Mechanical fluctuations of the membrane-skeleton are dependent on F-actin ATPase in human erythrocytes

Cell membrane fluctuations (CMF) of human erythrocytes, measured by point dark field microscopy, were shown to depend: to a large extent, on intracellular MgATP (Levin, S.V., and R. Korenstein. 1991. Biophys. J. 60:733-737). The present study extends that investigation and associates CMF with F-actin's ATPase activity. MgATP was found to reconstitute CMF in red blood cell (RBC) ghosts and RBC skeletons to their levels in intact RBCs, with an apparent K-d of 0.29 mM. However, neither non-hydrolyzable ATP analogues (AMP-PNP, ATP gamma S) nor hydrolyzable ones (ITP, GTP), were able to elevate CMF levels. The inhibition of ATPase activity associated with the RBC's skeleton, carried out either by the omission of the MgATP substrate or by the use of several inhibitors (vanadate, phalloidin, and DNase I): resulted in a strong decrease of CMF. We suggest that the actin's ATPase, located at the pointed end of the short actin filament, is responsible for the MgATP stimulation of CMF in RBCs.