Membrane Adenosine Triphosphatase from Streptococcus faecalis

Abstract

Abstract In the preceding paper we describe the purification to homogeneity of the membrane adenosine triphosphatase from Streptococcus faecalis. Here we report some of the molecular characteristics of this enzyme. The molecular weight of the native enzyme and its subunits was determined by the Yphantis high speed sedimentation equilibrium method. In addition, the amino acid composition was determined, and the enzyme was examined by gel electrophoresis in 8 m urea and by electron microscopy. The partial specific volume, calculated from the amino acid composition, is 0.735 ml per g; a value of 0.742 ml per g was obtained from the comparison of sedimentation equilibria in D2O and H2O. The native enzyme has a molecular weight of 385,000 and s020,w of 13.4. From these two values a frictional ratio of 1.35 could be calculated. From the specific activity of our most highly purified preparations and the molecular weight, a maximal turnover number of 18,000 per molecule per min could be estimated. The molecular weight of the subunits, determined by sedmentation equilibrium in 6 m guanidine hydrochloride, is 33,000. This value is in good agreement with a minimal molecular weight of 32,800 calculated from the amino acid composition. It is thus concluded that the enzyme consists of 12 subunits. Electrophoretic analyses in 7% polyacrylamide gels containing 8 m urea indicate that the enzyme consists of two kinds of subunits, designated α and β. No free NH2-terminal amino acids could be detected by the 3H-fluorodinitrobenzene method. Electron micrographs of a negatively stained preparation of the ATPase show particles of uniform size with a substructure that suggests a planar, hexagonal array of 6 globules. A model of the enzyme with a formula α6β6 is proposed, consisting of a hexagonal arrangement of 6 globules, each of which is made up of an α and a β subunit.