Phospholipid and guanine nucleotides sensitive properties of the smooth muscle adenylate cyclase catalytic unit.

The adenylate cyclase catalytic unit was partially purified from uterine smooth muscle by chromatography on columns of SM-2 Bio-Beads and Sepharose 6B. Stimulation of catalysis by forskolin was much greater in the presence of Mn2+ than in the presence of Mg2+. Neither NaF nor guanine nucleotide stimulated catalysis in the presence of Mg2+ or Mn2+.

Phospholipase A2 sensitivity of uterine smooth muscle membrane phospholipids and adenylate cyclase activity. Effect of temperature on the action of phospholipase present in excess.

Basal as well as GTP-dependent adenylate cyclase activity was partially resistant to porcine pancreatic phospholipase A2, although more activity was degraded at 16 than at 2 degrees C. In contrast, isoproterenol-dependent activity was completely destroyed regardless of the temperature. Snake venom phospholipase A2 destroyed approximately 90% of basal and GTP-dependent adenylate cyclase activity at all temperatures.

Adenylate cyclase activation. Characterization of guanyl nucleotide requirements by direct radioligand-binding methods.

Particulate adenylate cyclase preparations from rat uterine smooth muscle had a single class of [3H]guanyl-5'-yl imidodiphosphate ([3H]GMP.P(NH)P)-binding sites with all of the properties of the guanyl nucleotide-requiring enzyme activation sites (N) which couple hormone receptors and catalytic subunits. These sites bound the radioligand in a reversible manner at low temperature (less than 2 degrees C) but irreversibly at temperatures between 6 and 24 degrees C, properties characteristic of the activation of the enzyme by treatment with GMP.

Kinetics and thermodynamics of activation of pretreatment with guanosine 5'-[beta, gamma-imido]triphosphate of smooth-muscle adenylate cyclase.

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2 degrees C. Activation (C-->C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37 degrees C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37 degrees C.