STRUCTURE OF BEEF-HEART MITOCHONDRIAL F1-ATPASE - ARRANGEMENT OF SUBUNITS AS DISCLOSED BY CROSS-LINKING REAGENTS AND SELECTIVE LABELING BY RADIOACTIVE LIGANDS

The bifunctional reagents, dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate were used to produce dimers between the neighboring subunits of beef heart F1-ATPase. Treatment of beef heart F1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of 4 cross-linked products. Their MW determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 115,000, 105,000, 95,000 and 80,000, respectively. The products of MW 115,000 and 105,000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of MW 115,000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by .beta.-mercaptoethanol, the original gel electrophoresis pattern was restored. Cross-linking of beef heart F1-ATPase by dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate was accompanied by a loss of the ATPase activity. Cleavage of the cross-linked products obtained with methylmercaptobutyrimidate did not restore the original ATPase activity. Identification of subunits A and B in the products of MW 115,000 and 105,000 was achieved by specific labeling of subunit A with N-[14C]ethylmaleimide and of subunit B by chloronitro[14C]benzooxodiazole. Both products were able to bind N-[14C]ethylmaleimide; only the 105,000 dalton product was able to bind chloronitro[14C]benzooxodiazole. The product of MW 115,000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[14C]ethylmaleimide. Its cleavage, following N-[14C]ethylmaleimide binding, yielded 1 labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. The above results indicate that the product of MW 115,000 is a dimer containing 2 subunits A and that the product of MW 105,000 is a dimer containing 1 subunit A and 1 subunit B. Thus, in beef heart F1-ATPase, the A subunits are close to each other and subunit A is close to subunit B. In contrast the B subunits are probably too far from each other to be cross-linked by dimethylsuberimidate, dimethyladipimidate or methylmercaptobutyrimidate.