An alkylating ATP analogue, y-[4-(N-2-chlorethyl-N-methylamino)]henzylamide ATP (CIRATP), covalently binds to the catalytic a-subunit of Na+,K+-ATPase yielding a product resistant to hydrolysis by the enzyme and inhibiting the ATP-hydrolysing activity. The Naf-form of the membrane-bound Na+,K+-ATPase modified with ClRATP was hydrolysed by pepsin under conditions providing maximum stability of the modification product (4°C pH 1 S). The modified peptide was isolated by HPLC and its amino acid sequence was found to involve residues 706713 of the a-subunit polypeptide chain. This fragment located near the y-phosphate of ATP is a component of the active site. It is highly homologous with corresponding regions of the catalytic subunits of all the known E,-E, ATPases. In the Na+-(or E,-)enzyme form Asp-710 is the target of modification. Evidently E,-and E,-enzymes have different targets in ClRATP modification, i.e. the polypeptide chain regions near the ATP y-phosphate in the enzyme active site differ somewhat in their conformations.
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