Fluorescein 5'-isothiocyanate-modified Na+, K+ -ATPase, at Lys-501 of the alpha-chain, accepts ATP independent of pyridoxal 5'-diphospho-5'-adenosine modification at Lys-480.
The modification of Na+,K+-ATPase with increasing pyridoxal 5'-diphospho-5'-adenosine (AP2PL) concentrations resulted in saturation of the approximately 0.5 mol AP2PL probe incorporation into the Lys-480/mol catalytic alpha-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 approximately 0.9 mol FITC probe incorporation into the Lys-501/mol alpha-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 (approximately 0.8 mol/mol alpha-chain). This is the first direct demonstration that Na+-bound FITC-modified enzymes accept ATP with an affinity for ATP (K(1/2) > 150 microM) 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.
Tsuda
T
T
Biological Chemistry, Graduate School of Science, Hokkaido University, Sapporo.
Kaya
S
S
Funatsu
H
H
Hayashi
Y
Y
Taniguchi
K
K
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine