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.
Mangroo
D
D
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Steele
L
L
Rachubinski
R A
RA
Gerber
G E
GE
D000214:Acyl Coenzyme A / Q000378:metabolism; D042962:Acyl-CoA Oxidase; D000345:Affinity Labels; D001665:Binding Sites; D002175:Candida; D002352:Carrier Proteins / Q000032:analysis; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D008830:Microbodies / Q000737:chemistry*; D009363:Neoplasm Proteins; D010088:Oxidoreductases / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D011506:Proteins; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase