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) (Ea=144kJ/mol) is one-third of that calculated for below the transition temperature (Ea=408kJ/mol). 4. The outer-membrane ATPase (Km for MgATP=50μm) is inactive unless Mg2+is added, whereas the inner-membrane ATPase (Km 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+(Km=50μm) can be replaced effectively by Ca2+(Km=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+(Ki=90μm), Ni2+(Ki=510μm), Zn2+(Ki=680μm) and Co2+(Ki=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 (Ki=0.7mm) but not by the second product Pi or by 5′-AMP.

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