Giannini J L, Holt J S, Briskin D P
Department of Agronomy, University of Illinois, Urbana 61801.
Arch Biochem Biophys. 1988 Sep;265(2):337-45. doi: 10.1016/0003-9861(88)90136-1.
Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.
使用先前开发的用于植物质膜囊泡分离方法的改进方法，从植物致病真菌大豆疫霉中分离出密封囊泡。对钒酸盐敏感的质子泵微粒体膜囊泡在线性蔗糖密度梯度上进行分离，发现其与钒酸盐敏感的ATP酶共同迁移。这些囊泡的质子泵活性和ATP酶活性的最适pH均为6.5，并且在底物特异性和抑制剂敏感性方面表现出相似的特性。这些特性与先前发表的关于疫霉质膜ATP酶的数据一致。与先前的报道相反，质膜ATP酶没有受到K+的刺激，并且Mg:ATP（1:1浓度比）的Km更高（2.5 mM）。对阴离子（钾盐）对密封的疫霉质膜囊泡中ΔpH和Δψ形成的影响进行比较，结果显示阴离子刺激质子运输和降低Δψ的相对能力之间存在对应关系。这种效应的相对顺序为KCl＞KBr＞＞KMes、KNO3、KClO3、K2SO4。本研究提出了一种从疫霉菌丝中分离密封囊泡的方法。它还提供了关于质膜H+-ATP酶及其相关质子泵活性的基本信息。
