The compartmentation of CoA and fatty acid activating enzymes in rat liver mitochondria.

Abstract

The effect of disulphides on acyl‐carnitine formation and fatty acid oxidation in rat liver mitochondria has been studied. Tetrathionate, which does not penetrate the inner membrane, inhibited the carnitine dependent oxidation of long chain fatty acid oxidation almost completely. This oxidation was reactivated by CoA but not by other thiols. Carnitine increased the inhibition by tetrathionate, indicating that a small pool of preformed acyl‐CoA is present at the outer membrane. By means of a new, sensitive method for the determination of CoA, a leakage of CoA from the mitochondria during the incubations was demonstrated. The leakage was increased by Pi and 2,4‐dinitrophenol. The observations in paragraphs 3 and 4 explain why carnitine stimulates the oxidation of fatty acids in the absence of added CoA. It was confirmed that an ATP‐dependent fatty acid‐CoA synthetase with great capacity is localized in the outer mitochondrial compartment. A small residual capacity for ATP‐dependent long chain fatty acid activation was demonstrated even in the presence of tetrathionate, indicating an “internal” localization. This activity may depend on the medium chain synthetase or on a separate enzyme in the matrix compartment. The activation of short chain fatty acids was not inhibited by tetrathionate. Atractyloside inhibited both long chain acyl‐CoA synthetase and carnitine palmityltransferase. The inhibition of both enzymes was counteracted by CoA. It is concluded that atractyloside is unsuitable for the study of the compartmentation of fatty acid activating enzymes in mitochondria. From the studies with disulphides it is concluded that fatty acid oxidation in mitochondria can be explained by a simple two compartment model where the inner membrane is the permeability barrier. Such a functional model is in agreement with previous enzyme localization studies.