Actin-activated ATPase in human red cell membranes

The ability of human RBC and their ghosts to undergo reversible shape changes as well as endocytosis and exocytosis raised the likelihood that contractile proteins like actomyosin might be present. Actin, a component of actomyosin, is a constituent of the red cell membrane. Two generally accepted criteria for identifying a myosin include the ability of purified preparations to form arrow-head complexes with F actin fibers visible in the electron microscope, and the presence of an unusual enzyme activity where a low level of Mg⁺⁺-ATPase is markedly enhanced by addition of F actin, a reaction reflecting the physiologic interaction of actin and myosin. Our studies on a RBC myosin were therefore based on the use of the actin-activated ATPase (AA-ATPase) as a reliable marker of a myosin-like protein. RBC contain AA-ATPase, all of which was recoverable in the ghost or membrane fraction prepared by either hypotonic or isotonic lysis. AA-ATPase was optimally stimulated by human erythrocyte F actin as compared to rabbit muscle F actin or human erythrocyte G actin. In common with the myosin AA-ATPase of macrophages there was no saturation effect seen upon F actin addition. Myosin AA-ATPase of platelets and macrophages is inhibited when the protein is dephosphorylated. Dephosphorylation of the RBC membrane resulted in almost complete inhibition of AA-ATPase. Another myosin enzyme marker is a K⁺, EDTA-ATPase, which is also detectable in the red cell membrane. The AA-ATPase does not appear to be related to the other RBC membrane ATPases. Attempts to solubilize, isolate, and purify the AA-ATPase have thus far been unsuccessful; however, the AA-ATPase is not a peripheral membrane protein and no AA-ATPase is found in purified spectrin preparations. The presence of AA-ATPase and K⁺, EDTA-ATPase in the red cell membrane as well as several distinguishing characteristics of activation and inactivation of the AA-ATPase presumptively establishes the likelihood that there is a myosin-like (which is not spectrin) in the red cell membrane.