Effect of Trypsin on Mitochondrial and Microsomal Enzymes

Abstract

The rotenone‐insensitive NADH‐cytochrome c reductase system of rat liver mitochondria and isolated mitochondrial outer membrane preparations is inactivated by trypsin. The trypsin sensitivity of the system is considerably higher than that of liver microsomes when compared on equal protein basis. The trypsin sensitivity of the mitochondrial system decreases with increasing ionic strength of the incubating medium, whereas that of the microsomal system increases with increasing ionic strength, except at low protein concentrations (< 0.1 mg protein/ml), in which case the effect of ionic strength is similar to that found with mitochondria. Experiments with 2,6‐dichlorophenolindophenol and ferricyanide as electron acceptors indicate that trypsin does not inactivate the flavoprotein component of the mitochondrial system. The inactivation of the NADH‐cytochrome c reductase by trypsin is accompanied by a release of cytochrome b5, similar to that found with microsomes. Trypsin does not inactivate the adenylate kinase of intact mitochondria, but readily inactivates this enzyme after brief exposure of the mitochondria to a hypotonic medium containing EDTA. Respiration, respiratory control, and various translocator functions of the mitochondria which are not affected by this treatment, remain insensitive to trypsin. The results are discussed with regard to their bearing on the distinction between the mitochondrial (rotenone‐insensitive) and microsomal NADH‐cytochrome c reductase systems, as well as on the location of the mitochondrial NADH‐cytochrome c reductase system and adenylate kinase in relation to the outer mitochondrial membrane. Copyright © 1970, Wiley Blackwell. All rights reserved