Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

Abstract

(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTP gamma S as nucleotide activator, cholecystokinin as peptide hormone, and GDP beta S and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (EA/ETOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant kappa+1), and a deactivation process (rate constant kappa off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant kappa 2) and/or the dissociation of the intact nucleotide (rate constant kappa-1), so that EA/ETOT = kappa+1/(kappa+1 + kappa 2 + kappa-1). (2) The hormone CCK-8 increased the value of kappa+1 with GTP dose-dependently, from 0.2 to 10.9 min-1. The value of kappa-1 increased 0.01 to 0.3 min-1 but the value of kappa 2 was unaltered at 7 min-1, so that EA/ETOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 micrograms/ml allowed also a large increase in EA/ETOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the kappa off value of the GTP-activated enzyme (from 7 min-1 to 0.5 min-1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of kappa+1 (from 0.2 to 0.8 min-1) and the occurrence of a significant 0.3 min-1 value for kappa-1.

The differential detergent solubilization of adenylate cyclase and polypeptides ADP‐ribosylated with cholera toxin suggests an excess of G/F protein relative to adenylate cyclase in rat pancreatic plasma membranes

“…In [4] we used the whole erythrocyte cytosol and cholera toxin to activate adenylate cyclase in pancreatic plasma membranes. Using similar conditions with [c~-32P] NAD +, we observed that cytosolic proteins were labelled in addition to plasma membrane proteins (not shown).…”

“…One volume of this membrane suspension was added to an equal volume of the complete cytosol of packed human erythrocytes [4] or of a 'cytosolic factor' (purified as in [18]) and to 3 vol. reaction medium so as to obtain, at final concentration, 25/~M [a.32p].…”

“…The non-ionic detergent G 3707, a lauryl polyoxyethylene with 7 ethylenoxide groups/ molecule was from Atlas Chemical Industries (Everberg). The source of the other chemicals was as in [4].…”

“…Specificity of cholera toxin-dependent labelling and solubilization with G 3707 of ADP-ribosylated polypeptides of rat pancreatic plasma membranes. (A) Plasma membranes from rat pancreas [1,2] or human erythrocytes [3,4] were labelled in the absence [1,3] or presence [2,4] of 20 ug cholera toxin/ml, as in section 2. The medium contained the purified cytosolic factor and was buffered with 50 mM sodium phosphate (pH 7.4).…”

“…Cholate extracted, in addition, some 30 kM r labelled material (not shown). Lubrol PX (Sigma) and Lubrol 12A9 (Imperial Chemical Industries, Brussels), 2 non-ionic detergents, when tested at 1%, gave similar solubilization yields of labelled polypeptides as 1% G 3707; (C) Intoxication of adenylate cyclase with 20 ~g cholera toxin/ml was performed as in [4], except that at 0.1 mM [c~-32p]NAD (0.4 Ci/mmol) was used to obtain rapidly a quantitative intoxication of the enzyme Washed membranes were homogenized at 1 1.5 mg protein/ml in 10 mM Tes-Tris (pH 7.4), 5 mM MgC1 v 1 mM EDTA, 1 mM DTT, 0.25 M sucrose and the indicated concentration of detergent. After incubation for l h at 0°C with occasional stirring, the samples were centrifuged at 5 X 10 t° rad2/s in a 50 Ti rotor.…”