Adenylate cyclase from term human placenta and its regulation

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 K_m 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 μM. In the presence of GMP-P(NH)P (10 μm) the kinetics of the AC dependence on Mg⁺ ion concentration changed from sigmoidal to hyperbolic. Most of the AC activity (> 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 β-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 μm. The stimulatory effect of epinephrine was blocked by propranolol in a dose-dependent manner but was not blocked by phentolamine. Oestrone, oestradiol-17β, 2-hydroxyoestreone, 2-hydroxyoestradiol-17β, 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.