OBTAINING FUNCTIONAL MEMBRANE-VESICLES FROM LEUCONOSTOC-OENOS TO STUDY L-MALATE TRANSPORT MECHANISMS

Membrane vesicles from the malolactic bacterium Leuconostoc oenos were obtained by a modified version of the procedure of Kaback [Methods En zymol 22:99-120 (1971)]. Protoplasts were produced at frequencies grea ter than 95% by a method entailing mutanolysin digestion and osmotic s hock. Glycerol or polyethyleneglycol 600 was required as an osmotic st abilizer while the use of sucrose prevented closed vesicle formation d uring osmotic shock. The membrane vesicles retained their functional p roperties and accumulated L-malic acid in response to an ATPase induce d proton gradient across the membrane of ATP loaded vesicles. L-Malate uptake was strongly inhibited by dicyclohexylcarbodiimide, a specific inhibitor of membrane-bound ATPases. These data support the possibili ty of a Delta pH-dependent transport of L-malate. Vesicles not loaded with ATP were slightly permeable to malic acid with an initial uptake rate (0.5 nmol.mu l(-1).s(-1)) similar to the diffusion rate obtained previously in a L. oenos malate-transport-deficient strain. These resu lts confirm two simultaneous uptake mechanisms in L. oenos, a permease -mediated transport and a passive diffusion for the anionic and the un dissociated forms of L-malic acid respectively.