Binding of manganese ions to the Na+/K+-ATPase during phosphorylation by ATP.
The aim of the present work was to study the Mg2+-Na+/K+-ATPase interaction that was proposed to lead to the formation of a stable Mg-enzyme complex during phosphorylation from ATP. Instead of Mg we used Mn, which can replace Mg as essential activator of Na+/K+-ATPase activity. The amounts of steady-state Mn bound to the enzyme were estimated at 0 degree C on the basis of the 54Mn remaining in the effluent after passing the reaction mixture through a cation exchange resin column. As a function of the MnCl2 concentration, the amount of Mn retained by the enzyme in the absence and presence of ATP showed a saturable and a linear component; the slope of the linear component was the same in both instances (0.016 nmol/mg per microM). The ATP-dependent Mn binding could be adjusted to a hyperbolic function with a Km of 0.76 microM. The ratio [ATP-dependent E-Mn]/[E-P] measured at 5 microM MnCl2 and 5 microM ATP was not different from 1.0, both in native (Mn-E2-P) as well as in a chymotrypsin treated enzyme (Mn-E1-P). When the Mn.E-P complex was allowed to react with KCl (E2-P form) or ADP (E1-P form), the enzyme was dephosphorylated and simultaneously lost the strongly bound Mn in such a way that the ratio [ATP-dependent E-Mn]/[E-P] remained 1:1. These results show the existence of strongly bound Mn ions to Na+/K+-ATPase during phosphorylation by ATP. That binding is (i) of high affinity for Mn, (ii) probably on a single site, and (iii) with a stoichiometry Mn-Pi of 1:1.
Campos
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M
División de Biofísica, Instituto de Investigación Médica M. y M. Ferreyra, Córdoba, Argentina.
Beaugé
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L
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003035:Cobalt / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D003035:Cobalt; D008345:Manganese; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase