Roles of proteins from inner face of plasma membranes in susceptibility of of (Na+ + K+)-stimulated Mg2+ adenosinetriphosphatase to ouabain.

Purified right-side-out (RSO) and inside-out (IO) plasma membrane vesicles release 35% of the total plasma membrane proteins after EDTA treatment. After such a treatment both types of vesicles exhibited the same total activity of (Na+ + K+)-stimulated Mg2+ adenosinetriphosphatase (ATPase; ATP phosphohydrolase, EC 3.6.1.3) as in their native state. The EDTA treatment increases the enzyme sensitivity to ouabain by 350-fold in IO vesicles while being without any effect RSO vesicles. Thus, proteins released only from the IO vesicles led to a change in ouabain sensitivity of the (Na+ + K+)-stimulated Mg2+ ATPase. Moreover, only proteins released from IO vesicles, when added to treated IO vesicles with divalent cations, were able to restore the original resistance of the enzyme to ouabain; released proteins from RSO vesicles failed to make such a reconstitution. Thus, we assume that these proteins detach from the inner face of the plasma membrane upon EDTA treatment and are distinct from the enzyme. Polyacrylamide gel electrophoresis indicates that these inner face plasma membrane proteins are approximately 30,000 daltons.