Effects of Cerebroside and Cholesterol on the Reconstitution of Tonoplast H+-ATPase Purified from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

For an examination of the effects of cholesterol and cerebrosideon the rate and extent of proton-pumping across the membranesof proteoliposomes prepared with tonoplast H⁺-ATPase, the tonoplastH⁺-ATPase of mung bean (Vigna radiata L.) was purified by fastprotein liquid chromatography (FPLC) and incorporated into liposomesprepared from asolectin and cholesterol or cerebroside. Proteoliposomeswere formed after the removal of Triton X-100 from a mixtureof Triton X-100, asolectin and purified tonoplast H⁺-ATPaseby passage through an Ampure DT column. Proteoliposomes preparedfrom cholesterol and asolectin at a ratio of 45 : 55 (w/w) andat a ratio of lipid to protein of 200 : 1 (w/w) gave the largestpH gradient, as determined by the ATP-generated quenching ofquinacrine fluorescence. In the presence of cholesterol, thepH gradient formed across the membranes of proteoliposomes andthe average diameter of proteoliposomes increased about two-fold.The initial rate of proton-pumping decreased to 20% of thatobserved with proteoliposomes prepared from asolectin alone.The addition of cerebroside to asolectin at a ratio of 5 : 95(w/w) caused a 1.6-fold increase in the maximum pH gradientwithout any significant change in the initial rate of proton-pumpingor the diameter of proteoliposomes, but the maximum pH gradientdecreased greatly at ratios above 20 : 80 (w/w). The maximumpH gradient was transient and decreased spontaneously when onlyasolectin was used to prepare proteoliposomes, or when cerebrosideand asolectin were used together. Disappearance of the protongradient once it had formed and/or leakage of protons were suppressedby cholesterol at ratios above 30 : 70 (w/w). It was clear,therefore, that cholesterol and asolectin at ratios 30 : 70(w/w) to 45 : 55 (w/w) formed larger and more stable proteoliposomesthan did asolectin alone. ¹Present address: Laboratory of Climatic Stress Control, TohokuNational Agricultural Experiment Station, 4 Shimokuriyagawa,Morioka, Iwate, 020-01 Japan