Tonoplast-enriched vesicles were prepared from suspension-cultured Populus euphratica Oliv. cells by differential centrifugation and discontinuous sucrose density gradient centrifugation. The properties of the proton pumping activity of H+-ATPases in tonoplast vesicles were studied by acridine orange fluorescent quenching measured at 22 ℃. The proton pumping activity of ATPase was ATP-dependent with apparent Michaelis-Menten Constant (Km) for ATP about 0.65 mmol/L. The optimal pH for H+-ATPases activity was 7.5. The proton pumping activity of H+-ATPase could be initiated by some divalent cations, Mg2+ being highly efficient, much more than Fe2+; and Ca2+, Cu2+ and Zn2+ were inefficient under the experimental condition. The proton translocation could be stimulated by halide anions, with potencies decreasing in the order Cl-> Br->I->F-. The proton pumping activity was greatly inhibited by N-ethylmaleimide (NEM), N,N′-dicyclohexylcarbodiimide (DCCD), NO-3 and Bafilomycin A1, but not by orthovanadate and azide. These results demonstrated that the H+-ATPase in the tonoplast of Populus euphratica belonged to vacuolar type ATPase. This work was the first time that tonoplast-enriched vesicles were isolated from Populus euphratica cells.
胡杨液泡膜微囊H+-ATPase质子泵活性研究
刘群录1 张旭家2 李义3 王沙生1 蒋湘宁1*
（1. 北京林业大学生物学院森林生物学实验中心，国家林业局树木花卉育种生物工程重点实验室，北京100083； 2.中国科学院生物物理研究所生物大分子国家重点实验室，北京100101；3. 康涅狄克大学植物科学系，康涅狄克州06269-4067,美国）
摘要：将悬浮培养的胡杨(Populus euphratica Oliv.)细胞捣碎后，通过差速离心和不连续蔗糖密度梯度离心获得富集液泡膜的膜微囊。通过连续监测吖啶橙的荧光淬灭研究膜微囊上H+-ATPase的质子转运特性。结果表明，质子转运依赖于ATP，其表观米氏常数Km值为0.65mmol/L。质子泵活性受pH和温度的影响较大。测定液pH值为7.5时，质子泵的活性最高(测定温度选定为22℃)。一些二价阳离子可启动H+-ATPase的质子转运，其中Mg2+的作用远高于Fe2+。在实验条件下，Ca2+、Cu2+和Zn2+均不能启动H+-ATPase的质子转运。质子跨膜转运还可被一价阴离子激活，激活作用的顺序为：Cl－>Br－>I－>F－。质子泵活性受NEM（乙基马来酰亚胺）、DCCD(二环己基碳二亚胺)、NO-3和Bafilomycin A1的强烈抑制，但对Na3VO4和NaN3不敏感。这些性质说明胡杨液泡膜微囊上的H+-ATPase属于囊泡型的ATPase。
关键词： 胡杨；H+-ATPase；质子转运活性
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