Restoration of Oxidative Phosphorylation by Purified N,N′-dicyclohexylcarbodiimide-Sensitive Latent Adenosinetriphosphatase from Mycobacterium phlei

The N,N′-dicyclohexylcarbodiimide (DCCD)-sensitive latent adenosinetriphosphatase (ATPase) (EC 3.6.1.3; ATP phosphohydrolase) from Mycobacterium phlei has been purified to homogeneity and used to restore oxidative phosphorylation to detergent-extracted membranes. The phosphorylation was inhibited by DCCD and by tetraphenylboron and valinomycin. The enzyme was solubilized from the membrane vesicles by treatment with cholate followed by extraction with Triton X-100. After partial purification on a sucrose gradient, the enzyme was purified to homogeneity by affinity chromatography on Sepharose coupled to ADP. The DCCD-sensitive latent ATPase of coupling factor from M. phlei consists of two components, the latent ATPase (BCF4), which is insensitive to DCCD, and an intrinsic membrane component, BCF0. This hydrophobic portion of the DCCD-sensitive ATPase was partially purified on a sucrose gradient after solubilization with detergents from membrane vesicles that had been first depleted of the BCF4by washing with 0.25 M sucrose. When BCF0was combined with purified BCF4, the latent ATPase of the resulting complex was sensitive to DCCD. Moreover, like the purified DCCD-sensitive latent ATPase, the combined BCF4and BCF0restored coupled phosphorylation to detergent-extracted membranes.