Photoinactivation experiments

Such an approach to the preliminary evaluation of photoaffinity reagents goes one step beyond the reversible binding experiments discussed earlier. It is a useful approach in that, like competitive binding experiments, radiolabeled ligand is not required and therefore many potential reagents may be screened. However, many pitfalls severely reduce its utility. 

The not infrequent failure of the extent of attachment of photoaffinity labels to parallel receptor inactivation can be explained in several ways. First, in some membrane-bound receptor systems, receptor subunits are present in excess over the other components of the system. Second, the ligand might induce or take part in a photochemical reaction at the ligand binding site that does not culminate in covalent attachment. Ligand sensitized photooxidation can be prevented by irradiating the sample in the 

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. 

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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... 

Experiments were carried out which showed that the ratio of TT/UT (4 8) observed in the acid hydrolysate of a DNA was nearly the same as the ratio (5 2) found in the products of the more gentle enzymatic hydrolysis. Other experiments showed that the photoreactivating enzyme could excise all three dimers equally readily, that all three dimers were implicated in the photoinactivation of primer DNA, and that CT dimers were excised from the DNA of radiation-resistant bacteria. 

Having obtained a rough estimate of the irradiation time, the time dependence of labeling of the receptor should be measured directly. The optimal photolysis time (e.g. Fig. 4.2), determined by the incorporation of label or by photoinactivation (see below), will be used in many labeling and control experiments, and it is important that it be reproducible. For the results to be useful the sample must be irradiated at a fixed point relative to the lamp. For example, if a long arc is used, the intensity varies both with the distance from the center of the lamp and with the position along the... 

Figure 2. Photoinactivation of ADPGlc-synthetase at 1.5 mM 8-N3ADPGIC. The mixture containing 1 yM (subunits) enzyme, 10 mM HEPES (pH 7.5), 1 mM 3-PGA, 5 mM MgCl2, 1.5 mM 8-N3ADPGIC and indicated nucleotide, was irradiated in the wells of Coor 1 s plate with UVS-51 lamp, at a distance of 5 cm as previously described (1 1 ). The time of irradiation in experiment A was 3 min and in B it was varied as indicated. A - Effect of the varied concentrations of ADPGlc on photoinactivation. B - Effect of 10 mM ADPGlc (A), ADP (o) and UDPGlc ( ) on the rate of photoinactivation. (x) is a...

In the Ile-tRNA synthetase-tRNA system, addition of tRNA after reaching the plateau results in no further cross-linking however, addition of Ile-tRNA synthetase gives an increase in the percent of tRNA cross-linked. This experiment clearly suggests that the plateau in cross-linking is due to competing photoinactivation processes that prevent many... 

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