Characterization of Mg2+-ATPase from sheep kidney medulla: magnetic resonance and kinetic studies.

Abstract Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg 2+ or Mn 2+ indicate a biphasic response to divalent cations. Apparent K m values of 23 μ m for free Mg 2+ and 3.3 μ m for free Mn 2+ are obtained at low levels of added metal, while K m values of 0.50 m m for free Mg 2+ and 0.43 m m for free Mn 2+ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg 2+ -ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent K m values for free ATP of 6.0 and 1.4 μ m with Mg 2+ and Mn 2+ , respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that K m values for free ATP of 8.0 and 2.0 m m are obtained for Mg 2+ and Mn 2+ , respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg 2+ -ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with K i values of 10.5 μ m and 4.5 m m . Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn 2+ per 469,000 g of protein. The Mn 2+ binding studies yield a K D for Mn 2+ at the single high affinity site of 2 μ m , in good agreement with the kinetically determined activator constant for Mn 2+ at low Mn 2+ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn 2+ per single high affinity Mn 2+ site. The K D for Mn 2+ at these sites is 0.55 m m , in good agreement with the kinetic activator constant for Mn 2+ of 0.43 m m , consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn 2+ in the presence of the enzyme is also consistent with the tight binding of a single Mn 2+ ion per 469,000 M r protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn 2+ at the single tight site, ϵ b , of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn 2+ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10 −9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg 2+ -ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg 2+ -ATPase described here are discussed.