Photoaffinity labeling of a protein kinase from bovine brain with 8-azidoadenosine 3',5'-monophosphate

Abstract

8-Azidoadenosine 3',5'-monophosphate (8-N3-cAMP) containing 32P has been used as a photoaffinity label specific for the adenosine 3',5'-monophosphate (cAMP) binding site(s) present in a partially purified preparation of soluble protein kinase from bovine brain. 8-N3-cAMP and cAMP were found to compete for the same binding site(s) in this preparation, as determined by a standard filter assay. When this protein preparation was equilibrated with [32P]-8-N3-cAMP, and then irradiated at 253.7 nm, the incorporation of radioactivity was predominantly into a protein with an apparent molecular weight of 49,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. This labeled protein comigrated in the gel with the only protein which is endogenously phosphorylated by [gamma-32P]ATP, a protein which has been shown to be the regulatory subunit of the protein kinase (H. Maeno, P. L. Reyes, T. Ueda, S. A. Rudolph, and P. Greengard (1974), Arch. Biochem. Biophys. 164, 551). The incorporation of [32P]-8-N3-cAMP into this protein was half-maximal at a concentration of 7 x 10(-8) M. In accordance with a proposed mechanism involving the formation of a highly reactive nitrene intermediate upon irradiation of the azide, the incorporation of radioactivity into protein was maximal within 10 min of irradiation, and was almost eliminated by preirradiation of the photolabile ligand. Moreover, this incorporation was virtually abolished by a 50-fold excess of cAMP, but not by AMP, ADP, ATP, or adenosine. We suggest that 8-N3-cAMP may prove to be a useful molecular probe of the cAMP-binding site in receptor proteins and report its use in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a highly sensitive and selective radiochemical marker for cAMP-binding proteins.

Differential interaction of glimepiride and glibenclamide with the β-cell sulfonylurea receptor II. Photoaffinity labeling of a 65 kDa protein by [3H]glimepiride

“…However, this does not explain the discrepancy between the higher potency of glimepiride in inhibiting photolabeling and its lower potency in competing equilibrium binding compared to glibenclamide. Besides other factors, the exchange rate of the ligand with the receptor determines the efficiency of photoaffinity labeling and the inhibitory effect of competing ligands (Pomerantz et al [27]). In fact, glimepiride binding to/t-cell membranes is characterized by drastically higher dissociation and association rates compared to glibenclamide.…”

Interaction of the photoaffinity label 8‐azido‐ADP with glutamate dehydrogenase

“…Recently, the synthesis of the 8-azidoadenine analogs of 3',5'-cyclic AMP [8] , .5'-ATP [9 3 , NAD' and FAD [IO] has been reported and some of these nucleotides have been used in photoaffinity labeling experiments [9,11,12]. We, therefore, synthesized 8-azido-ADP as a photoaffinity reagent for glutamate dehydrogenase.…”

An amphitropic cAMP-binding protein in yeast mitochondria. 1. Synergistic control of the intramitochondrial location by calcium and phospholipid

“…Published procedures were used for the determination of the following enzyme activities: succinate:cytochrome c oxidoreductase (Lang et al, 1974), fumarase (Racker, 1950), and cytochrome c peroxidase (Djavadi-Ohaniance et al, 1978). Cytochrome c1 was assayed by immunodecoration with specific antibodies and 14C-labeled protein A (Towbin, 1979;Muller & Bandlow, 1987a) and densitometry of autoradiograms with an LKB Ultrascan XL laser densitometer.…”