We have investigated the localization of palmitoyl-CoA (hexadecanoyl-CoA) synthetase (EC 6.2.1.3) and cerotoyl-CoA (hexacosanoyl-CoA) synthetase in peroxisomes isolated from rat liver. Palmitoyl-CoA and cerotoyl-CoA synthetases, like acyl-CoA: dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), are present in the peroxisomal membrane. Trypsin treatment of intact peroxisomes led to the disappearance of both palmitoyl-CoA and cerotoyl-CoA synthetase activities but had little, if any, effect on L-alpha-hydroxy-acid oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3) or acyl-CoA:dihydroxyacetone phosphate acyltransferase. The latter three enzymes were inactivated if the trypsin treatment was preceeded by disruption of the peroxisomes by sonication. These results show that the active site, or at least domains essential for the activity of cerotoyl-CoA synthetase, like that of palmitoyl-CoA synthetase, is located on the cytosolic face of the peroxisomal membrane.
