Isolation of plasma membrane ofPhytophthora megasperma f. sp.glycinea and some properties of the associated ATPase

Plasma membrane vesicles were isolated fromPhytophthora megasperma f. sp.glycinea using conventional methods of mechanical disruption followed by differential and density gradient centrifugation. The validity of presumed biochemical markers was confirmed using electron microscopy and the phosphotungstic acid-chromic acid staining procedure, which was judged to be specific for plasma membrane when performed under suitable conditions. The plasma membrane fraction showed a peak equilibrium density of 1.14 g/ml and was identified by its vanadate-sensitive Mg²⁺-dependent ATPase with an optimum temperature of 42°C and a pH optimum of 6.0 to 6.5. The activity was weakly stimulated by K⁺ and strongly inhibited by Ca²⁺. The enzyme showed a marked specificity for ATP as a substrate compared to other nucleoside mono-, di-, and triphosphate substrates or other general phosphatase substrates. The divalent cation requirement could be met equally well by Mg²⁺ and Co²⁺ and, to a lesser extent, by Mn²⁺, but not by Ni²⁺, Ba²⁺, Zn²⁺, Sr²⁺, Ca²⁺, Hg²⁺, Cu²⁺, or Fe²⁺ (in decreasing order of preference). Contamination by intact mitochondria (density 1.21 g/ml) or mitochondrial fragments (density 1.16 g/ml) was minimal and could be monitored by measuring cytochromec oxidase or oligomycin-sensitive pH 8.5 ATPase.