Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei

• Published in: The Journal of biological chemistry 1975-05, Vol.250 (10), p.3690-3698
• Main Authors: Prasad, R, Kalra, V K, Brodie, A F
• Format: Article
• Language: English
• Subjects: Anilino Naphthalenesulfonates - metabolism; Animals; Binding Sites; Cattle; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Electron Transport; Hydrogen-Ion Concentration; Kinetics; Liposomes - metabolism; Mycobacterium - metabolism; Mycobacterium phlei - drug effects; Mycobacterium phlei - metabolism; Mycobacterium phlei - ultrastructure; Oxidative Phosphorylation - drug effects; Oxygen Consumption - drug effects; Phospholipases - pharmacology; Phospholipids - analysis; Protein Binding; Serum Albumin, Bovine; Snake Venoms; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)41453-1

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.