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.
Romero
P J
PJ
Instituto de Biología Experimental, Fac. Ciencias, Universidad Central de Venezuela, Caracas.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D004357:Drug Synergism; D004533:Egtazic Acid; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000201:enzymology; D005454:Fluorescence Polarization; D006801:Humans; D008274:Magnesium / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D014638:Vanadates / Q000494:pharmacology*
D002147:Calmodulin; D011480:Protease Inhibitors; D014638:Vanadates; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium