R. Tourdotmarechal et al., OBTAINING FUNCTIONAL MEMBRANE-VESICLES FROM LEUCONOSTOC-OENOS TO STUDY L-MALATE TRANSPORT MECHANISMS, Applied microbiology and biotechnology, 41(5), 1994, pp. 603-607
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.