Couplage de la corticotropine‐β1–24 avec le système adénylate cyclase des adipocytes de rat
Authors: PAIRAULT Jacques 
Published: March 2005

The Coupling of β1‐24‐Corticotropin to the Adenylate‐Cyclase System in Rat Adipocytes.

Evidence for Hormone‐Nucleotides Interaction

The general aim was to define some of the most important parameters involved in the coupling step between the synthetic analog of adrenocorticotropin hormone (β1−24‐corticotropin tetracosa peptide) and the catalytic unit of the adenylate‐cyclase system of fat cells. These studies were performed with a purified plasma membrane fraction from rat adipose tissue. In this regard, some effects of ions, pH, and nucleotides (ATP and GTP) on this hormone‐sensitive system were studied. A simple model based on a random association process of reactants yielded a satisfactory approximation of the kinetic data.

In contrast to results obtained by two other groups, which were analyzed by De Haën [J. Biol. Chem. (1974) 249, 2756–2762], no evidence was found for a regulation of the adenylate‐cyclase activity by the adenosine triphosphate which was not complexed to magnesium. The inhibition observed by an excess of substrate, the ATP‐magnesium complex, was independent of the concentration of Mg2+ and free ATP4−.

Increasing the pH of the incubation medium from 7.0 to 9.6 resulted in a sigmoidalcurve of basal and β1−24‐corticotropin‐stimulated activity without affecting the response to the hormone. In contrast, the adenylate‐cyclase activity, in the presence of fluoride ion, showed an optimum at pH 8.0 and then dramatically decreased at higher pH values. This observation suggested dissociation of the two stimulation processes.

The addition of 1 mM ethylene glycol bis (β‐aminoethylether)‐N,N′‐tetracetic acid to the incubation mixture led to inhibition of the response of adenylate cyclase to ß1−24‐corticotropin. While the activation ratio (the ratio of the activity at stimulated and basal levels) was unchanged in the range of 0.05 – 5 mM Ca2+, the hormonal response was progressively inhibited from 0.1 mM La3+ and disappeared completely at 1 mM La3+. From these data it was apparent that La3+ ion, a Ca2+ antagonist, competed with critical sites of Ca2+ binding required for β1−24‐corticotropin stimulation of the adenylate cyclase.

In the range of substrate concentration which gave a good correlation with Michaelis‐Menten kinetics (0.006–0.3 mM ATP) we calculated the apparent Km values for the substrate; they were 2 × 10−5 M for basal, 2.5 × 10−5 M for fluoride ion‐stimulated and 4 × 10−5 to 6 × 10−5 M for β1‐24‐corticotropin‐stimulated activity. The activators of the adenylate cyclase increased essentially its V.

The measurement of β1−24‐corticotropin‐sensitive adenylate‐cyclase activity as a function of the ATP concentration in the medium showed that ATP increased the activation ratio to a maximum value. The plot of the activation ratio due to hormone versus ATP concentration was simulated in the rate equations derived from the reaction scheme presented; the curve obtained from the experimental data closely approximated the theoretical curve. Therefore, it was concluded that the stimulatory effect of ATP on the adenylate cyclase did not require a hypothetical regulatory site for this nucleotide.

A thermodependent effect of guanosine triphosphate was observed on the response of fat‐cell adenylate cyclase to β1−24‐corticotropin. Moreover, at 37 °C, two independent effects of GTP were observed; they were concentration dependent. At low concentrations, GTP enhanced both the basal and hormone‐stimulated adenylate‐cyclase activity resulting in an enhanced activation ratio.