Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

Journal Article · Tue Aug 12 00:00:00 EDT 1986 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00364a017· OSTI ID:6847404

Verkman, A S; Ausiello, D A; Jung, C Y; Skorecki, K L

Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

• Research Organization: Univ. of California, San Francisco
• OSTI ID: 6847404
• Journal Information: Biochemistry; (United States), Vol. 16
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
CYCLASES
CHEMICAL ACTIVATION
CHEMICAL RADIATION EFFECTS
ENZYME ACTIVITY
IN VITRO
INACTIVATION
RECEPTORS
SOMATIC CELLS
VASOPRESSIN
ANIMAL CELLS
CHEMISTRY
ENZYMES
HORMONES
LYASES
MEMBRANE PROTEINS
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PITUITARY HORMONES
PROTEINS
RADIATION CHEMISTRY
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560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture