Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase.
With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na+ + K+)-ATPase, a number of properties of that reaction differ from those with the common substrate p-nitrophenyl phosphate (NPP): the Km is 2 orders of magnitude less and the Vmax is two times greater, and dimethyl sulfoxide (Me2SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both the Km and Vmax for K+-activated 3-OMFP hydrolysis as well as the K0.5 for K+ activation. However, reducing the incubation pH increases inhibition by Pi and the Vmax for 3-OMFP hydrolysis in the absence of K+. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K+-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5-30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K+, however, NaCl stimulates increasingly over the range 5-100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na+, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na+ + K+)-phosphatase pathway, most apparent at suboptimal K+ concentrations, that is also more readily available to 3-OMFP.
Davis
R L
RL
Department of Pharmacology, SUNY Health Science Center, Syracuse 13210.
Robinson
J D
JD
D000818:Animals; D002412:Cations; D004285:Dogs; D005452:Fluoresceins / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
C032079:3-O-methylfluorescein phosphate; D002412:Cations; D005452:Fluoresceins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000254:Sodium-Potassium-Exchanging ATPase