Identification and characterization of high-affinity Ca2(+)-ATPase associated with axonal plasma membranes of dog mesenteric nerves.

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca2(+)-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with Km for Ca ions of 0.4 microM and Vmax = 12.5 +/- 2.4 mumol Pi.mg-1h-1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive 3H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with Ki = 0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg greater than Mn greater than Zn greater than or equal to Co greater than Ni. In contrast to low affinity (high Km) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 microM) and N-ethylmaleinimide (100 microM). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.