Structure of beef heart mitochondrial F 1-ATPase Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands

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1. 1. The following bifunctional reagents, dimethylsuberimidate, dimethyladipimidate, methylmercaptobutyrimidate have been used to produce dimers between the neighboring subunits of beef heart F 1-ATPase. 2. 2. Treatment of beef heart F 1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of four cross-linked products. Their molecular weights determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis were 115 000, 105 000, 95 000 and 80 000, respectively. The products of molecular weight 115 000 and 105 000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F 1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of molecular weight 115 000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by β-mercaptoethanol, the original gel electrophoresis pattern was restored. 3. 3. Cross-linking of beef heart F 1-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. 4. 4. Identification of subunits A and B in the products of molecular weight 115 000 and 105 000 was achieved by specific labeling of subunit A with N-[ 14 C] ethylmaleimide and of subunit B by chloronitro[ 14C]benzooxodiazole. Bothe products were able to bind N-[ 14 C] ethylmaleimide ; only the 105 000 dalton product was able to bind chloronitro[ 14C]benzooxodiazole. 5. 5. The product of molecular weight 115 000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[ 14 C] ethylmaleimide . Its cleavage, following N-[ 14 C] ethylmaleimide binding, yielded one labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. 6. 6. The above results indicate that the product of molecular weight 115 000 is