Partial characterization of two (Ca+2 + Mg+2)-dependent ATPase activities from bovine brain synaptic membrane homogenates.
Synaptic plasma membranes isolated from bovine brain exhibited a low and high affinity (Ca+2 + Mg+2)-dependent ATPase as evidenced by kinetic constants for ATP. One activity which hydrolyzed ATP maximally at pH 7.4 and 7.8 exhibited an 8-fold higher affinity when compared to the second or lower affinity activity which hydrolyzed ATP maximally at pH 7.0. Both activities exhibited submicromolar kinetic constants for Ca+2 (Km = 0.24 micromolar). Km values for magnesium differed significantly; the lower affinity activity being approximately 6.5 times higher (120 microM) than that observed for the high affinity activity (18 microM). Vmax values obtained under optimal assay conditions (low and high) were 110-135 and 43-55 nmol/min/mg protein, respectively. Both activities were KCN, NaN3 and ruthenium red insensitive. Only slight inhibition was observed in the presence of rotenone and oligomycin. Although both activities were observed to be trifluoperazine sensitive, they differed significantly with regard to other parameters. Na+1 and NH4+1 ions preferentially inhibited the low affinity activity greater than 90%. Cs+1 ions completely inhibited the high affinity activity while reducing the low affinity only 22%. Li+1, Al+3 and Mn+2 significantly inhibited the high affinity activity while reducing the low activity only moderately. Both the low and high activity were inhibited by vanadate with half maximum inhibition occurring at 2 and 5 microM, respectively indicating the plasma membrane origin of these activities. Thermal denaturation studies indicated the high affinity activity to be stable for 2 minutes at 45 degrees C after which 50% of the activity is lost at 2.5 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001921:Brain / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000502:physiology; D002417:Cattle; D004110:Diltiazem / Q000494:pharmacology; D006358:Hot Temperature; D007473:Ion Channels / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D013570:Synaptic Membranes / Q000201:enzymology*; D014700:Verapamil / Q000494:pharmacology
D002147:Calmodulin; D007473:Ion Channels; D007527:Isoenzymes; D014700:Verapamil; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D004110:Diltiazem