Fluorescein 5′-isothiocyanate-modified Na+,K+-ATPase, at Lys-501 of the α-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 (AP(2)PL) concentrations resulted in saturation of the similar to 0.5 mol AP(2)PL 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 AP(2)PL fluorescence caused by ATP and AcP. Further modification with fluorescein 5'-isothiocyanate (FITC) resulted in similar to 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 AP(2)PL-FITC enzyme was shown to be at least 50% of that of the control enzyme (similar to 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 mu M) reduced by two orders of magnitude, The data also suggest half site reactivity of Lys-480 as to AP(2)PL and all site reactivity of Lys-501 as to FITC in the catalytic subunits.