大豆液泡膜H^+-ATPase的纯化和重组

Purification and Reconstitution of Tonoplast H^+-ATPases from Soybean

通过不连续蔗糖密度梯度离心得到的液泡膜微囊 ,先由胆酸钠和 OG分步破膜抽提、经阴离子交换柱 ( Q- Sepharose)层析分离 .纯化后的酶含 V型 H+ - ATPase的主要亚基 ,与大豆磷脂重组 ,获得了有较高泵活性的脂酶体 .脂酶体的质子泵活性受 Valinomycin激活 ,说明它是致电性的 ,受NO-3 ,DCCD以及特异性的 V型 ATPase抑制剂 Bafilomycin的抑制 .脂酶体的泵活性不受 F型和P型 ATPase抑制剂抑制 ,表明质子转运是由 V型 H+ - ATPase引起的 .

The tonoplast vesicle preparation was obtained by sucrose gredient. To solubilize the H + ATPase from the vesicle membrane, four detergents (Na Cholate, CHAPS, Triton X 100 and OG) were tested. OG was chosen because of its good performance in maintaining the ATPase activity. Vesicle membrane was first treated by 0 5% Na Cholate to remove the other membrane proteins. The H + ATPase still on the membrane was solubilized by 30 mmol/L OG. The solubilized holoenzyme was further purified by ion exchange chromatograph(Q Separose) in an HPLC system. The purified soybean H + ATPase consisted of 67, 58, 45, 38, 35, 33 and 16 kD subunits indicated by SDS PAGE. They were similar to the subunits of H + ATPase from other sources. It also indicated there was difference between these H + ATPases from plants. After removing detergent by gel filtration, the purified holoenzyme was reconstituted into liposomes and proton pumping activity was recovered. The proton pumping activity at 22℃ was measured by quenching of acridine orange fluorecence. Transmembrane electrical potential was dissipated in the presence of K + and valinomycin, indicating an electrogenic ATP dependent proton pump. The proton pumping activity of proteoliposomes was inhibited by bafilomycin A 1, DCCD and nitrate but not by azide and vanadate. These results may indicate that vacuolar H + ATPase with high proton pumping activity has been successively reconstituted.