Purification and characterization of a Ca2+- or Mg2+-stimulated ATPase from plasma membrane enriched fractions of Dictyostelium discoideum.
Evidence is presented for the presence of both diethylstilbestrol (DES)-sensitive and DES-insensitive Mg2+-ATPase activities in plasma membrane enriched fractions of Dictyostelium discoideum. When removed from the membrane, the DES-sensitive activity is markedly less stable than the DES-insensitive activity, and the two activities display a number of quite distinct properties. The DES-sensitive enzyme has a decided preference for Mg2+ over Ca2+, displays saturation kinetics in response to ATP as substrate (Km = 0.2 mM) and has a narrow pH optimum range. In contrast, the DES-insensitive activity is stimulated equally by Mg2+ or Ca2+, is not saturable by ATP within the mM concentration range and has a much broader pH optimum. The DES-insensitive activity has been purified extensively. The purified enzyme is inhibited by vanadate and fluoride, but is insensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide and thimerosal. In the absence of divalent cations, the enzyme displays a sigmoidal activity curve in response to substrate concentration, which is abolished by addition of either Mg2+ or Ca2+, suggesting a binding site for a divalent cation and a positive cooperative interaction. The enzyme is capable of hydrolyzing other nucleotide triphosphates and ADP, but is without activity on AMP, p-nitrophenyl phosphate and pyrophosphate. The enzyme has an apparent molecular weight of approximately 64,000.
MacDonald
J I
JI
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Weeks
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D003902:Detergents; D004023:Dictyostelium / Q000201:enzymology*; D004054:Diethylstilbestrol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013379:Substrate Specificity
D002413:Cations, Divalent; D003902:Detergents; D004054:Diethylstilbestrol; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases