A pitfall in the interpretation of data on adenylate cyclase inactivation by irradiation

Abstract

St6phane SWlLLENS and Jacques E. DUMONT Institut de Recherche lnterdisciplinaire, School of Medicine, Free University of Brussels, 1000 Brussels, Belgium Received 17 September 1981 1. Introduction Enzyme inactivation by irradiation has been recog- nized as a valuable technique for the determination of enzyme size (review [1]). This approach has been used by some groups for elucidating the dynamic structure of hormone-responsive adenylate cyclase [2-5]. These authors described the organization of the known components of the system (hormone receptor (R), guanine nucleotide binding protein (G), adenylate cyclase enzyme (E)) prior to and subsequent to activation of the system by different effectors as hormones, guanine nucleotides and ions. For example, on the basis of target size analysis, it was proposed [5] that the ground state of the turkey erythrocyte adenylate cyclase system consists of a tightly bound ternary complex RGE which dissociates upon isopro- terenol action and releases an active E unit (pre- coupled model). This description is in complete dis- agreement with the model derived from kinetic exper- iments (review [6]) which suggests that hormone binding to the uncoupled receptor leads to the for- mation of a transient ternary complex HRG necessary for the activation of the G-unit which in turn activates the adenylate cyclase E. The latter model (preuncou- pled model) is conceptually compatible with the cyclic model in [7], the collision coupling model [8], the ternary complex model [9]. In our opinion, it is impossible to reconcile these contradictory concepts as defined in fig.1. Here, we show that the target size analysis applied to the adenylate cyclase system is misleading as this method cannot discriminate between the 2 models at least on the basis of the data reported so far. RGE Precoupled Model H ) Activation of E through dis- sociation of the inactive holo- enzyme Preuncoupled Model R+G+E ) Activation of E through transient association of R and G, and through inter- action between E and active G Fig.1. Definitions of two hypothetical models describing adenylate cyclase activation by hormone: R, hormone recep- tor; G, guanine nucleotide binding protein; E, adenylate cyclase. 2. Rationale The rationale of the target size analysis was pre- viously described (review [1 ]). Briefly, irradiation of a sample leads to the deposition of ionization energy. If ionization occurs inside an enzyme molecule, the enzymatic activity is completely lost. As the ioniza- tion occurs randomly, the larger the enzyme size, the more likely it will be destroyed. The concentration of survival active enzyme [E] obeys a simple exponential law: [E] = [E]o e -vED (I) where D is the radiation dose (rads), [E]o is the enzyme concentration prior to irradiation and #E is a Published by Elsevier/North-Holland Biomedical Press 00145793/81/0000-0000[$02.75 © 1981 Federation of European Biochemical Societies 29