| Co-Investigator(Kenkyū-buntansha) |
SVEN Mardh スウェーデン、リンシェピング大学, 生命科学部, 教授
FROERHIC Jeffrey Maryland University・Professor, 老年学研究センター・兼メリーランド大学・部長, 教授
SCHONER Wilhelm Justus-Liebig University・Professor, 生化学・内分泌学, 教授
KAYA Shunji Hokkaido University school of Dentistry, ・Associate Professor, 大学院理学研究科, 助教授 (90186023)
MARDH Sven Linkoping University・Professor
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| Research Abstract |
The fluorescent probe, (BIPM), was used to monitor Na^+ translocation coupled to the conversion of E1P to EP2 in pig kidney Na, K-ATPase. The addition of 10 muM ATP to BIPM-labeled Na, K-ATPase gave an increase in fluorescence intensity with an apparent rate of 137/s and the phosphorylation with an apparent rate of 119/s. These results show that the BIPM signal and phosphoenzyme formation have similar kinetics and that, consequently, the conversion of E1P to E2P is very fast. At physiological[ATP], Na^+ release from E2P is rate-limited by phosphorylation and/or the conformational transitions involving Na^+ deocclusion.
When pig stomach membrane H^+, K^+-ATPase preparations were incubated with[gamma-^<32>P]ATP,Mg^<2+> and Ca^<2+>, ^<32>P were incorporated into Tyr and Ser residues in the alphachain of H^+, K^+-ATPase. The radioactivity incorporated was shown to turn over. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatmentifollwed by a reverse-phase column chromatography gave th
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ree radio active peptide peaks. The first and the second peaks were assigned, respectively, to be the same amino-terminal phospho peptides, containing boty Tyr^<10>(^<32>P) and Tyr^7(^<32>P) and Tyr^<10>(^<32>P), both of which had been also obtained by the trypsin treatment of the preparations incubated with[gamma-^<32>P]ATP,Mg^<2+> and vanadate (Togawa K., Ishiguro T., Kaya S., Shimada A., Imagawa T., and Taniguchi K.(1995)J.Biol. Chem. 270,15475-15478). The third peak was also obtained after the trypsin treatement of partially purified H^+, K^+-ATPase preparations incubated with[gamma-^<32>P]ATP,Mg^<2+> and protein kinase-C + Ca^<2+> or protein kinase-A.Addition of endoproteinase ASP-N to each third peak fraction gave a major ^<32>P peptide peak as shown to be Asp^<21>-Met-Ala-Lys-Met-Ser(^<32>P)-Lys-Lys-Lys^<30> in the alpha-chain. These data and others indicate that amino-terminal domain of the alpha-chain of H^+, K^+-ATPase containing Tyr^7, Tyr^<10> and Ser^<27> is a hot spot for protein kinases dependent phosphorylation. The data also suggest participation of Ca^<2+> and cAMP in these phosphorylation reactions. Less
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