Molecular and Kinetic Properties of the Purified Plasma Membrane ATPase of the Yeast Schizosaccharomyces pombe

Abstract

This paper reports the properties of the purified plasma-membrane ATPase of the yeast Schizo-saccharomyces pombe which appears to transport H+ across the cellular membrane. The rate of sedimentation of the purified ATPase, estimated by the computation model of McEwen, yields a sedimentation coefficient s0.78420, w of 24 S. Calculation of the corresponding molecular weight shows that the ATPase is purified in an oligomeric form of molecular weight 800000 to 1000000 corresponding to 8 to 10 monomers of molecular weight 100000. Eadie-Hofstee plots for the purified ATPase activity are linear when tested with or without lysophosphatidylcholine showing that strict Michaelis-Menten kinetics apply within a large range of ATP and Mg2+ concentrations. Addition of egg lyso-phosphatidylcholine increases V from 1.4 to 26.8 μmol × min−1× mg−1 whereas Km is less modified. The lipid-reconstituted purified ATPase activity is strictly specific for ATP and is inhibited competitively by ADP with a Ki of 2.5 mM. The optimum pH of 5.6 for the purified enzyme is shifted to pH 6.0 in the presence of lysophosphatidylcholine. The optimum temperature of the purified ATPase is 30 and 32.5 °C in absence or presence of lysophosphatidylcholine, respectively. The lipid-reconstituted purified ATPase activity is cation-dependent with the following specificity: Zn2+ > Co2+ > Mg2+ > Mn2+ > Ni2+ for cation concentrations below 2 mM (M2+/ATP < 0.6). However above 2 mM (M2+/ATP > 1), Mg2+ is the most efficient cofactor followed by Co2+ > Mn2+ > Zn2+ > Ni2+. These properties are qualitatively similar to those of the membrane-bound ATPase activity indicating that the integrity of the lipid-reconstituted purified ATPase is not appreciably modified by the extraction, purification and reconstitution procedures.