Abstract
Publisher Summary This chapter describes the preparation and properties of the enzymes and enzyme complexes of the mitochondrial oxidative phosphorylation system. The enzymes concerned with electron transport from NAD(P)H and succinate to molecular oxygen, energy conservation and transduction, and adenosine triphosphate (ATP) synthesis and hydrolysis are located in the mitochondrial inner membrane. Systematic fractionation of this membrane has shown that these enzymes are contained mainly in five protein–lipid complexes. This results in precipitation of complexes I, II, and III, which are separated by centrifugation from complex V in the supernatant. Then, the binary complex I–III is separated from II–III by precipitation with ammonium acetate, and finally I–III and II–III are resolved and separated into I, II, and III, using cholate and ammonium sulfate. The fractionation procedure involves the use of deoxycholate and cholate in conjunction with KCI, a neutral salt of low ionic strength, for membrane solubilization, and of ammonium acetate and ammonium sulfate for precipitation of the desired fragments.
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