Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin.
Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.
Lowndes
J M
JM
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
Hokin-Neaverson
M
M
Ruoho
A E
AE
D000345:Affinity Labels; D001345:Autoradiography; D001386:Azides / Q000097:blood* / Q000138:chemical synthesis; D002855:Chromatography, Thin Layer; D004073:Digitoxigenin / Q000031:analogs & derivatives* / Q000097:blood / Q000138:chemical synthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D010782:Photolysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000097:blood*
D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D004073:Digitoxigenin; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase