Kinetic properties of a magnesium ion- and calcium ion-stimulated adenosine triphosphatase from the outer-membrane fraction of rat spleen mitochondria

1. Isolated outer membranes from rat spleen mitochondria can be stored in liquid N₂ for several weeks without significant loss of ATPase (adenosine triphosphatase) activity. 2. The ATPase reaction has a broad pH optimum centering on neutral pH, with little significant activity above pH9.0 or below pH5.5. 3. A sigmoidal response of the ATPase activity to temperature is observed between 0 and 55°C, with complete inactivation at 60°C. The Arrhenius plot shows that the activation energy above the transition temperature (22°C) (E_a=144kJ/mol) is one-third of that calculated for below the transition temperature (E′_a=408kJ/mol). 4. The outer-membrane ATPase (K_m for MgATP=50μm) is inactive unless Mg²⁺is added, whereas the inner-membrane ATPase (K_m for ATP=11μm) is active without added Mg²⁺unless the mitochondria have been depleted of all endogenous Mg²⁺(by using ionophore A23187). 5. The substrate for the outer-membrane ATPase is a bivalent metal ion–nucleoside triphosphate complex in which Mg²⁺(K_m=50μm) can be replaced effectively by Ca²⁺(K_m=6.7μm) or Mn²⁺, and ATP by ITP. Cu²⁺, Co²⁺, Sr²⁺, Ba²⁺, Ni²⁺, Cd²⁺and Zn²⁺support very little ATP hydrolysis. 6. Univalent metal ions (Na⁺, K⁺, Rb⁺, Cs⁺and NH₄⁺, but not Li⁺) stimulate the MgATPase activity (<10%) at low concentrations (50mm), but, except for K⁺, are slightly inhibitory (20–30%) at higher concentrations (500mm). 7. The Mg²⁺-stimulated ATPase activity is significantly inhibited by Cu²⁺(K_i=90μm), Ni²⁺(K_i=510μm), Zn²⁺(K_i=680μm) and Co²⁺(K_i=1020μm), but not by Mg²⁺, Ca²⁺, Ba²⁺or Sr²⁺. 8. The outer-membrane ATPase is insensitive to the inhibitors oligomycin, NN′-dicyclohexylcarbodiimide, NaN₃, ouabain and thiol-specific reagents. A significant inhibition is observed at high concentrations of AgNO₃ (0.5mm) and NaF (10mm). 9. The activity towards MgATP is competitively inhibited by the product MgADP (K_i=0.7mm) but not by the second product P_i or by 5′-AMP.