Differential photoaffinity labeling of catalytic subunits of NaK-ATPase with carrier-free 125I-cardiac glycosides

The authors have obtained evidence for structural differences in the cardiac glycoside binding site between the α and α(+) forms of the catalytic subunit of NaK-ATPase, using three closely related photoaffinity derivatives of the cardiotonic steroid, digitoxigenin. [125I]N-(p-azido-m-iodo-o-hydroxybenzoyl)-4-amino-4,6-dideoxy-galactosyl digitoxigenin (IA-GaD), [125I]N-(3-(p-azido-m-iodophenyl)-propionyl)-4-amino-4,6-dideoxy-ga-lactosyl digitoxigenin (AIPP-GaD) and [125I]N-(3-(p-azido-m-iodophenyl)-propionyl)-4-amino-4,6-dideoxy-glucosyl digitoxi-genin (AIPP-GluD) were synthesized. AIPP-GaD and AIPP-GluD are stereoisomers. Eel electroplax and dog kidney NaK-ATPase (α form) and rat brain synaptosomes (rich in α(+) form) were photolabelled and then analyzed by SDS-PAGE and autoradiography. Photolysis with either carrier-free IA-GaD or AIPP-GluD gave ouabain-protectable labelling of NaK-ATPase catalytic subunit from all three tissues. However, photolysis with AIPP-GaD showed protectable labelling of the enzyme from eel and kidney but not from brain. This suggests a structural difference in the α(+) form which results in either an inability to bind AIPP-GaD, or, perhaps more likely, an absence of a photoinsertion site in the correct location in the α(+) form, as compared with the α form. It is of interest that the labelling pattern of the enzyme in the human erythrocyte resembles that of the brain enzyme