Adenylate cyclase of human articular chondrocytes. Responsiveness to prostaglandins and other hormones.

Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1.     

Stimulation of hormone-responsive adenylate cyclase activity by a factor present in the cell cytosol.

1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E.     

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.     

Stimulation of rat platelet adenylate cyclase by an endogenous calcium-dependent protease-like activity

The stimulation of adenylate cyclase by various exogenous proteases has been described in several tissues. In this study, we describe a 2 to 7-fold increase of adenylate cyclase activity in a particulate preparation from rat platelets following prior exposure of the homogenate to calcium. Calmodulin alone was unable to increase the adenylate cyclase activity and trifluoperazine only partially inhibited the calcium-dependent activation. On the other hand, calcium had a slight stimulatory effect on the particulate preparation but this activation was greatly enhanced by the addition of supernatant. Only the combined addition of calcium, supernatant and calmodulin to washed particulate preparations reconstituted the activation seen in homogenates. The activation was significantly inhibited by leupeptin and thiol reagents. It is concluded that platelets contain a calcium-dependent protease-like activity that is able to increase adenylate cyclase activity in membrane fractions. This phenomenon may be involved in the regulation of adenylate cyclase activity in platelets.     

Endogenous GTP and the regulation of epinephrine stimulation of adenylate cyclase.

Epinephrine increased adenylate cyclase activity 10 to 15 fold in lysates of the cultured human astrocytoma cell line 132-1N1. GTP had little effect on adenylate cyclase activity of lysed cell preparations either with or without added epinephrine. However, the epinephrine stimulation of adenylate cyclase was essentially lost (less than 90%) when a washed nuclei-free membrane preparation of the cyclase was assayed. A 10 to 15 fold epinephrine stimulation of the membrane adenylate cyclase could be demonstrated if cytosol of GTP were added to the assay with the hormone. The criteria of anion exchange, cation exchange, gel exclusion and paper chromatography indicated that the cytosolic agents which acted synergistically with hormones were GTP and GDP. The apparent Kact's for the synergistic action of GDP and GTP were essentially identical (1.0 muM) and of all the other nucleotides examined only GDP had a potency similar to GTP. However, the effect of GDP was apparently due to its rapid conversion to GTP even in the absence of a regenerating system. With epinephrine pretreatment of the intact 132-1N1 cells there was a specific loss of epinephrine stimulation of adenylate cyclase activity. The hormone pretreatment did not alter the capacity of the cytosol from these desensitized cells to potentiate epinephrine stimulation of the cyclase. Rather, the alteration was in the particulate fraction of the lysate. The desensitization of the membranous cyclase was stable and not reversed by GTP.     

Mn2+-sensitive, soluble adenylate cyclase in rat testis. Differentiation from other testicular nucleotide cyclases.

In subcellular fractions prepared from homogenate of adult rat testis adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity was found in the particulate, primarily 600 X g for 10 min, fractions, as well as in the cytosol. The properties of the adenylate cyclase in the cytosol differs substantially from the adenylate cyclase system associated with the 600 X g for 10 min particulate fraction. The cytosol enzyme, in contrast to the particulate adenylate cyclase, was found to be fluoride- and gonadotropin hormone-insensitive. The cytosol adenylate cyclase appears to be located in the germ cell while the particulate enzyme system in the non-germ cell component of the seminiferous tubules, The cytosol adenylate cyclase was found to be distinct also from the guanylate cyclase present in the rat testis cytosol. The adenylate cyclase appears to be located in the germ cell component while the guanylate cyclase, in the non-germ cell tubular component. Furthermore, it was found that the cytosol guanylate cyclase develops at an earlier stage of spermatogenesis, and precedes the development of the cytosol adenylate cyclase.     

Effects of noradrenaline on the activation and the stability of brain adenylate cyclase.

At constant 1 mM-ATP, the Mg2+-saturation curves for adenylate cyclase (EC 4.6.1.1) particulate preparations obtained from corpus striatum and cortex tissues of rat brain show that addition of 0.1 mM-noradrenaline increases the apparent Vmax. for Mg2+ by 300% in corpus striatum particles, and by 280% in cortex particles. At 10 mM-MgCl2, the addition of 0.1 mM-noradrenaline increased by 800% the adenylate cyclase activity of corpus striatum particles. At all Mg2+ concentrations, the addition of 0.3 mM-CaCl2 suppressed the noradrenaline-induced stimulation of adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase activity of cortex particles. The addition of noradrenaline during a 3 h preincubation of particle preparations of brain cortex at 38 degrees C decreased by more than 4-fold the half-life of the decay of adenylate cyclase activity. The addition of MgATP protected against noradrenaline-induced inactivation.     

Calcium and calmodulin in the regulation of human thyroid adenylate cyclase activity.

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.     

Concanavalin A binding to brain particulate preparations and its effect on adenylate cyclase.

[³H] -Concanavalin A binding to brain particulate preparations measured by a filtration technique was found to show a characteristic regional specificity with the caudate-putamen area exhibiting the greatest density of concanavalin A (con A) binding sites. The synaptic membranes were shown to be the most highly enriched of the subcellular fractions examined in terms of lectin-binding glycoproteins. Con A was also shown to inhibit the basal adenylate cyclase activity of cerebral, cerebellar, and caudate-putamen particulate preparations in a concentration-dependent manner. This lectin sensitivity of the adenylate cyclase is apparently an intrinsic property of the enzyme complex since a detergent dispersed preparation of the cerebral cortical enzyme was equally inhibited by con A. It is proposed that one of the membrane con A binding sites in brain tissue is a component of the adenylate cyclase system.     

Properties of prostaglandin-sensitive adenylate cyclase system of a murine macrophage-like cell line (P388D1)

The properties of basal and prostaglandin (PG)-stimulated adenylate cyclase of membrane preparations of P388D1 cells were investigated. Three partially purified membrane fractions were obtained by sucrose density gradient centrifugation at the final step of purification from crude homogenate. About 96% of the basal and 89% of PGE2-stimulated adenylate cyclase activity in the homogenate were recovered in three membrane fractions. Two lighter membrane fractions (I and II), which were enriched 11-fold and 8.4-fold in adenylate cyclase activity over crude homogenate, were pooled and subjected to various studies. Results suggested that the basal activity of the membrane preparations has, as in many other cell types, a relatively broad pH optimum (pH 7.5 to 8.5), requires Mg2+, which must be present in excess ATP, and is inhibited by Ca2+. Highly reactive sulfhydryl group(s), which may be present in the lipid bilayer, is required for the adenylate cyclase activity. Because both fluoride ions and GTP augment the enzymatic activity, P388D1 cell membrane adenylate cyclase must possess stimulatory guanine nucleotide-binding protein. The membrane preparations respond to exogeneously added PG by 1.5-fold to 3-fold increase in adenosine 3'-5' cyclic monophosphate (cAMP) production. The magnitude of PG-responsiveness was dependent on the types of PG and the order of potency in stimulation was PGE1 greater than PGE2 greater than PGI2. PGA1, B1, B2, F1 alpha, and F2 alpha stimulated adenylate cyclase only at the highest concentration tested.     

DEVELOPMENTAL AND REGIONAL VARIATIONS IN NEUROTRANSMITTER-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN CELL-FREE PREPARATIONS FROM RAT BRAIN

Investigations have been carried out on regional and developmental variations in the properties of adenylate cyclase systems in participate preparations from rat brain. EGTA was routinely included in the assay medium to minimize differences in the state of activation of these systems resulting from variations in their exposure to endogenous Ca²⁺. At birth, adenylate cyclase activity was much higher in the hindbrain-medullary preparations than in comparable fractions from cerebellum, cerebral cortex or subcortex (including midbrain, corpus striatum, hypothalamus and hippocampus). Adenylate cyclase activity increased during early development in preparations from all areas of the brain. Maximal levels were reached at 14 days of age or later. These levels were not greatly altered in the young adult animal, except in the hindbrain-medullary area, where a decrease in activity was observed. Adenylate cyclase systems in cerebral cortical and subcortical preparations were activated by norepinephrine and dopamine throughout development. Serotonin also stimulated adenylate cyclase activity in these preparations from young animals but was much less effective in comparable fractions from adult rats. The response to dopamine was diminished with age in cerebral cortical preparations, but not in subcortical fractions. The responses to norepinephrine increased in both brain regions during early development. Adenylate cyclase systems in particulate preparations from the cerebellum and hindbrain-medullary areas exhibited relatively poor responses to the biogenic amines. Detailed studies of the properties of the cerebral cortical adenylate cyclase systems revealed enhancement of activity by Ca²⁺ and F^? at all stages of development with the maximal activation at 2–3 weeks of age. The results suggest that developmental differences in hormonal sensitivity of adenylate cyclase systems from diverse areas of the brain are related to changes in the proportions of the receptor-enzyme complexes responsive to the different biogenic amines.     

Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.     

Partial purification from rat and pig liver of cytosolic stimulators of hormone-sensitive adenylate cyclase: quantitation and resolution of two components

Procedures were carried out to isolate from liver cytosol the protein activators of hormone-sensitive adenylate cyclase. A method for quantifying amounts of activator protein was used to monitor recovery after each isolation step. The activator proteins were precipitable by ammonium sulfate (30-60% saturation) and partially recoverable from the precipitate. On gel filtration of cytosol, stimulatory activity for glucagon-sensitive adenylate cyclase was recovered in two peaks representing proteins with molecular weights of 49,000 and 25,500. Exposure to GTP-Sepharose reduced liver cytosol's content of stimulatory factors for glucagon-sensitive adenylate cyclase by up to 70%. However, soluble protein adenylate cyclase activators distinct from GTP could not be subsequently eluted from the affinity matrix. Purification efforts were thwarted by factor instability and large losses during simple and conventional steps despite the use of a variety of protein stabilizers and protease inhibitors. If the problem of stimulator instability can be overcome, large-scale purification should be possible using pig liver as a starting material.     

