Samarão Solange S, Teodoro Carlos E S, Silva Flavia E, Ribeiro Camila C, Granato Thais M, Bernardes Natalia R, Retamal Cláudio A, Façanha Arnoldo R, Okorokova-Façanha Anna L, Okorokov Lev A
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociência e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, Campos dos Goytacazes-RJ, 28013-600, Brazil.
Biochim Biophys Acta. 2009 Feb;1788(2):303-13. doi: 10.1016/j.bbamem.2008.11.006. Epub 2008 Nov 19.
H+ transport driven by V H+-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca2+-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H+ transport activity of P H+-ATPase by more than 75% while that of V H+-ATPase remained unchanged. ER H+ ATPase exhibits higher resistance to bafilomycin (I50=38.4 nM) than Golgi and vacuole pumps (I50=0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H+ transport mediated by V H+-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H+-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.
根据包括液泡Ca2+-ATPase、Pmc1::HA等各自标记物的测定，在通过蔗糖密度梯度从液泡膜中有效纯化的富含内质网/质膜以及高尔基体/类高尔基体膜的膜组分中，发现了由V型H+-ATP酶驱动的H+转运。用伴刀豆球蛋白A修饰质膜以从质膜中去除内质网，可使P型H+-ATP酶的H+转运活性降低75%以上，而V型H+-ATP酶的活性保持不变。内质网H+ ATP酶对巴弗洛霉素的抗性（I50 = 38.4 nM）高于高尔基体和液泡泵（I50 = 0.18 nM）。内质网、比内质网重的膜、液泡和高尔基体中泵的偶联效率之比分别为1.0、2.1、8.5和14，其中高尔基体的偶联效率最高。对内质网、高尔基体和液泡膜囊泡中V型H+-ATP酶介导的H+转运初始速度以及催化亚基A和调节亚基B的免疫反应性进行比较分析，进一步支持了V型H+-ATP酶是酵母内质网和高尔基体的固有酶且可能以不同形式存在和/或受到选择性调节的结论。
