Synergistic activation of the human red cell calcium ATPase by magnesium and vanadate.

The Ca(2+)-ATPase activity of human red cells was studied on calmodulin-free membrane fragments after previous incubation with Mg2+ and vanadate. In the presence of EGTA (5 mM), the activity was slightly affected by either ion alone. However, when added together, both Ca2+ affinity and Vmax were increased up to levels found with calmodulin (0.3 microM). This synergistic activation was not abolished by proteinase inhibitors (iodoacetamide, 10 mM; leupeptin, 200 microM; pepstatin A, 100 microM; phenylmethanesulfonyl fluoride, 100 microM), neomycin (200 microM), washing with EDTA (5 mM) or by both incubating and washing with delipidized serum albumin (1 mg/ml). During preincubation under optimal Mg2+ and vanadate conditions, the replacement of K+ by Na+ or Li+ was without effect. Co2+ or Zn2+ (10 mM) could not substitute for Mg2+, whereas Mn2+ almost replaced it at equimolar amounts. By contrast, addition of ATPMg (2 mM) decreased the activation by about one-half. Like calmodulin, pretreatment with Mg2+ plus vanadate also increased the affinity for ATP and elicited appearance of a second (low) affinity site (apparent Km = 120 microM). The fluorescence depolarization of 1,6-diphenyl- and 1-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating with Mg2+, vanadate or Mg2+ plus vanadate. The results show that Mg2+ and vanadate are acting neither via proteolysis or fatty acid production nor by facilitating phospholipid metabolism or altering membrane fluidity. They may be enhancing the Ca(2+)-ATPase activity by stabilizing the E1 conformer or promoting an enzyme conformation which facilitates the E2-E1 transition.