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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033710-03
Application #
3283641
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-08-01
Project End
1987-07-31
Budget Start
1986-08-01
Budget End
1987-07-31
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Hebrew University of Jerusalem
Department
Type
DUNS #
600044978
City
Jerusalem
State
Country
Israel
Zip Code
91904
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