J. Biochem, 1998, Vol. 123, No. 1 169-174
© 1998 Japanese Biochemical Society
research-article |
Fluorescein 5'-Isothiocyanate-Modified Na+, K+-ATPase, at Lys-501 of the 
-Chain, Accepts ATP Independent of Pyridoxal 5'-Diphospho-5'-Adenosine Modification at Lys-4801
*Biological Chemistry, Graduate School of Science, Hokkaido University Kita-ku, Sapporo 060
Department of Biochemistry, School of Medicine, Kyorin University Mitaka, Tokyo 181
2To whom correspondence should be addressed. Fax/Tel: +81-11-736-2074, E-mail: ktan{at}hucc.hokudai.ac.jp
The modification of Na+, K+-ATPase with increasing pyridoxal 5'-diphospho-5'-adenosine (AP2PL) concentrations resulted in saturation of the 
0.5 mol AP2PL probe incorporation into the Lys-480/mol catalytic 
-chain and reduced the Na+, K+-ATPase activity to around half without affecting the phosphorylation by acetyl phosphate (AcP), and led to increases in the AP2PL fluorescence caused by ATP and AcP. Further modification with fluorescein 5'-isothiocyanate (FITC) resulted in 0.9 mol FITC probe incorporation into the Lys-501/ mol -chain and reduced the activity to below 5% without affecting the phosphorylation by AcP and these fluorescence increases. The ATP binding capacity of the AP2PL-FITC enzyme was shown to be at least 50% of that of the control enzyme (0.8 mol/mol -chain). This is the first direct demonstration that Na+-bound FITC-modified enzymes accept ATP with an affinity for ATP (K1/2 > 150 µM) reduced by two orders of magnitude. The data also suggest half site reactivity of Lys-480 as to AP2PL and all site reactivity of Lys-501 as to FITC in the catalytic subunits.
1This work was supported in part by Grants for Scientific Research (07558220, 09257201 and 09878131) and for the International Scientific Research Program (08044047) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Japan Private School Promotion Foundation.
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