The effect of dimethylsulfoxide on the substrate site of Na+/K(+)-ATPase studied through phosphorylation by inorganic phosphate and ouabain binding.
To obtain further information on the role of H2O at the substrate site of Na+/K(+)-ATPase, we have studied the enzymes reaction with P(i) and ouabain in 40% (v/v) Me2SO (dimethylsulfoxide). When the enzyme (E) was incubated with ouabain (O) for 5 min in a 40% (v/v) Me2SO-medium with 5 mM MgCl2 and 0.5 mM KCl (but no phosphate), ouabain was bound (as EO). Subsequent incubation with P(i) showed that E, but not EO, was rapidly phosphorylated (to EP). Long-time phosphorylation revealed that EO is also phosphorylated by P(i) albeit very slowly (t1/2 about 60 min) and that binding of ouabain to EP also is very slow. The EOP complex is stable, i.e., the t1/2 for the loss of P(i) is >> 60 min in contrast to about 1 min in water. These results in 40% Me2SO are distinctly different from what would be obtained in a watery milieu: ouabain would bind slowly and inefficiently in the absence of P(i), and ouabain would catalyse phosphorylation from P(i) rather than retard it. Equilibrium binding of [3H]ouabain to E and EP in water or 40% Me2SO confirmed these observations: Kdiss in water is 11 microM and 12 nM for EO and EOP, respectively, whereas in Me2SO they are 112 nM and 48 nM. It is suggested that the primary effect of the lowered water activity in 40% Me2SO is a rearrangement of the substrate site so that it also in the absence of P(i) attains a transition state configuration corresponding to the phosphorylated conformation. This would be sensed by the ouabain binding site and lead to high affinity ouabain binding in the absence of P(i).
Fontes
C F
CF
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
Scofano
H M
HM
Barrabin
H
H
Nørby
J G
JG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D010042:Ouabain / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D014867:Water / Q000378:metabolism*
D010710:Phosphates; D014867:Water; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D004121:Dimethyl Sulfoxide