Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin.
A radioiodinated, photoactive cardiac glycoside derivative, 4'-(3-iodo-4-azidobenzene sulfonyl)cymarin (IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC inhibited the activity of (Na+K+)-ATPase in electroplax microsomes from Electrophorus electricus with the same I50 as cymarin. [125I]IAC binding, in the presence of Mg2+ and Pi, was specific, of high affinity (KD = 0.4 microM), and reversible (k-1 = 0.11 min-1) at 30 degrees C. At 0 degree C, the complex was stable for at least 3 h, thus permitting washing before photolysis. Analysis of [125]IAC photolabeled electroplax microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7-14%) showed that most of the incorporated radioactivity was associated with the alpha (Mr = 98,000) and beta (Mr = 44,000) subunits of the (Na+K+)-ATPase (ratio of alpha to beta labeling = 2.5). A higher molecular weight peptide (100,000), similar in molecular weight to the brain alpha(+) subunit, and two lower molecular weight peptides (12,000-15,000), which may be proteolipid, were also labeled. Two-dimensional gel electrophoresis (isoelectric focusing then SDS-PAGE, 10%) resolved the beta subunit into 12 labeled peptides ranging in pI from 4.3 to 5.5. When (Na+K+)-ATPase in synaptosomes from monkey brain cortex was photolabeled and analyzed by SDS-PAGE (7-14%), specific labeling of the alpha(+), alpha, and beta subunits could be detected (ratio of alpha(+) plus alpha to beta labeling = 35). The results show that [125I]IAC is a sensitive probe of the cardiac glycoside binding site of (Na+K+)-ATPase and can be used to detect the presence of the alpha(+) subunit in crude membrane fractions from various sources.
D000345:Affinity Labels / Q000037:antagonists & inhibitors*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000494:pharmacology; D003529:Cymarine / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000494:pharmacology; D004557:Electric Organ / Q000378:metabolism; D004593:Electrophorus; D007425:Intracellular Membranes / Q000201:enzymology; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010782:Photolysis; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013328:Strophanthins / Q000138:chemical synthesis*
D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D013328:Strophanthins; C042526:4'-(3-iodo-4-azidobenzene sulfonyl)cymarin; D000254:Sodium-Potassium-Exchanging ATPase; D003529:Cymarine