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 Enzymol 22:99-120 (1971)]. Protoplasts were produced at frequencies greater than 95% by a method entailing mutanolysin digestion and osmotic shock. Glycerol or polyethyleneglycol 600 was required as an osmotic stabilizer while the use of sucrose prevented closed vesicle formation during osmotic shock. The membrane vesicles retained their functional properties and accumulated L-malic acid in response to an ATPase-induced 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 possibility 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.microliter-1.s-1) similar to the diffusion rate obtained previously in a L. oenos malate-transport-deficient strain. These results confirm two simultaneous uptake mechanisms in L. oenos, a permease-mediated transport and a passive diffusion for the anionic and the undissociated forms of L-malic acid respectively.