Isolation and partial characterization of magnesium ion-and calcium ion-dependent adenosine triphosphatase activity from bovine brain microsomal fraction

摘要

pMicrosomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mgsup2+/sup (6.4μmol of Psubi/sub/h per mg of protein) and Casup2+/sup (3.4μmol of Psubi/sub/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Casup2+/sup resulted in the demonstration of two apparent iK/isubm/sub values for Casup2+/sup (6.0×10sup?8/supm and 1.2×10sup?6/supm). 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 sup45/supCasup2+/sup an ATP-dependent uptake of Casup2+/sup was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mgsup2+/sup- and Casup2+/sup-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 Casup2+/sup. Kinetic analysis of the results for peak 4.8 demonstrated an apparent iK/isubm/sub value for Casup2+/sup of 4.1×10sup?8/supm. The enzyme isolated at pH6.3 had an apparent iK/isubm/sub value of 3.8×10sup?6/supm. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mgsup2+/sup the ATPase could not be activated by Casup2+/sup./p