Adenylate cyclase from term human placenta and its regulation

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The activation of placental AC by either Mg2+ or Mn2+, in the presence and absence of NaF, followed sigmoidal saturation kinetics. Mn2+ enhanced maximally the NaF-stimulated Mg2+-dependent AC activity. The apparent Km of Mg2+-dependent AC for ATP was 0.4 mM, with and without NaF addition. GTP and GMP-P(NH)P stimulated the Mg2+-dependent AC in a dose-dependent manner with half-maximal stimulation taking place at concentrations of approximately 2 microM. In the presence of GMP-P(NH)P (10 microM) the kinetics of the AC dependence on Mg2+ ion concentration changed from sigmoidal to hyperbolic. Most of the AC activity (greater than 83 per cent) was associated with the particulate fractions of placental homogenate. For better reproducibility, the AC assay was performed using sonicated particulate fraction preparations; sonication did not alter the response of AC to stimuli to a variety of agents used in these experiments; freezing and thawing, however, obliterated the stimulation by beta-adrenergic agonists. Placental AC activity was inhibited by p-hydroxymercuriphenyl sodium sulphonate in a dose-dependent fashion, and the inhibition was reversed by dithiothreitol. Mg2+-dependent AC was inhibited by 0.5 mM phenylhydrazine (95 per cent). Mg2+-dependent AC activity was responsive to stimulation by epinephrine, without and with GTP addition, with half-maximal stimulation taking place at a concentration of 2 microM. The stimulatory effect of epinephrine was blocked by propranolol in a dose-dependent manner but was not blocked by phentolamine. Oestrone, oestradiol-17 beta, 2-hydroxyoestreone, 2-hydroxyoestradiol-17 beta, dehydroepiandrosterone sulphate, and progesterone, as well as oxytocin, did not alter either the basal or GMP-P(NH)P-stimulated Mg2+-dependent AC activities. Preincubation of 20 000 g particulate fraction with either NaF or GMP-P(NH)P, followed by washing, resulted in preparations that remained stimulated without the requirement of any further additions.