Affinity labeling of the digitalis receptor with p-nitrophenyltriazene-ouabain, a highly specific alkylating agent.
Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitrophenyltriazene (NPT) ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, II, and III) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na+,K+)-ATPase) purified from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NPT-ouabain I to the enzyme, and (iii) by the inhibition of [3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25 degrees C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not significantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as that observed with the purified enzyme. [3H]NPT-ouabain I was also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, [3H]NPT-ouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a Mr = 93,000 is specifically labeled by [3H]NPT-ouabain I.
D000345:Affinity Labels; D000477:Alkylating Agents; D000818:Animals; D004557:Electric Organ / Q000201:enzymology; D004593:Electrophorus; D007700:Kinetics; D010042:Ouabain / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D011955:Receptors, Drug / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014226:Triazenes
C027083:4-nitrophenyltriazene ouabain; D000345:Affinity Labels; D000477:Alkylating Agents; D011955:Receptors, Drug; D014226:Triazenes; C027082:digitalis receptor; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase