Purification of the synaptosomal plasma membrane (Ca(2+) + Mg(2+))-ATPase from pig brain.
The Ca(2+)-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca(2+)-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr140 000 that can be phosphorylated by [gamma-(32)P]ATP in the presence of La(3+) and recognized by specific antibodies against the plasma membrane Ca(2+)-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5-1.8-fold in the presence of phosphatidylcholine but not in the presence of phosphatidylserine.
Salvador
J M
JM
Departamento de Bioquímica y Biología Molecular y Genética, Facultadde Ciencias, Universidad de Extremadura, Badajoz, Spain.
Mata
A M
AM
D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure; D017301:Ca(2+) Mg(2+)-ATPase / Q000302:isolation & purification*; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D013574:Synaptosomes / Q000201:enzymology*
D012967:Sodium Dodecyl Sulfate; D017301:Ca(2+) Mg(2+)-ATPase