Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.
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.
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D009161:Mycobacterium / Q000378:metabolism*; D009168:Mycobacterium phlei / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D010740:Phospholipases / Q000494:pharmacology*; D010743:Phospholipids / Q000032:analysis; D011485:Protein Binding; D012710:Serum Albumin, Bovine; D012910:Snake Venoms; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; D008081:Liposomes; D010743:Phospholipids; D012910:Snake Venoms; D012710:Serum Albumin, Bovine; D010740:Phospholipases