Specific labeling of Candida tropicalis peroxisomal proteins with photoreactive fatty-acid derivatives

The labeling of Candida tropicalis peroxisomal proteins with photoreactive fatty-acid derivatives was investigated. Proteins having molecular masses of 70 kDa, 48 kDa and 15 kDa were labeled with 11-m-diazirinophenoxy-[11-3H]undecanoate while 11-m-diazirinophenoxy-[11-3H]undecanoyl-CoA labeled proteins of 70 kDa and 55 kDa. The 70 kDa protein labeled with both photoreactive probes was resolved into two bands by electrophoresis on a gradient polyacrylamide gel; immunoprecipitation with anti-fatty acyl-CoA oxidase showed that these proteins are fatty-acyl-CoA oxidases. In purified peroxisomal membranes, two proteins of 36 kDa and 25 kDa were labeled with the photoreactive fatty-acid probe, whereas very little labeling of the above proteins or other proteins was observed with the fatty-acyl-CoA probe. The photoaffinity labeling method described is, thus, clearly capable of identifying and distinguishing between proteins having an affinity for fatty acid or fatty-acyl-CoA. The labeling also identified a fatty-acid-binding site on the 16 kDa peroxisomal matrix protein as well as on two peroxisomal acyl-CoA oxidases. This approach thus provides a general means for the identification of fatty-acid metabolizing enzymes, as well as for the identification of fatty-acid-binding sites on known enzymes.