[64] ATP formation in mitochondria, submitochondrial particles, and F0F1 liposomes driven by electric pulses
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Submitochondrial particle
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The NADH-cytochrome c reductase activity of bovine heart submitochondrial particles was found to be slowly (half-time of 16 min) and progressively lost upon incubation with the Fe2(+)-adriamycin complex. In addition to this slow progressive inactivation seen on incubation, a reversible fast phase of inhibition was also seen. However, if EDTA was added to the incubation mixture within 15 s, the slow progressive loss in activity was largely preventable. Separate experiments indicated that EDTA removed about one-half of the iron from the Fe2(+)-adriamycin complex in about 40 s. These results indicated the requirement for iron for the inactivation process. Since the Vmax. for the fast phase of inhibition was decreased by the inhibitor, the inhibition pattern was similar to that seen for uncompetitive or mixed-type inhibition. The direct binding of both Fe3(+)-adriamycin and adriamycin to submitochondrial particles was also demonstrated, with the Fe3(+)-adriamycin complex binding 8 times more strongly than adriamycin. Thus binding of Fe3(+)-adriamycin to the enzyme or to the inner mitochondrial membrane with subsequent generation of oxy radicals in situ is a possible mechanism for the Fe3(+)-adriamycin-induced inactivation of respiratory enzyme activity.
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Using conventional polarographic and spectrophotometric methods, the effect of sodium oleate on isolated mitochondria dn submitochondrial particles was analysed. A loose coupling was developed in isolated mitochondria in the presence of about 10 nmoles oleate per mg protein. Oleate stimulates and activity of bovine heart mitochondrial Mg-ATPase, and it was found to be dependent on substrate concentration. Investigation of the membrane potential showed that the delta psi is abolished at high concentration of oleate. The deterioration of the morphological appearance of mitochondria, occurring on addition of oleate, is also described.
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Polarography
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Declines in the rate of mitochondrial electron transport and subsequent increases in the half-life of reduced components of the electron transport chain can stimulate O2•- formation. We have previously shown that, in solubilized cardiac mitochondria, Ca2+ mediates reversible free radical-induced inhibition of complex I. In the study presented here, submitochondrial particles prepared from rat heart were utilized to determine the effects of Ca2+ on specific components of the respiratory chain and on the rates of electron transport and O2•- production. The results indicate that complex I is inactivated when submitochondrial particles are treated with Ca2+. Inactivation was specific to complex I with no alterations in the activities of other electron transport chain complexes. Complex I inactivation by Ca2+ resulted in the reduction of NADH-supported electron transport activity. In contrast to the majority of electron transport chain inhibitors, Ca2+ suppressed the rate of O2•- production. In addition, while inhibition of complex III stimulated O2•- production, Ca2+ reduced the relative rate of O2•- production, consistent with the magnitude of complex I inhibition. Evidence indicates that complex I is the primary source of O2•- released from this preparation of submitochondrial particles. Ca2+ therefore inhibits electron transport upstream of site(s) of free radical production. This may represent a means of limiting O2•- production by a compromised electron transport chain.
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Inhibitor protein
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Submitochondrial particle
Nigericin
Inhibitor protein
Valinomycin
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1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.
Submitochondrial particle
Inhibitor protein
Adenosine triphosphate
Adenosine triphosphatase
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