RAPID PURIFICATION AND RECONSTITUTION OF A PLANT VACUOLAR ATPASE USING TRITON X-114 FRACTIONATION - SUBUNIT COMPOSITION AND SUBSTRATE KINETICS OF THE H+-ATPASE FROM THE TONOPLAST OF KALANCHOE-DAIGREMONTIANA

A rapid procedure for the purification and reconstitution into proteoliposomes of the H+-translocating ATPase of plant vacuolar membranes is reported. It involves fractionation of the tonoplast with Triton X-114, resolubilization of the ATPase with octyl glucoside in the presence of a mixture of phosphatidylcholine, phosphatidylserine and cholesterol (27:53:20, by weight), and removal of the detergent by gel-filtration. Starting with partially purified vacuolar membranes, the procedure can be accomplished in about 2 hours. It has been applied to the H+-ATPase from the crassulacean plant Kalanchoe daigremontiana, from which it yields vesicles with a specific ATPase activity of about 3-mu-mol/min per mg protein. The purified enzyme contains polypeptides of apparent molecular mass 72, 57, 48, 42, 39, 33 and 16 kDa; these polypeptides also co-sediment on centrifugation of the solubilized ATPase through glycerol gradients. The 16-kDa subunit is labelled with [C-14]dicyclohexylcarbodiimide. There is no evidence for a larger ATPase subunit in this preparation. The reconstituted ATPase proteoliposomes undergo ATP-dependent acidification, which can be measured by quenching of the fluorescence of 9-aminoacridine. The initial rate of fluorescence quenching is a measure of the rate of H+ translocation, and is directly proportional to the vesicle protein concentration, so the preparation is suitable for studying the kinetics of the tonoplast H+-ATPase. The dependence of the rate of fluorescence quenching on the concentration of MgATP is well fitted by the Michaelis equation, with a K(m) value about 30-mu-M. ATP can be replaced by dATP, ITP, GTP, UTP or CTP, and Mg2+ by Mn2+ or Ca2+; kinetic parameters for these substrates are reported In contrast, hydrolysis of MgATP shows complex kinetics, suggestive either of negative cooperativity between nucleotide-binding sites, or of two non-interacting catalytic sites. Both the hydrolytic and the H+-translocating activities of the proteoliposomes are inhibited by nitrate, though not in parallel, the latter activity being the more sensitive. Both activities are inhibited in parallel by bafilomycin A1, which does not produce complete inhibition; the bafilomycin-insensitive component has complex ATPase kinetics similar to those of the uninhibited enzyme.