A CL--TRANSLOCATING ADENOSINE-TRIPHOSPHATASE 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 35SO42−, no ATP-dependent 35SO42− efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9-ACMA was observed. © 1990, American Chemical Society. All rights reserved.