Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity

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 K_m for Mg:ATP (1:1 concentration ratio) was higher (2.5 mm). A comparison of anion (potassium salts) effects upon ΔpH and Δψ formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce Δψ. The relative order for this effect was KCl > KBr ≫ 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.