Abstract
Human erythrocyte cytoskeletal ATPase was extracted with 0.2mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90 % of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein•h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KC1 and was almost completely inhibited by 10-5M free calcium in the presence of 0.2mM MgCl2. The K1 for Ca2+ was 7 × 10-7 M. Phalloidin and DNase 1, which affect actin, inhibited this K, Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP > ITP>CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg•h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.
