Localization of enzymes by means of proteases

A method is described which makes it possible to determine whether a given enzyme is located on the outer surface of a mitochondrial membrane, or wether it is localized within a mithondrial compartment. The method combines the use of proteases with digitonin. Depending on the concentration of digitonin, enzymes behind the microsomal membranes and the outer mitochondrial membrane may be successively exposed to the action of proteases. Thus, enzymes located within microsomes contaminating the mitochondrial fraction may be easily distinguished from true intramitochondrial enzymes.Applying this technique to the mitochondrial fraction of rat liver, it is shown that lactate dehydrogenase (l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27) is located on the outer surface of the mitochondria and within microsomal contaminants.