Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation: XVII. Further Resolution of the Rutamycin-Sensitive Adenosine Triphosphatase

Abstract

1. A mitochondrial fraction from beef heart (TU-particles) was shown to confer rutamycin sensitivity to added mitochondrial ATPase (F1). After sonic oscillation at an alkaline pH, the resulting particles (TUA-particles) no longer had this activity. A rutamycin sensitivity factor (Fc), purified from crude preparations of coupling factor 4, restored the capacity of the particles to confer rutamycin sensitivity to F1. Highly purified preparations of coupling factor 5 contained Fc, and the relationship between Fc and F5 is discussed. 2. The binding of F1 to TUA-particles was shown to take place with Mg++ (0.8 m M ) alone, but the bound ATPase was insensitive to rutamycin. Other divalent cations were less effective at these low concentrations; higher concentrations of monovalent cations (20 to 50 m M ) substituted for Mg++. The particulate ATPase activity was rendered rutamycin-sensitive by addition of Fc. 3. Exposure of either TUA-particles or Fc to heat or to trypsin resulted in complete loss of rutamycin sensitivity of the reconstituted system. This finding indicates that two labile factors contribute to the phenomenon. In contrast to the lability of the individual components, the Fc-TUA-F1 particle complex was resistant to heat and trypsin treatment under the same conditions. This observation represents another example of allotopy. 4. Dicyclohexylcarbodiimide was shown to inhibit particle-bound ATPase in a manner similar to rutamycin, except that no reversibility could be demonstrated. The site of action of dicyclocarbodiimide was shown to be associated with the particles and not with Fc.