Photoaffinity labeling protection experiments

Several controls related to those required in photoaffinity labeling experiments with radiolabeled ligands (see Section 4.7) must also be performed in studies of photoinactivation. In particular inactivation should not occur on irradiation in the absence of the photoaffinity label, and the receptor site should be protected against photoinactivation if it is first blocked with a photochemically inert molecule. 

If the photoaffinity reagent has been designed well and there are a measurable number of tight binding sites for it, the protection experiment will usually succeed. Occasionally it will not for example, four polypeptides of sarcoma cells were labeled with low concentrations of 8-N3-CAMP but only three of the sites could be protected by cAMP. Presumably, the fourth site is not part of a cAMP binding protein but a different nucleotide binding site that just happens to have affinity for the reagent but not for cAMP. 

Another test for specific labeling is to determine whether the ligand binding site is blocked. But, as 1 pointed out in the discussion of photoinactivation experiments, there are several other possible causes of apparent binding site occupation besides the covalent attachment of a ligand. It has also been noted that specific labeling as defined by a protection experiment may not always yield a blocked receptor when the photoaffinity label is a macromolecule. When sodium channels in tissue culture cells were labeled... 

A final possibility is to label in the presence of a molecule that bears a strong structural resemblance to the reagent but is not photoactivatable. This is analogous to a protection experiment in photoaffinity labeling. 

In unpublished experiments it has been shown that irradiation with each of the two photoaffinity labeling reagents resulted in the irreversible inactivation of peptidyltransferase. The presence of erythromycin in the irradiation mixture with p-azidochloramphenicol protected against inactivation. It thus appears that these analogs react w ith the ribosome at functionally significant sites. The modified ribosomal components in these two cases have not been identified, however. 

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