Isolation and partial characterization of magnesium ion- and calcium ion-dependent adenosine triphosphatase activity from bovine brain microsomal fraction.
Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg(2+) (6.4mumol of P(i)/h per mg of protein) and Ca(2+) (3.4mumol of P(i)/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca(2+) resulted in the demonstration of two apparent K(m) values for Ca(2+) (6.0x10(-8)m and 1.2x10(-6)m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing (45)Ca(2+) an ATP-dependent uptake of Ca(2+) was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg(2+)- and Ca(2+)-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca(2+). Kinetic analysis of the results for peak 4.8 demonstrated an apparent K(m) value for Ca(2+) of 4.1x10(-8)m. The enzyme isolated at pH6.3 had an apparent K(m) value of 3.8x10(-6)m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg(2+) the ATPase could not be activated by Ca(2+).
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D007525:Isoelectric Focusing; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000494:pharmacology
D008081:Liposomes; D011092:Polyethylene Glycols; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium