Photoaffinity Labeling of Cyclic AMP-Dependent and Cyclic GMP-Dependent Protein Kinases
Photoaffinity labeling is a useful technique for the study of ligand-receptor interactions. Irradiation of chemically modified ligands with light produces highly reactive intermediates which are capable of forming covalent bonds with the receptors. Photoaffinity labeling has been used for many different ligands and has been especially successful for cyclic nucleotides. Several photoaffinity analogues of cAMP and cGMP are available. 8-Azido-cAMP (8-N3-cAMP) has been the one most widely and successfully used. 8-N3-cAMP binds to the regulatory subunit of a Type II cAMP-dependent protein kinase with a slightly lower affinity than cAMP and is able to label all cAMPbinding sites under saturating conditions. The data indicate that 8-N3-cAMP is an effective analogue of cAMP and a very efficient photoaffinity label for cAMP-binding sites. Regulatory subunits of Type I and Type II cAMP-dependent protein kinases are the only proteins present in all tissues studied which are specifically labeled by 8-N3-cAMP. Other minor labeled proteins appear to be proteolytic fragments of the two types of regulatory subunits. The finding that most, if not all, specific cAMP-receptor proteins are regulatory subunits of cAMP-dependent protein kinase strongly supports the hypothesis that cAMP-dependent protein kinases and their subunits mediate all the physiological effects of cAMP. Photoaffinity labeling has been used to detect and characterize proteolytic fragments of cAMP-dependent protein kinases and to study the regulation of the amounts of cAMP-dependent protein kinases and their subunits. Similarly, photoaffinity analogues of cGMP have been used to label cGMP-dependent protein kinases. Photoaffinity analogues of cyclic nucleotides are powerful tools for the investigation of cAMP- and cGMP-receptor proteins. Studies with these analogues should contribute substantially to an understanding of the mechanism of action of cyclic nucleotides as second messengers.
KeywordsEstrogen Lysine Trypsin Prostaglandin Thiol
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