Rat liver mitochondria essentially free of endogenous Ca 2+ show low initial rate of energy‐dependent Ca 2+ uptake. Preincubation of mitochondria under de‐energized conditions in the presence of small amounts of external Ca 2+ results in a 8–10‐fold time‐dependent increase of energy‐dependent Ca 2+ uptake. Ca 2+ ‐dependent activation of the Ca 2+ ‐transporting system follows first‐order kinetics ( in the presence of 5 μM Ca 2+ at 20°C). Ca 2+ ‐activated mitochondria demonstrate a simple hyperbolic initial rate‐Ca 2+ concentration dependence, whereas strong apparent cooperativity is observed in the velocity‐substrate curves for Ca 2+ ‐depleted mitochondria. It is concluded that apparent cooperativity of the energy‐dependent Ca 2+ uptake is due to slow (as compared with the ‘turnover number’) activation of a Ca 2+ ‐specific uniporter which is inactive in the absence of external Ca 2+ .
Cytochrome bd is a terminal quinol oxidase in Escherichia coli . Mitochondrial respiration is inhibited at cytochrome bc 1 (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol‐inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone‐sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.
1. The initial rapid phase of ATP hydrolysis by bovine heart submitochondrial particles or by soluble F1-ATPase is insensitive to anion activation (sulphite) or inhibition (azide). 2. The second slow phase of ATP hydrolysis is hyperbolically inhibited by azide (Ki approximately 10(-5) M); the inosine triphosphatase activity of submitochondrial particles or F1-ATPase is insensitive to azide or sulphite. 3. The rate of interconversion between rapid azide-insensitive and slow azide-sensitive phases of ATP hydrolysis does not depend on azide concentration, but strongly depends on ATP concentration. 4. Sulphite prevents the interconversion of the rapid initial phase of the reaction into the slower second phase, and also prevents and slowly reverses the inhibition by azide. 5. The presence of sulphite in the mixture when ADP reacts with ATPase of submitochondrial particles changes the pattern of the following activation process. 6. Azide blocks the activation of ATP-inhibited ATPase of submitochondrial particles by phosphoenolpyruvate and pyruvate kinase. 7. The results obtained suggest that the inhibiting effect of azide on mitochondrial ATPase is due to stabilization of inactive E*.ADP complex formed during ATP hydrolysis; the activation of ATPase by sulphite is also realized through the equilibrium between intermediate active E.ADP complex and inactive E*.ADP complex.
The steady-state kinetics of the NADH dehydrogenase activity of the three-subunit flavo-iron-sulfur protein (FP, Type II NADH dehydrogenase) in the presence of the one-electron acceptor hexammineruthenium(III) (HAR) were studied. The maximal catalytic activities of FP with HAR as electron acceptor calculated on the basis of FMN content were found to be approximately the same for the submitochondrial particles. Complex I and purified FP. This result shows that the protein structure responsible for the primary NADH oxidation by FP is not altered during the isolation procedure and the lower (compared with Complex I) catalytic capacity of the enzyme previously reported was due to the use of inefficient electron acceptors. Simple assay procedures for NADH dehydrogenase activity with HAR as the electron acceptor are described. The maximal activity at saturating concentrations of HAR was insensitive to added guanidine, whereas at fixed concentration of the electron acceptor, guanidine stimulated oxidation of low concentrations of NADH and inhibited the reaction at saturating NADH. The inhibitory effect of guanidine was competitive with HAR. The double-reciprocal plots 1 /v vs. 1 /[NADH] at various HAR concentrations gave a series of straight lines intercepting on the ordinate. The plots 1 /v vs. 1 /[HAR] at various NADH concentrations gave a series of straight lines intercepting in the fourth quadrant. The kinetics support the mechanism of the overall reaction where NADH is oxidized by the protein-Ru(NH3)63+ complex in which positively charged electron acceptor is bound at the specific site close to FMN, thus stabilizing the flavosemiquinone intermediate.
NADH and succinate oxidase activities of inside‐out submitochondrial particles treated with excess oligomycin are inhibited by lanthanides (La 3+ and Dy 3+ ). Inhibition by both oligomycin and oligomycin plus lanthanides is completely relieved by an uncoupler. The respiratory control, measured as the stimulation of NADH or succinate oxidation caused by the addition of uncoupler to the oligomycin‐treated particles, is thus increased in the presence of lanthanides. The coupling effect of lanthanides is completely prevented and rapidly reversed by excess of EDTA. La 3+ increases the extent of the aerobic energy‐linked succinate‐supported NAD + reduction catalyzed by the oligomycin‐treated submitochondrial particles. Lanthanides seem to be a useful tool to increase the energy coupling capacity of the submitochondrial particles.
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