Adenyl cyclase activity was measured in gently homogenised fat cells using 3H-ATP as substrate, and in intact fat cells prelabelled with 3H-adenine. Km for ATP of the homogenised preparation was 3–5 × 10–4 m and the preparation showed a requirement for 5 mM Mg++ for maximum activation. The accumulation of cyclic AMP was increased in the presence of theophylline but very high concentrations of this compound inhibited the formation of cyclic AMP by adenyl cyclase in homogenised or intact fat cells. Cells incubated with 3H-adenine for 15 min converted 60 % of the adenine taken up to ATP and most of the remaining radioactivity was associated with ADP and AMP. Incubation of labelled cells with norepinephrine and theophylline caused the accumulation of cyclic AMP. The initial rate of accumulation proportional to the catecholamine concentration reached a maximum rate at around 3 × 10–7m, a concentration of norepinephrine known to produce almost maximum rates of lipolysis and maximum cyclic AMP formation in unlabelled cells. The data suggest that the accumulation of 3H-cyclic AMP in cells prelabelled with adenine is remarkably similar to the accumulation of cyclic AMP in unlabelled cells and may, with care, be used as a model for the formation of cyclic AMP within the cell.
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Schönhöfer et al. (2008) studied this question.