Modulation of the Activity of Purified Tonoplast H+-ATPase from Mung Bean (Vigna radiataL.) Hypocotyls by Various Lipids

摘要

H+-ATPase was solubilized from the tonoplast of mung bean (Vigna radiataL.) hypocotyls and purified by fast protein liquid chromatography on a Mono Q ion-exchange column. The purified ATPase hardly contained any phospholipid, but it did contain 10 to 15 molecules of sterol and 25 to 30 molecules of glycolipid per ATPase molecule, and it had little activity without exogenously added phospholipids. Each individual polar head group, acylglyceride and fatty acid that constituted a phospholipid was incapable by itself of activating the ATPase. Sterols and cerebroside had little activating effect. Maximal activation of ATPase was noted with asolectin or various molecular species of phosphatidylcholine (PC) at 0.005%to 0.01%(w/v). The activation by the various molecular species of PC was dependent on the length and degree of unsaturation of fatty acyl chains. PC with two saturated and long fatty acyl chains of more than 18 carbon atoms failed entirely to activate the ATPase. PC, PS and PG with 1-palmitoyl (16:0)-2-oleoyl(18:1) fatty acyl chains all activated ATPase to nearly the same extent as asolectin, but the activation by PE and PA with the same fatty acyl composition was 52%and 15%of that by asolectin, respectively. The molecular species of PC with phase-transition temperatures below 50°C activated ATPase, as determined at 38°C. The dependence on temperature of the activation by the molecular species of PC indicated that the activation of the ATPase began close to the temperature of the phase transition of the PC added. These data indicate that phospholipids in the liquid-crystalline phase are essential for the catalytic activity of the ATPas