Mangroo D, Steele L, Rachubinski R A, Gerber G E
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Biochim Biophys Acta. 1993 Jul 1;1168(3):280-4. doi: 10.1016/0005-2760(93)90183-a.
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.
对热带假丝酵母过氧化物酶体蛋白用光反应性脂肪酸衍生物进行标记的情况进行了研究。分子量为70 kDa、48 kDa和15 kDa的蛋白质用11 - m - 重氮苯氧基 - [11 - ³H]十一烷酸进行标记，而11 - m - 重氮苯氧基 - [11 - ³H]十一烷酰辅酶A标记的是70 kDa和55 kDa的蛋白质。用两种光反应性探针标记的70 kDa蛋白质在梯度聚丙烯酰胺凝胶上电泳后可分离成两条带；用抗脂肪酰辅酶A氧化酶进行免疫沉淀表明这些蛋白质是脂肪酰辅酶A氧化酶。在纯化的过氧化物酶体膜中，36 kDa和25 kDa的两种蛋白质用光反应性脂肪酸探针进行了标记，而用脂肪酰辅酶A探针观察到上述蛋白质或其他蛋白质的标记很少。因此，所描述的光亲和标记方法显然能够识别和区分对脂肪酸或脂肪酰辅酶A有亲和力的蛋白质。该标记还在16 kDa过氧化物酶体基质蛋白以及两种过氧化物酶体酰基辅酶A氧化酶上鉴定出了一个脂肪酸结合位点。因此，这种方法为鉴定脂肪酸代谢酶以及鉴定已知酶上的脂肪酸结合位点提供了一种通用手段。
