The localization of enzymes in cells may be studied by the differential centrifugation of tissue homogenates. This method has been used to study the distribution of L-malic and L-glutamic dehydrogenases, choline oxidase, adenosinetriphosphatase, and also of the nucleic acids and nitrogen between the fractions of the homogenate. L-Glutamic dehydrogenase is entirely m itochondrial, and malicdehydrogenase is shared almost equally, between the mitochondria and the supernatant although the true mitochondrial activity is not apparent unless unmasked, e. g. by water disruption. In the absence of this precaution intact mitochondria show only a small proportion of the activity of the whole homogenate. Choline oxidase is almost entirely mitochondrial, and adenosine triphosphatase has a large representation in all the particulate fractions. In our experiments we have found that the ribonucleic acid content of the mitochondria is higher than previously reported. Distribution studies of enzymes are misleading unless it can be shown that the methods employed are valid for all the fractions studied, and our evidence shows that serious errors of in terpretation may arise unless more than one method of determination is used. In particular, the physical state of the mitochondria affects their apparent enzyme content, as shown by the; investigation of malic and glutamic dehydrogenases by manometric and optical methods. This anomaly is due to an ‘accessibility barrier’ or ‘permeability barrier’ present in intact mitochondria, which hinders the entry of coenzyme I. In an integrated system composed of several enzymes, the rate of the whole reaction may be limited by the rate of any one of the intermediate steps. Thus intact mitochondria fail to develop their maximum oxygen up take with several substrates unless a continuous supply of phosphate acceptor is ensured. Otherwise the rate of transfer of phosphate limits the whole reaction, reducing the apparent activity of the mitochondrial fraction relative to that of the whole homogenate, and hence the apparent distribution of enzyme. Similarly, the activity of the choline oxidase of the mitochondrial fraction is more sensitive to pH changes than that of the whole homogenate so that at pH 6·8, 80% of the homogenate activity may be recovered in the mitochondria, whereas at pH 7·8, the recovery is only 50%. At pH 7·8 full activity of the mitochondria, and a recovery of over 80%, may be achieved by the addition of coenzyme I.
