Determination of the cellular location of cyclic nucleotide binding sites using 8-azidoadenosine-3′,5′-monophosphate, a photoaffinity probe

Abstract

A photoaffinity analog of cyclic AMP, 8-azidoadenosine-3′,5′-monophosphate (8-N 3cAMP), and a linear gradient sodium dodecyl sulfate-polyacrylamide gel system have been used to photolabel and separate possible cyclic AMP binding sites in whole cells of the sarcoma 37 line. At least four sites within the whole sarcoma 37 cell bind [ 32P]8-N 3cAMP such that they are saturated at 1.0 μ m concentration. These four binding moieties appear to have measurably different affinities for 8-N 3cAMP and the number of total sites labeled per cell differs with each binding moiety. Cell fractionation allows separation of some of the photolabeled sites and gives an indication as to the geographical location of these binding sites within the whole cell. Enrichment by cell fractionation allows additional photolabeled sites to be observed.