein that is absent from the fibers that exert high affinity for ADP (i.e., fast-twitch skeletal muscle gastrocnemius). Partial proteolysis, which increases the affinity of mitochondria of the heart and slow-twitch skeletal muscles for ADP, results in the disappearance of this protein. The results suggest that this protein may be an intracellular factor that controls the permeability of the outer mitochondrial membrane for ADP.

Phosphocreatine pathway for energy transport: ADP diffusion and cardiomyopathy.

Chemically skinned (by treatment with saponin, 40 micrograms/ml) isolated cardiomyocytes were used to study the intracellular diffusion of ADP and creatine (Cr). Stimulation of respiration was studied in these cardiomyocytes without intact sarcolemma and in isolated heart mitochondrial by addition of ADP and Cr in the presence of 0.2 mM ATP (via mitochondrial creatine kinase reaction: Cr + MgATP = MgADP + PCr). The Michaelis constant (Km) for Cr was similar in both cases, 5.67 +/- 0.11 (SD) mM in skinned myocytes and 6.9 +/- 0.2 mM in mitochondria, showing that there is no significant restriction to the diffusion of this substrate. However, the apparent Km for external ADP increased from 17.6 +/- 1.0 microM for mitochondria to 250 +/- 38 microM for skinned cardiomyocytes, showing decreased diffusivity of ADP as a result of binding to cellular structures. In the presence of 25 mM Cr, the Km for ADP for myocytes decreased to 35.6 +/- 5.6 microM due to the coupling of the creatine kinase and oxidative phosphorylation reactions. Provision of substrate for the creatine kinase reaction amplified the weak ADP signal in the regulation of respiration. The activity of the mitochondrial creatine kinase was decreased by a factor of two in cardiomyopathic hamsters and human hearts and was associated with a twofold decrease in creatine-stimulated respiration. These data show a potentially key role of mitochondrial creatine kinase in the regulation of cellular respiration and the possible importance of changes in its activity for the functional disturbances of the cardiomyopathic heart.

In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP.

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.

Regulation of succinate dehydrogenase and tautomerization of oxaloacetate.

Highly purified succinate-ubiquinone reductase catalyzes the oxidation of L- or D-malate with a Km and initial Vmax equal to approximately 10(-3) M and approximately 100 nmol/min/mg of protein, respectively. The malate dehydrogenase activity of succinate dehydrogenase rapidly decreases regardless of the presence of glutamate plus glutamate-oxaloacetate transaminase. The inhibitor trapping system, however, prevents the inactivation of succinate dehydrogenase under the conditions when the rate of tautomeric oxaloacetate enol in equilibrium oxaloacetate ketone interconversion is high. These results suggest that enol oxaloacetate is an immediate product of malate oxidation at the succinate dehydrogenase active site. Two proteins (Mr 37 and 80 kD) which catalyze the oxaloacetate tautomerase reaction were isolated from the mitochondrial matrix. Some physico-chemical and kinetic properties of these enzymes were characterized. The larger protein was identified as inactive aconitase. The system containing succinate dehydrogenase, L-malate, glutamate plus transaminase and oxaloacetate tautomerase was reconstituted. Such a system is capable of oxidizing malate to aspartate without rapid inactivation of succinate dehydrogenase. Taken together, the data obtained emphasize a significant role of enzymatic oxaloacetate tautomerization in the control of the succinate dehydrogenase activity in the mitochondrial matrix.

Isolation and properties of oxaloacetate keto-enol-tautomerases from bovine heart mitochondria.

Two highly purified proteins with quite different properties capable of oxaloacetate keto-enol-tautomerase activity (oxaloacetate keto-enol-isomerase, EC 5.3.2.2) were isolated from the bovine heart mitochondrial matrix. The first protein has an apparent molecular mass of 37 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-200 gel filtration. It is quite stable upon storage at 40 degrees C and reaches the maximal catalytic activity at pH 8.5 with a half-maximal activity at pH 7.0. The enzyme is specifically inhibited by oxalate and diethyloxaloacetate. When assayed in the enol----ketone direction at 25 degrees C (pH 9.0), the enzyme obeys a simple substrate saturation kinetics with Km and Vmax values of 45 microM and 74 units per mg of protein, respectively; the latter value corresponds to the turnover number of 2700 min-1. The second protein has an apparent molecular mass of 80 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-300 gel filtration. The enzyme is rapidly inactivated at 40 degrees C and shows a sharp pH optimum of activity at pH 9.0. The enzyme can be completely protected from thermal inactivation by oxaloacetate and dithiothreitol. The kinetic parameters of the enzyme as assayed in the enol----ketone direction at 25 degrees C (pH 9.0) are: Km = 220 microM and Vmax = 20 units per mg of protein; the latter corresponds to the turnover number of 1600 min-1. The enzyme activity is specifically inhibited by maleate and pyrophosphate. About 30% of the total oxaloacetate tautomerase activity in crude mitochondrial matrix is represented by the 37 kDa enzyme and about 70% by the 80 kDa protein.

Oxidation of malate by the mitochondrial succinate-ubiquinone reductase.

The purified succinate-ubiquinone reductase catalyzes the L- (or D-) malate: acceptor oxidoreductase reaction with Km for malate of about 2.10(-3) M and initial Vmax of 50 and 100 nmol per min per mg of protein for L- and D-stereoisomers, respectively (25 degrees C, pH 7.0). The reaction rate rapidly decreases both in the absence and presence of L-glutamate and L-glutamate-oxaloacetate transaminase added for trapping of oxaloacetate. Both keto and enol forms of oxaloacetate were found to be strong, slowly dissociating inhibitors of succinate dehydrogenase; the first-order rate constant for the enzyme inhibition by the enol form is about 3 times as high as that by the keto form. Oxidation of malate by succinate dehydrogenase in the presence of the oxaloacetate trapping system occurs at an indefinitely constant rate when enoloxaloacetate, which is an immediate product of the reaction, is rapidly converted into the keto isomer--a substrate for transaminase. A quantitative kinetic scheme for malate oxidation by succinate dehydrogenase which includes two kinetically distinct enzyme-oxaloacetate complexes is proposed, and the specific role of the mitochondrial oxaloacetate keto-enol-tautomerase (EC 5.3.2.2) in the regulation of succinate dehydrogenase is suggested.