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-480

Abstract

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 α-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 α-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 α-chain). This is the first direct demonstration that Na⁺-bound FITC-modified enzymes accept ATP with an affinity for ATP (K_1/2 > 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.