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. 1999 Aug;77(2):934–942. doi: 10.1016/S0006-3495(99)76944-4

Rate determination in phosphorylation of shark rectal Na,K-ATPase by ATP: temperature sensitivity and effects of ADP.

F Cornelius 1
PMCID: PMC1300384  PMID: 10423438

Abstract

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na(+) was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E(2)-P, which follows formation of E(1)-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) approximately 90 s(-1) and a minor slow phase with a k(obs) of approximately 9 s(-1) (20 degrees C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of approximately 3.1 x 10(7) M(-1) and 1. 8 x 10(5) M(-1), respectively. Increasing [ADP] decreased k(obs) for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E(1)-P followed by a conformational change to E(2)-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K(+) the highly fluorescent E(2)-P is slowly transformed into the "K(+)-insensitive" E'(2)-P subconformation forming a side branch of the main cycle.

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Selected References

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  1. Albers R. W. Biochemical aspects of active transport. Annu Rev Biochem. 1967;36:727–756. doi: 10.1146/annurev.bi.36.070167.003455. [DOI] [PubMed] [Google Scholar]
  2. Bühler R., Stürmer W., Apell H. J., Läuger P. Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements. J Membr Biol. 1991 Apr;121(2):141–161. doi: 10.1007/BF01870529. [DOI] [PubMed] [Google Scholar]
  3. Campos M., Beaugé L. Na(+)-ATPase activity of Na(+),K(+)-ATPase. Reactivity of the E2 form during Na(+)-ATPase turnover. J Biol Chem. 1994 Jul 8;269(27):18028–18036. [PubMed] [Google Scholar]
  4. Clarke R. J., Kane D. J., Apell H. J., Roudna M., Bamberg E. Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase. Biophys J. 1998 Sep;75(3):1340–1353. doi: 10.1016/S0006-3495(98)74052-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cornelius F., Fedosova N. U., Klodos I. E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase. Biochemistry. 1998 Nov 24;37(47):16686–16696. doi: 10.1021/bi981571v. [DOI] [PubMed] [Google Scholar]
  6. Cornelius F. Phosphorylation/dephosphorylation of reconstituted shark Na+,K(+)-ATPase: one phosphorylation site per alpha beta protomer. Biochim Biophys Acta. 1995 May 4;1235(2):197–204. doi: 10.1016/0005-2736(95)80005-z. [DOI] [PubMed] [Google Scholar]
  7. Cornelius F., Skou J. C. The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve. Biochim Biophys Acta. 1987 Nov 13;904(2):353–364. doi: 10.1016/0005-2736(87)90385-3. [DOI] [PubMed] [Google Scholar]
  8. Cornelius F., Skou J. C. The sided action of Na+ on reconstituted shark Na+/K+-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. II. Transmembrane allosteric effects on Na+ affinity. Biochim Biophys Acta. 1988 Oct 6;944(2):223–232. doi: 10.1016/0005-2736(88)90435-x. [DOI] [PubMed] [Google Scholar]
  9. Fedosova N. U., Cornelius F., Klodos I. E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate. Biochemistry. 1998 Sep 29;37(39):13634–13642. doi: 10.1021/bi980703h. [DOI] [PubMed] [Google Scholar]
  10. Fedosova N. U., Cornelius F., Klodos I. Fluorescent styryl dyes as probes for Na,K-ATPase reaction mechanism: significance of the charge of the hydrophilic moiety of RH dyes. Biochemistry. 1995 Dec 26;34(51):16806–16814. doi: 10.1021/bi00051a031. [DOI] [PubMed] [Google Scholar]
  11. Frank J., Zouni A., van Hoek A., Visser A. J., Clarke R. J. Interaction of the fluorescent probe RH421 with ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments. Biochim Biophys Acta. 1996 Apr 3;1280(1):51–64. doi: 10.1016/0005-2736(95)00277-4. [DOI] [PubMed] [Google Scholar]
