Factors affecting the activity of mitochondrial and soluble aconitase.

Considerable information on the intracellular localization of enzymes has been obtained in recent years through the technique of differential centrifugation of sucrose homogenates of tissues and subsequent testing of the fractions for enzyme activity. One of the results of this work has been the demonstration that rat liver mitochondria (Mw) are capable of completely oxidizing a wide variety of substrates by way of the reactions of the tricarboxylic acid cycle (1). Consequently, mitochondria should contain an active aconitase. This supposition has beei! verified. The aconitase associated with mitochondria is released into solution after freezing and thawing. It therefore became possible to compare the properties of the particulate-bound enzyme with its released (soluble) c0unterpart.l A number of interesting points have been brought out by this comparison. Some factor, presumably the mitochondrial membrane, is responsible for a difference between the pH optimum of the particulate aconitase and that of the soluble enzyme. It has also been observed that appreciable amounts of aconitate (AA) are retained within the particle in the conversion of isocitrate (ICA) to citrate (CA). These differences in the activity of mitochondrial aconitase, which probably are not. directly related to the properties of the protein itself, point up the necessity of considering permeability factors in the measurement of particle-bound enzymes. Thus the data on the intracellular distribution of aconitase have been found to be markedly affected by the pH of the assay medium. The results invalidate the assumption that the physical environment of an enzyme has no effect on its relative activity under varying assay conditions.