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Abstract
In the present study we have investigated the conditions for optimal adenylate cyclase (AC) activity in preparations of human myocardial biopsies, with emphasis on both basal enzyme activity and isoproterenol response.
Different preparation procedures (homogenates, membrane particles) of the same biopsy showed no difference in relative response to isoproterenol, although absolute activities, using protein concentration for normalization, showed some variance. The AC-receptor complexes of the preparations were also stable when stored on ice for 3 h, and both basal and stimulated AC activities were constant at a wide range of protein concentrations (2.9-31.9 μg/tube), and throughout 92 min incubation.
The effects of varying Mg2+, guanyl nucleotides (GTP, GMP-P(NH)P), and ATP concentrations on myocardial AC activities were also investigated under both basal conditions as well as after isoproterenol stimulation. The apparent Km, for the substrate (Mg ATP) binding to the AC was approximately 0.1 mmol/l. Isoproterenol stimulated the AC activity by increasing Vmax (41 to 142 pmol/mg protein. min) without any change in the apparent Km Maximal relative activation by isoproterenol was achieved at pH 6.5-7.0. The concentration of isoproterenol causing half maximal AC stimulation was approximately 0.1 μg/ml (2× mmol/l). Half maximal inhibition of isoproterenol (4 μg/ml) stimulated AC activity was obtained by 0.025 μg/ml propranolol (8× 10−5 mmol/l).
The sensitivity and precision of this assay should make it possible to measure AC activity as well as isoproterenol response in very small quantities of myocardial tissue. This could provide method for studying receptor functions of sick hearts by endomyocardial biopsies.