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. 1977 Aug 1;165(2):355–365. doi: 10.1042/bj1650355

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

Elur K Vijayakumar 1, Maurice J Weidemann 1,*
PMCID: PMC1164908  PMID: 21656

Abstract

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 (E′a=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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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