Postsynthetic stabilized modification of adenylate cyclase by metabolites.

Membrane preparations of adenylate cyclase of cat cerebral particles (Klainer et al., 1962) and those of rat cerebral-cortex particles (Drummond et al., 1971) have been reported to be susceptible to stimulation by adrenaline. However, considerable variations in the efforts of catecholamines were also reported (Klainer et al., 1962). This suggested the possibility that environmental effects were producing stabilized modifications of adenylate cyclase with the potential to alter the normal thresholds of adenylate cyclase response to hormones. Particulate preparations of adenylate cyclase from rat brain cerebral cortex were obtained from male Sprague-Dawley rats (weighing about 300g) which were stunned and decapitated at 4°C. Mgz++-treated particles were prepared as follows: the cerebral cortex of each brain was sliced and collected under the cover of 3 ml of a 2 m ~ Tricine*Tris-1 mM-MgSO, buffer, pH7.4. The tissue was weighed and then homogenized in a glass motor-driven homogenizer at low speed for 2min. The homogenates were centrifuged in a cold-room (4°C) at 1700revJmin for 20min in head 809 of a CL International centrifuge. The pellet was washed twice and finally suspended in 3 ml of the same homogenizing medium and stored at -70°C. After thawing for 1 h at room temperature, portions of the particulate preparations were incubated under the conditions described under Fig. 1 for the assay of their adenylate cyclase activity. The cyclic AMP formed by adenylate cyclase was measured by the Gilman (1970) method with the use of the saturation assay procedure of Brown et al. (1972). Fig. 1 shows theeffect of noradrenaline on the activity of adenylatecyclaseon theMgZ+treated particles. Noradrenaline-dependent stimulation of adenylate cyclase was maximal in the range 0.025-0.25 m~ substrate concentrations. The data were replotted in terms of the Hill equation and the concentration of ATP for half-maximal velocity (K,,,) was found to decrease from 0 . 2 m ~ , in the absence of noradrenaline, to 0 . 0 7 m ~ in the presence of 1 mwnoradrenaline. Hence, catecholamines in high concentration exert a modifying force on the membrane that changes adenylate cyclase from a high-K,,, form to a low-K,,, form. On the other hand, high concentrations of noradrenaline are found in the urine of acute psychotic patients (Bunney, 1968), which appears to indicate that under pathophysiological stress the adrenals or the central nervous system may respectively allow for the persistence for long periods of time of increased amounts of catecholamines in the blood or in the synapse. It has been proposed (Bennum, 1974a) that high concentrations of catecholamines persisting for long periods of time would cause disruption of the chelated structures which allow the activation and regulation of enzymes within a membrane (Bennun, 1971, 19746). Studies on the stability of adenylate cylase should therefore indicate whether the properties of the enzyme can be altered by its prolonged exposure to hormones and/or metabolites. In order to discover if stabilized conformations could be obtained experimentally, adenylate cylase preparations were pre-incubated under the conditions described in Table 1. The specific activities without noradrenaline are reported in percentage of the preincubated control as noradrenalineindependent activity, which indicates inactivating or activating effects. The noradrenaline-dependent effect, indicating sensitivity to the hormone, was calculated by expressing activity with 1 m-noradrenaline as a percentage of that in its absence. Table 1 shows that preincubation of Mp3+-treated particles with ATP-Mgactivated the adenylate cyclase, but also desensitized the enzyme to its stimulation by noradrenaline. Preincubation with noradrenalinein activated the enzyme. However, if noradrenaline and ATP-Mg were jointly present during preincubation, only a 10% decrease in noradrena-