Restoration of oxidative phosphorylation by purified N , N ' - dicyclohexylcarbodiimide - sensitive latent adenosinetriphosphatase from Mycobacterium phlei ( membrane / electron transport particles / affinity chromatography / reconstitution )

The NN-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, BCFo. This hydrophobic portion of the DCCDsensitive ATPase was partially purified on a sucrose gradient after solubilization with detergents from membrane vesicles that had been first depleted of the BCF4 by washing with 0.25 M sucrose. When BCFo was 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 BCF4 and BCFo restored coupled phosphorylation to detergent-extracted membranes. The coupling factor-latent ATPase (BCF4) from Mycobacterium phlei can be solubilized from electron transport particles by washing with buffers of low ionic strength or with 0.25 M sucrose in the absence of cations (1, 2). The enzyme has been purified to homogeneity by affinity chromatography on Sepharose coupled to ADP (3, 4). The ATPase of BCF4 is latent, requiring trypsin treatment to express the hydrolytic activity. The BCF4 is similar to the coupling factor ATPase, F1, which has been purified from mammalian mitochondria (5, 6), yeast mitochondria (7), and other bacteria (8-10). In contrast to the membrane-bound BCF4 from M. phlei (11), the solubilized BCF4 was insensitive to N,N'-dicyclohexylcarbodiimide (DCCD). DCCD inhibits the membrane-bound ATPase and phosphorylation in mammalian mitochondria and submitochondrial preparations (12-14) and in M. phlei (11). With the help of detergents, a DCCDor oligomycin-sensitive ATPase has been isolated from beef heart (15, 16) and yeast (17, 18) mitochondria. It has been purified from yeast (17) and from a thermophilic bacterium (19, 20). In addition to the F1 moiety, the DCCD-sensitive complex contains an intrinsic membrane protein called Fo (19-22). Fo contains several polypeptidvs, some of which have been isolated and purified as the DCCD-binding protein and the oligomycin sensitivity conferring protein (23-25). Recently, Sone et al. (20) have purified the complete Fo protein from a thermophilic bacterium. This communication describes the isolation of both the DCCD-sensitive latent ATPase (BCFo-BCF4 complex) and the hydrophobic protein component (BCFo) from M. phlet, and the reconstitution of oxidative phosphorylation using these preparations. The BCF0-BCF4 complex was purified to homogeneity by affinity chromatography and was capable of restoring coupled phosphorylation to membrane vesicles depleted of the complex. Some energy-dependent reactions, such as 32Pi-ATP exchange, ion translocation, and light-driven ATP synthesis, have been reconstituted with purified Fo-C, complex (19, 20, 26). We now report the reconstitution of oxidative phosphorylation in detergent-extracted membranes by purified DCCD-sensitive ATPase. MATERIALS AND METHODS Preparation of DCCD-Sensitive Latent ATPase (BCFoBCF4 Complex) M. phlei (ATCC 354) was grown as described (27), and the electron transport particles were prepared by sonication of cells as described by Brodie (28). The BCFo-BCF4 complex was isolated essentially according to the method of Sone et al. (20), by suspending the electron transport particles in a solution of 0.5 M Tris-HCl (pH 8.0) containing 1% sodium cholate (Sigma Chemical Co.) and 0.15 M KC1, at a protein concentration of 10 mg/ml. After incubation at 250 for 60 mi, the mixture was centrifuged at 105,000 X g for 45 min. The cholate-washed pellets were resuspended by 2 min of sonication in the same volume of 50mM Tris-HCl (pH 8.0) containing 0.25 M sucrose, 0.15 M KC1, and 2% Triton X-100 (Sigma Chemical Co.) and incubated for 30 min at 25°. The BCFO-BCF4 complex remained in the supernatant after centrifugation of the Triton X-100 mixture at 144,000 X g for 60 min. Preparation of the Hydrophobic Protein (BCFo). Electron transport particles were depleted of BCF4 by washing with 0.25 M sucrose in the absence of cations (1, 3). The BCFO was solubilized from these depleted particles by the procedures described above. Preparation of BCF4. BCF4 was purified to homogeneity by affinity chromatography as described by Higashi et al. (3). This preparation had coupling factor-latent ATPase activity and was not contaminated with respiratory components or other proteins. Preparation of Sepharose-ADP. CH-Sepharose 4B (Pharmacia Fine Chemicals) was coupled to ADP for affinity chromatography by the procedure described (3, 4). Analytical Procedures. Latent ATPase activity was assayed as described (3). For the estimation of DCCD sensitivity, the samples were incubated with DCCD (0.6 mM) for 10 min at 30° before trypsin treatment. Oxygen consumption was measured manometrically with a Gilson differential respirometer at 300, and phosphorylation 3050 Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide; ATPase, adenosinetriphosphatase; BCF4, bacterial coupling factor-latent ATPase; BCF0-BCF4 complex, DCCD-sensitive latent ATPase; BCFo, intrinsic membrane protein component of BCFo-BCF4 complex. Proc. Nati. Acad. Sci. USA 73 (1976) 3051 was estimated by the procedures described (27). Protein concentration was determined either by the method of Lowry et al. (29), with bovine serum albumin as standard, or spectrophotometrically at 280 nm (30). Disc gel electrophoresis was performed on 7.5, 8, and 10% polyacrylamide gels in a Trisglycine buffer, pH 8.3 (31). Protein on the gels was stained with 0.05% Coomassie blue. Phospholipids were extracted by the method of Folch et al. (32), and inorganic phosphates were removed by gel filtration on Sephadex G-25, as described by Wells and Dittmer (33). Phospholipid phosphorus was determined by the method of King (34).