HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yamasaki, K. Articles by Kanazawa, T. Search for Related Content PubMed PubMed Citation Articles by Yamasaki, K. Articles by Kanazawa, T. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 269, Issue 6, 4129-4134, Feb, 1994

3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP exclusively labels Lys-492 at the active site of the sarcoplasmic reticulum Ca(2+)-ATPase

K Yamasaki, T Daiho and T Kanazawa
Department of Biochemistry, Asahikawa Medical College, Japan.

Sarcoplasmic reticulum vesicles were labeled with 40 microM 3'-O-(5- fluoro-2,4-dinitrophenyl)-ATP (FDNP-ATP) at 25 degrees C and pH 7.0 for 4 h. The Ca(2+)-ATPase was inhibited strongly. The enzyme was almost completely protected either by 20 mM Mg.ATP or by 50 mM acetyl phosphate against this inhibition. Pi gave no protection. There was a linear relationship between the extent of this inhibition and the Mg.ATP-sensitive part of the content of bound FDNP-ATP. Extrapolation showed that the enzyme is completely inhibited by Mg.ATP-sensitive binding of 3.6 nmol of FDNP-ATP/mg of the vesicle protein. This value is in good agreement with the content of the phosphorylation site (3.3 nmol/mg of the vesicle protein) in the vesicles used. These findings indicate that binding of 1 mol of FDNP-ATP per mol of the active sites leads to a complete inhibition of the enzyme. The acetylphosphatase activity and phosphorylation with ATP were also strongly inhibited by this labeling, whereas phosphorylation with Pi was not inhibited. The labeled vesicles were solubilized in SDS, and the Ca(2+)-ATPase was purified by size exclusion high performance liquid chromatography. Mapping the labeled peptides in the tryptic digest by reversed-phase high performance liquid chromatography and sequencing showed that Lys- 492 was exclusively labeled with FDNP-ATP. These results show that Lys- 492 is located in or near the ATP binding site and apart from the phosphorylation site and Pi binding site. Molecular modeling of FDNP- ATP suggests that this Lys-492 residue is situated on the 3'-OH side of the ribose moiety of bound ATP and is close to the alpha-phosphoryl group.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K. Yamasaki, T. Daiho, T. Saino, and T. Kanazawa Modification of Histidine 5 in Sarcoplasmic Reticulum Ca2+-ATPase by Diethyl Pyrocarbonate Causes Strong Inhibition of Formation of the Phosphoenzyme Intermediate from Inorganic Phosphate J. Biol. Chem., December 5, 1997; 272(49): 30627 - 30636. [Abstract] [Full Text] [PDF]

				K. Kimura, H. Suzuki, T. Daiho, K. Yamasaki, and T. Kanazawa Identification of Arginyl Residues Located at the ATP Binding Site of Sarcoplasmic Reticulum Ca2+-ATPase. MODIFICATION WITH 1,2-CYCLOHEXANEDIONE J. Biol. Chem., November 15, 1996; 271(46): 28933 - 28941. [Abstract] [Full Text] [PDF]

				D. B. McIntosh, D. G. Woolley, B. Vilsen, and J. P. Andersen Mutagenesis of Segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of Sarcoplasmic Reticulum Ca2+-ATPase Produces Pumps Defective in ATP Binding J. Biol. Chem., October 18, 1996; 271(42): 25778 - 25789. [Abstract] [Full Text] [PDF]

				H. Suzuki and T. Kanazawa Reduction in Water Activity Greatly Retards the Phosphoryl Transfer from ATP to Enzyme Protein in the Catalytic Cycle of Sarcoplasmic Reticulum Ca[IMAGE]-ATPase J. Biol. Chem., March 8, 1996; 271(10): 5481 - 5486. [Abstract] [Full Text] [PDF]