Biology Department, University of California, Santa Cruz, Santa Cruz, California 95064.
Plant Physiol. 1985 May;78(1):104-9. doi: 10.1104/pp.78.1.104.
Vacuoles were isolated from corn coleoptile protoplasts and ATP-dependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of [(14)C]methylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATP-stimulated methylamine uptake and alpha-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, the activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl(-), and inhibited by NO(3) (-), carbonyl cyanide-m-chlorophenylhydrazone, N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. We conclude that the microsomal proton pump previously characterized in corn coleoptiles (Mettler et al. 1982 Plant Physiol 70: 1738-1742) is derived from the tonoplast.
液泡从小麦苗原生质体中分离出来，并用吖啶橙荧光猝灭或[¹⁴C]甲基胺的摄取来测量其 ATP 依赖性质子转运。根据荧光染色用荧光素二乙酸酯，完整的液泡被判断为没有周围的质膜。基本上，所有可检测到的完整原生质体中存在的 ATP 刺激的甲基胺摄取和α-甘露糖苷酶活性都在分离的液泡中得到回收。相比之下，质膜、高尔基体、内质网和线粒体的标记酶的活性在液泡制剂中降低到 5 到 17%。与可能来源于质膜的光面微粒体膜相比，分离液泡的质子泵送特性进行了比较。ATP 依赖性质子泵送由分离的液泡和光面微粒体囊泡均由 Cl(-)刺激，并被 NO(3) (-)、羰基氰化物 m-氯苯腙、N，N'-二环己基碳二亚胺、N-乙基马来酰亚胺、4,4'-二异硫氰基-2,2'-联苯二磺酸、己烯雌酚和 7-氯-4-硝基苯并-2-氧代-1,3-二唑抑制，但不被钒酸盐抑制。这两种活性也表现出对 Mg-ATP 的底物特异性。最后，在线性葡聚糖梯度中浮选后，液泡和微粒体部分的质子转运活性表现出相似的图谱。我们得出结论，先前在玉米 coleoptiles 中表征的微粒体质子泵（Mettler 等人，1982 年植物生理学 70：1738-1742）来源于质膜。
