Microenvironment of two different extrinsic fluorescence probes in Na+,K+-ATPase changes out of phase during sequential appearance of reaction intermediates.
Na+,K+-ATPase from pig kidney was sequentially modified with N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) at Cys-964 and fluorescein isothiocyanate (FITC) at Lys-501. The resulting preparation showed little Na+,K+-ATPase activity with retention of nearly 90% of phosphorylation capacity from acetyl phosphate. The addition of acetyl phosphate to the preparation induced phosphoenzyme formation with a sequential decrease in the fluorescence intensities in the presence of 2 M NaCl and 4 mM MgCl2; the BIPM fluorescence decreased with a simultaneous increase in the amount of phosphoenzyme; there was a significant delay in a decrease in the FITC fluorescence. The extent of the decrease in the BIPM fluorescence and the increase in the amount of phosphoenzyme both showed monophasic kinetics with a similar dependence on the concentration of acetyl phosphate (K0.5 = 4 mM), while that of FITC fluorescence showed a biphasic decrease (K 0.5 greater than 10 mM). The phosphoenzyme formed was insensitive to ADP but sensitive to acetate (K0.5 = 2 M). These data and those of others (Taniguchi, K., Suzuki, K., Kai, D., Matsuoka, I., Tomita, K., and Iida, S. (1984) J. Biol. Chem. 259, 15228-15233) showed that the extent of the decrease in the BIPM fluorescence reflects an increase in the amount of a precursor of E1P and E1P, irrespective of the FITC treatment. They also suggest the presence of at least two conformationally different E1Ps; one gave little and the other gave a large FITC fluorescence decrease.
Taniguchi
K
K
Department of Pharmacology, School of Dentistry, Hokkaido University, Sapporo, Japan.
Tosa
H
H
Suzuki
K
K
Kamo
Y
Y
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D005456:Fluorescent Dyes; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008301:Maleimides / Q000378:metabolism*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D000085:Acetates; D005456:Fluorescent Dyes; D008301:Maleimides; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D000244:Adenosine Diphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D019342:Acetic Acid