Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion-activated adenosine triphosphatase induced by energy transfer

Abstract

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity was synthesized from ouabain and used as a photolabel for the (Na+,K+)-activated ATPase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produce covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was in the large MW .alpha.-chain of the kidney enzyme (MW 93,000). The same specific labeling of the .alpha.-subunit was obtained with a crude microsomal fraction from E. electricus. A mild tryptic fragmentation of the subunit into 2 peptide fragments of MW 58,000 and 41,000, respectively, shows that the digitalis receptor is located in the N-terminal 41,000 fragment.