Studies on conformational changes in Na,K-ATPase labeled with 5-iodoacetamidofluorescein.
The rates of individual steps in the reaction cycle of dog kidney Na,K-ATPase labeled with iodoacetamidofluorescein (IAF) were measured using the fluorescence stopped-flow technique. The maximal rate of the fluorescence quenching accompanying ATP hydrolysis at 20 degrees C in the presence of K+ is 66.3 s-1, while the turnover rate in the same conditions is 15.5 s-1. The rate without K+ is slightly lower. Unexpectedly, at very high ionic strength, K+ accelerates the rate 2-fold. The fluorescence change appears to be associated with the E1P----E2P transition. The results are consistent with the classical Albers-Post scheme but do not support recent criticisms that E1P is kinetically incompetent in the presence of Na+ plus K+. As expected, in the absence of ATP the rate of E2(K)----E1Na was very slow (0.2 s-1) but was greatly accelerated by ATP (maximal rate 15.9 s-1) with low affinity (K0.5 = 196 microM). It was concluded that E2(K)----E1 is the slowest step of the cycle, even at nonlimiting ATP concentrations. The rate of E1K----E2(K) for both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme was stimulated by K+ acting with low affinity, but not at all by ATP at 5 microM. Whereas the maximal rate with IAF-enzyme (271 s-1) was similar to previous work, the K+ affinity was significantly higher. Fluorescence signals accompanying hydrolysis of acetyl phosphate with both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme have similar rates, 5.25 s-1 and 4.06 s-1, respectively. A species difference was observed between dog and pig kidney Na,K-ATPase in that both enzymes are labeled with IAF but only in dog enzyme were conformational transitions associated with fluorescence changes. Therefore, the IAF-labeled dog kidney enzyme is the preparation of choice for measuring fluorescence changes accompanying ATP hydrolysis.
Steinberg
M
M
Department of Pharmacology, State University of New York Health Science Center, Syracuse 13210.
Karlish
S J
SJ
D000818:Animals; D004285:Dogs; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011487:Protein Conformation / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; C015296:5-iodoacetamidofluorescein; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate