The Analysis of Interactions between Hormone Receptors and Adenylate Cyclase by Target Size Determinations Using Irradiation Inactivation

Abstract

The irradiation inactivation is a method of protein size determination in which a protein molecule being hit will depend upon its size and, accordingly, upon the molecular weight. There is no requirement for the protein to be pure or for the protein to be in solution. All that is required is a measure of the activity of the protein under study. Thus, the method offers the opportunity to examine the size of proteins in intact biological membranes. Where multisubunit enzymes are examined by irradiation inactivation the target size sometimes corresponds to the whole enzyme, and sometimes corresponds to the monomer molecular weight. There are two types of approach to the study of protein-protein interaction by target size analysis. In the first case, the preparation is irradiated in the absence of any effectors or in the basal state. The activity under study, for example, the catalytic activity of adenylate cyclase, is then determined in the usual way, and the target size for the basal state can be calculated. The activity can also be determined in the presence of an activator such as a hormone. In the second type of general approach, the membranes are preincubated with the effector prior to irradiation. In this case, the preincubations should, so far as possible, be identical to the conditions which are normally employed in the assay of the activity which is under study, although minor modifications may be necessary.