Photoaffinity labeling of the digitalis receptor in the (sodium + potassium) -activated adenosinetriphosphatase

Rogers, T.B.; Lazdunski, M.

Biochemistry 18(1): 135-140


ISSN/ISBN: 0006-2960
PMID: 217402
DOI: 10.1021/bi00568a021
Accession: 068524432

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Two photoaffinity labels were synthesized from ouabain and strophanthidin. The photosensitive derivatives were formed through the reductive amination of N-(2-nitro-4-azidophenyl)ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain (NAP-ouabain) and the C-19 aldehyde side chain of strophanthicin (NAP-strophanthidin). The binding of these photoaffinity labels to the digitalis binding sites was followed in the dark by 2 methods: the ability of the NAP-ouabain and NAP-strophanthidin to inhibit the enzyme activity of the (Na + K)-activated ATPase purified from the tissue of the Electrophorus electricus electric organ, the inhibition of [3H] ouabain binding to a microsomal fraction of the same tissue. The results of photoaffinity labeling experiments with the purified (Na + K)-activated ATPase indicate that only the large MW protein (MW = 93,000) of the enzyme is labeled with either [3H]NAP-ouabain or [3H]NAP-strophanthidin. Therefore the large chain contains both the sugar and the steroid binding sites of the digitalis binding center of this enzyme. When either photoaffinity label was incubated with a membrane preparation from the electric organ and the solution irradiated with UV light, sodium dodecyl sulfate gel electrophoresis of the membrane proteins indicated that the large chain of the (Na + K)-activated ATPase was the major protein labeled. About 40% of the total radioactivity incorporated was found in this band in the gel. As with the purified enzyme preparation, the small chain (MW = 47,000) was not labeled significantly. There were 2 other proteins in the gels which were labeled in these experiments with MW of approximately 45,000 and 50,000. Protection experiments with 1.0 mM ouabain added to the solution indicated that this labeling was a specific affinity process. These results suggest there may be several proteins involved in the binding of digitalis in 1 or more specific sites on the membrane.