The goal of this research is two-fold: (a) to characterize the chemical structure of the Beta-adrenergic receptor by purifying this receptor's subunit to homogeneity and analyzing its amino acid composition and sequence. Beta-Receptor purification will be achieved by: (i) affinity chromatography which already gave us an essentially purified receptor; (ii) purification of the affinity labeled receptor subunit. For this purpose we have already prepared a 125I-bromoacetyl derivative of cyanopindolol. It is proposed to purify the 125I-affinity labeled Beta-receptor subunit, using anti-cyanopindolol antibodies, HPLC, isoelectric focusing, ion exchange chromatography (HPLC), and gel permeation HPLC. Once pure, the subunit will be analyzed for its amino acid composition and its amino acid sequence; (b) to analyze in detail the kinetic parameters of the coupling between the purified Beta-adrenergic receptor from the turkey erythrocyte and the purified GTP regulatory protein Ns as well as the partially GppNHp activated adenylate cyclase, Ns(GppNHp). C' in center-how do we key? The efficiency of Beta-receptor to Ns coupling will be examined by a number of criteria: 1. the ability to induce GTPase activity in Ns, and to determine its kinetic and catalytic properties; 2. to follow the kinetics of Ns activation by the Beta-receptor in the presence of Beta-agonists and partial agonists; 3. to establish the kinetics of activation and its dependence on the concentration of the reactant: the receptor, Ns, Beta-agonist and GppNHp (or GTPGammaS); 4. to examine dependence of Beta-receptor to Ns interaction on the phospholipid composition and on the fluidity of the lipid milieu; 5. the effect of glycophorin and of spectrin on the kinetics of receptor to Ns interaction will inform us about the influence of non-lipid membrane components on the interaction between the receptor and the other components of adenylate cyclase; 6. to examine the mode of coupling between the Beta-receptor and the partially purified adenylate cyclase. These experiments will enable us to define the reconstitution of the hormone sensitive adenylate cyclase from its components in quantitative terms.
| Almagor, H; Levitzki, A (1990) Analytical determination of receptor-ligand dissociation constants of two populations of receptors from displacement curves. Proc Natl Acad Sci U S A 87:6482-6 |
| Marbach, I; Shiloach, J; Levitzki, A (1988) Gi affects the agonist-binding properties of beta-adrenoceptors in the presence of Gs. Eur J Biochem 172:239-46 |
| Kashles, O; Levitzki, A (1987) Characterization of the beta 2-adrenoceptor-dependent adenylate cyclase of A431 epidermoid carcinoma cells. Biochem Pharmacol 36:1531-8 |
| Feder, D; Im, M J; Pfeuffer, T et al. (1986) The hormonal regulation of adenylate cyclase. Biochem Soc Symp 52:145-51 |
