Probing the topography of the intramembrane part of Na⁺,K⁺-ATPase by photolabelling with 3-(trifluoromethyl)-3_(m[¹²⁵I]iodophenyl)diazirine Analysis of the hydrophobic domain of the β-subunit

The mutual disposition of the α-helical intramembrane rods in Na⁺,K⁺-ATPase subunits was studied by photolabelling of the membrane-bound enzyme with 3-(trifluoromethyl)-3-(m-[¹²⁵I]iodophenyl)diazirine ([¹²⁵I]TID). Under the chosen conditions for modification, the ratio of label incorporated into the subunits was found to be α/β = 2.2, demonstrating the peripheral location of the β-subunit in the oligomeric membrane complex. The [¹²⁵I]TID-labelled β-subunit was subjected to tryptic hydrolysis and the modified fragment (Thr²⁷-Arg⁷¹) was isolated. The labelled amino acid residues are located predominantly on one side of the helix, which is helpful in unravelling the spatial orientation of the β-subunit relative to the α-subunit.