  12. Heyse S., Wuddel I., Apell H. J., Stürmer W. Partial reactions of the Na,K-ATPase: determination of rate constants. J Gen Physiol. 1994 Aug;104(2):197–240. doi: 10.1085/jgp.104.2.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hobbs A. S., Albers R. W., Froehlich J. P. Potassium-induced changes in phosphorylation and dephosphorylation of (Na+ + K+)-ATPase observed in the transient state. J Biol Chem. 1980 Apr 25;255(8):3395–3402. [PubMed] [Google Scholar]
  14. Kane D. J., Fendler K., Grell E., Bamberg E., Taniguchi K., Froehlich J. P., Clarke R. J. Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase. Biochemistry. 1997 Oct 28;36(43):13406–13420. doi: 10.1021/bi970598w. [DOI] [PubMed] [Google Scholar]
  15. Kane D. J., Grell E., Bamberg E., Clarke R. J. Dephosphorylation kinetics of pig kidney Na+,K+-ATPase. Biochemistry. 1998 Mar 31;37(13):4581–4591. doi: 10.1021/bi972813e. [DOI] [PubMed] [Google Scholar]
  16. Keillor J. W., Jencks W. P. Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer. Biochemistry. 1996 Feb 27;35(8):2750–2753. doi: 10.1021/bi951370g. [DOI] [PubMed] [Google Scholar]
  17. Klodos I., Fedosova N. U., Cornelius F. Fluorescent styryl dyes as probes for Na,K-ATPase reaction. Enzyme source and fluorescence response. Ann N Y Acad Sci. 1997 Nov 3;834:394–396. doi: 10.1111/j.1749-6632.1997.tb52280.x. [DOI] [PubMed] [Google Scholar]
  18. Kuzmic P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem. 1996 Jun 1;237(2):260–273. doi: 10.1006/abio.1996.0238. [DOI] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Mårdh S., Zetterqvist O. Phosphorylation and dephosphorylation reactions of bovine brain (Na+-K+)-stimulated ATP phosphohydrolase studied by a rapid mixing technique. Biochim Biophys Acta. 1974 Jun 18;350(2):473–483. doi: 10.1016/0005-2744(74)90523-3. [DOI] [PubMed] [Google Scholar]
  21. Ottolenghi P. The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochem J. 1975 Oct;151(1):61–66. doi: 10.1042/bj1510061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Post R. L., Kume S., Tobin T., Orcutt B., Sen A. K. Flexibility of an active center in sodium-plus-potassium adenosine triphosphatase. J Gen Physiol. 1969 Jul 1;54(1):306–326. doi: 10.1085/jgp.54.1.306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Post R. L., Toda G., Rogers F. N. Phosphorylation by inorganic phosphate of sodium plus potassium ion transport adenosine triphosphatase. Four reactive states. J Biol Chem. 1975 Jan 25;250(2):691–701. [PubMed] [Google Scholar]
  24. Pratap P. R., Robinson J. D. Rapid kinetic analyses of the Na+/K(+)-ATPase distinguish among different criteria for conformational change. Biochim Biophys Acta. 1993 Sep 5;1151(1):89–98. doi: 10.1016/0005-2736(93)90075-b. [DOI] [PubMed] [Google Scholar]
  25. Skou J. C., Esmann M. Preparation of membrane Na+,K+-ATPase from rectal glands of Squalus acanthias. Methods Enzymol. 1988;156:43–46. doi: 10.1016/0076-6879(88)56006-8. [DOI] [PubMed] [Google Scholar]
  26. Skou J. C., Esmann M. The effects of Na+ and K+ on the conformational transitions of (Na+ + K+)-ATPase. Biochim Biophys Acta. 1983 Jul 28;746(1-2):101–113. doi: 10.1016/0167-4838(83)90016-x. [DOI] [PubMed] [Google Scholar]
  27. Sokolov V. S., Apell H. J., Corrie J. E., Trentham D. R. Fast transient currents in Na,K-ATPase induced by ATP concentration jumps from the P3-[1-(3',5'-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP. Biophys J. 1998 May;74(5):2285–2298. doi: 10.1016/S0006-3495(98)77938-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stürmer W., Bühler R., Apell H. J., Läuger P. Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face. J Membr Biol. 1991 Apr;121(2):163–176. doi: 10.1007/BF01870530. [DOI] [PubMed] [Google Scholar]
  29. Wuddel I., Apell H. J. Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments. Biophys J. 1995 Sep;69(3):909–921. doi: 10.1016/S0006-3495(95)79965-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

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