Photoaffinity Labeling with Cyclic Nucleotides

Ross S Antonoff,James J Ferguson

doi:10.1016/s0021-9258(19)42677-x

Ross S Antonoff, James J Ferguson

Open Access

https://doi.org/10.1016/s0021-9258(19)42677-x

Copy DOI

Abstract

Abstract Upon irradiation with ultraviolet light radioactivity from labeled cyclic adenosine 3' : 5'-monophosphate was incorporated into high affinity macromolecular receptors in extracts of testis and adrenal cortex. The bond formed was stable in both 10 % trichloroacetic acid and 1 % sodium dodecyl sulfate, and is consequently thought to be covalent. This incorporation was linearly proportional to the amount of cyclic adenosine 3' : 5'-monophosphate bound by these extracts. Several proteins lacking the ability to bind the cyclic nucleotide did not incorporate significant radioactivity upon irradiation. Incorporation was inhibited by unlabeled cyclic adenosine 3' : 5'-monophosphate and by analogues which compete for its binding sites. Radioactivity from tritiated cyclic guanosine 3' : 5'-monophosphate was similarly incorporated into testicular extracts upon irradiation. These observations indicate that covalent labeling of receptors for cyclic nucleotides can be accomplished by ultraviolet irradiation in the presence of the unmodified ligand.

Highlights

Upon irradiation with ultraviolet light radioactivity from labeled cyclic adenosine 3’: 5’-monophosphate was incorporated into high affinity macromolecular receptors in extracts of testis and adrenal cortex
When [%]cAhIP was added to an extract of lamb testis and esposed to ultraviolet light, significant radioactivity became precipitable with trichloroacetic acid (Fig. 1)
The above results demonstrate that radioactive CAMP can be covalently incorporated into a macromolecule as the result of a photochemical reaction

Summary

Introduction

Upon irradiation with ultraviolet light radioactivity from labeled cyclic adenosine 3’: 5’-monophosphate was incorporated into high affinity macromolecular receptors in extracts of testis and adrenal cortex. The bond formed was stable in both 10% trichloroacetic acid and 1% sodium dodecyl sulfate, and is thought to be covalent This incorporation was linearly proportional to the amount of cyclic adenosine 3’:5’-monophosphate bound by these extracts. Radioactivity from tritiated cyclic guanosine 3’: 5’-monophosphate was incorporated into testicular extracts upon irradiation These observations indicate that covalent labeling of receptors for cyclic nucleotides can be accomplished by ultraviolet irradiation in the presence of the unmodified ligand. The molecule to be attached to the receptor site contains a functional group which, upon irradiation with light, yields a reactive intermediate capable of forming a covalent bond with molecular suhstituents in its immediate vicinity This method requires that a natural ligand be modified chemically by the attachment of a group that can be activated by irradiation (2). WC here &scribe experiments which indicate that cX~II’,’ without prior modification, can form covalent bonds with receptors in tissue cstracts upon csposure to ultraviolet light. c:\111’ can serve directly as an affinity label for macromolecules which bind it and presumably mediate its physiological effects (4). cGI\IP can be stably bound to tissue proteins by ultraviolet irradiation, National Institutes of Health and Grant GI 324985 from the National Science I~ountl:~tion

Methods

Results

Conclusion