Partial characterization of two (Ca +2 + Mg +2)-dependent ATPase activities from bovine brain synaptic membrane homogenates

Abstract

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.O. Both activities exhibited submicromolar kinetic constants for Ca +2 ( K m = 0.24 micromolar). K m values for magnesium differed significantly; the lower affinity activity being approximately 6.5 times higher (120 μM) than that observed for the high affinity activity (18 μM). V max values obtained under optimal assay conditions (low and high) were 110–135 and 43–55 nmol/min/mg protein, respectively. Both activities were KCN, NaN 3 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 NH 4 +1ions 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 μM, 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°C after which 50% of the activity is lost at 2.5 minutes. In contrast, the low affinity activity gradually decreased over the time course and retains greater than 60% activity at 2.5 minutes. The positive allosteric calcium channel modulator, diltiazem, stimulates both low and high affinity activities, 10 and 40% at 10 and 30 μM, respectively. In contrast, the negative allosteric calcium channel modulator, verapamil, has no effect upon the high affinity activity while slightly inhibiting the low affinity activity. These observations are suggestive of either (1) a direct interaction with the (Ca +2 + Mg +2)-dependent ATPase or (2) a close spatial relationship of either a specific catalytic site or conformation of the ATPase with that of the diltiazem binding site or resultant conformation of the calcium channel following diltiazem administration. Although kinetic data are consistent with other findings indicating the presence of different kinetic conformations of a single synaptic membrane protein, the presence of two different proteins maximally operational at two different ATP concentrations cannot be ruled out.