Biochemical and Biophysical Research Communications
Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the α-chain of pig gastric H+/K+-ATPase
Section snippets
Materials and methods
Peptide preparation. Non-phospho- and phospho-peptides containing the 33 N-terminal amino acids of pig gastric H+/K+-ATPase (Gly2–Gly34) based on the sequence of the pig H+/K+-ATPase α-chain, i.e., N33np (GKAENYELYQVELGPGPSGDMAAKMSKKKAGRG; 2–34), and N33pY10 that had been phosphorylated at Tyr 10 of N33np, were synthesized by Sawady Technology (Tokyo, Japan).
Circular dichroism. All measurements were performed on a Jasco J-725 spectropolarimeter (Jasco, Japan). Spectra were recorded at 25 °C and
Structure determination of the N-terminal fragment of the α-chain of pig gastric H+/K+-ATPase
In order to reveal the conformation of the N-terminal region of the α-chain of pig gastric H+/K+-ATPase, two peptides, N33np and N33pY10, were synthesized. The former contained the N-terminal region of pig gastric H+/K+-ATPase (Gly 2–Gly 34) without the phosphate group at Tyr 10, and the latter contained the same region but with the phosphate group at Tyr 10. Because the phosphorylation of Tyr 10 occurs before the phosphorylation of Tyr 7 via an endogenous Tyr-kinase [6], we designed a
Acknowledgements
We thank Professor Shin-ichi Tate of Japan Advanced Institute of Science and Technology for offering NMR experimental time and their useful discussions. We also thank Dr. Yoshihiro Mori of Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University for supporting CD experiments. This work was supported by Grants-in-Aid for Scientific Research (10308028) and the International Scientific Research Program (10044048) from the Ministry of Education, Science and Culture of Japan,
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