Partial purification and characterization of a novel Mg2+-dependent ATPase present in the cytosol from human erythrocytes.
Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SD gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44 degrees C. It was stable for several months at -20 degrees C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000255:Adenosine Triphosphate / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase; D002147:Calmodulin / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D011863:Radioimmunoassay; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D002147:Calmodulin; D002413:Cations, Divalent; D009711:Nucleotides; D013439:Sulfhydryl Reagents; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase