A Cl(-)-translocating adenosinetriphosphatase in Acetabularia acetabulum. 2. Reconstitution of the enzyme into liposomes and effect of net charges of liposomes on chloride permeability and reconstitution.
The Mono Q-III fraction, a Mg2(+)-ATPase, isolated from Acetabularia acetabulum was reconstituted into liposomes of various net charges prepared by the reversed-phase method and tested for a Cl(-)-translocating activity. The liposomes from a mixture of egg lecithin, dicetyl phosphate, and cholesterol (63:18:9 mole ratio, negative liposomes) and from a mixture of egg lecithin and cholesterol (63:9 mole ratio, neutral liposomes) were less leaky than positive liposomes from asolectin, and from a mixture of egg lecithin, stearylamine, and cholesterol (63:18:9 mole ratio). A significant increase in 36Cl- efflux from the negative and neutral liposomes was observed by addition of ATP in the presence of valinomycin after incorporation of the enzyme by short-term dialysis. The ATP-driven 36Cl- efflux was inhibited by addition of azide, an inhibitor of the ATPase. The preincubation of the enzyme with phenylglyoxal, an arginine-modifying reagent, inactivated ATP-mediated 36Cl- efflux, but the ATPase activity of the preparation was not affected. When chloride was replaced by 35SO4(2)-, no ATP-dependent 35SO4(2)- efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9-ACMA was observed.
Ikeda
M
M
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Oesterhelt
D
D
D000076:Acetabularia / Q000187:drug effects / Q000201:enzymology*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001386:Azides / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D000460:Chlorophyta / Q000201:enzymology*; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D010658:Phenylglyoxal / Q000494:pharmacology
D001386:Azides; D002712:Chlorides; D007476:Ionophores; D008081:Liposomes; D000251:Adenosine Triphosphatases; D010658:Phenylglyoxal