Activation of adenylate cyclase in cultured fibroblasts by trypsin

Adenylate cyclase activity measured in membranes of cultured normal rat kidney (NRK) fibroblasts was markedly increased by prior treatment of the intact cells with trypsin. Cell population density influenced the extent of activation observed. Trypsin treatment of sparse cells significantly enhanced adenylate cyclase activity, whereas similar treatment of confluent cells caused only a slight increase in adenylate cyclase activity. The degree of activation noted after trypsin treatment also varied depending on the adenylate cyclase function measured. Activity determined in the presence of GTP alone showed the greatest increase after trypsin treatment. Similar enhancement of adenylate cyclase activity of a washed cell membrane preparation was achieved by the addition of low concentrations of trypsin directly to the adenylate cyclase reaction mixture. The membranes of confluent NRK fibroblasts initially exhibited higher adenylate cyclase activity than did membranes of sparse cells. The present results suggest that this change in adenylate cyclase activity at cell confluence is not due to an increase in the amount of adenylate cyclase in the cell membrane but rather to a change in membrane components that regulate its activity. Proteolytic activation of adenylate cyclase appears to result from degradation of cell membrane proteins that modulate the activity of this enzyme.     

Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs alpha

An antibody (RM) raised against the carboxyl-terminal decapeptide of the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Gs alpha) has been used to study the interaction of Gs alpha with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-gamma-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-gamma-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gs alpha protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gs alpha detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gs alpha protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Go alpha, Gi alpha, and G beta. The G beta protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of G beta was about the same in adenylate cyclase from preactivated membranes and from control membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myocardial guanylate cyclase: properties of the enzyme and effects of cholinergic agonists in vitro.

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.     

The mode of action of chloride on rabbit heart adenylate cyclase

The mechanism by which chloride stimulates adenylate cyclase was investigated. Depletion of GDP increased basal adenylate cyclase activity and reduced the stimulation by isoprenaline. Restoration of bound GDP partially reversed these effects. Chloride stimulated cyclase activity by the same proportion in control, GDP-depleted and GDP-restored preparations, as did Gpp(NH)p. Fluoride increased adenylate cyclase activity to the same final level in both GDP-depleted and GDP-restored membranes; addition of Gpp(NH)p as well as fluoride had no further effect. Solubilisation of adenylate cyclase reduced the stimulatory effect of Gpp(NH)p only slightly, but greatly attenuated the activation by chloride. We conclude that chloride does not stimulate cyclase activity by an action on GDP exchange. Activation by chloride may be due to a disrupting or chaotropic effect on membrane/protein interactions.     

Magnesium-sensitive guanylate cyclase and its endogenous activating factor in Tetrahymena pyriformis.

Guanylate cyclase [EC 4.6.1.2] activity in Tetrahymena pyriformis cells was associated with particulate fractions, but not with soluble fractions. Mg2+ was much more effective than Mn2+ in activating the cyclase activity. Both specific and total cyclase activities with Mg2+ in the particulate fraction were very much lower than those in the original homogenate. The addition of the soluble fraction resulted in a marked enhancement of the particulate-bound cyclase activity, while the adenylate cyclase [EC 4.6.1.1] activity was not enhanced. The enhancement was dependent on Ca2+, and the activating factor is suggested to be a protein.     

Solubilization of calcitonin-responsive renal cortical adenylate cyclase.

Purification of pork renal cortex membranes yielded a particulate adenylate cyclase retaining good sensitivity to stimulation by parathyroid hormone and glucagon and a modest but significant response to porcine calcitonin. Treatment of this partially purified membrane fraction with 0.5% Lubrol PX and 5 mM NaF released adenylate cyclase activity into a fraction which was not sedimented by centrifugation for 20 min at 37,000 X g or for 2 hours at 100,000 X g and passed through a Millipore filter (0.22 mum pore). This solubilized adenylate cyclase was stimulated by porcine calcitonin and NaF but not by parathyroid hormone or glucagon. On gel filtration (Sephadex G-200) in the presence of 1mM dithiothreitol and 5mM NaF, the major portion of the adenylate cyclase activity eluted with the void volume of the column and showed 2.0-fold stimulation with 10 muM calcitonin. Binding of 125I-labeled porcine calcitonin was demonstrated in the 37,000 X g and the 100,000 X g supernatants. From 74 to 86% of the observed binding could be blocked by the addition of unlabeled porcine calcitonin to the reaction mixture. Addition of salmon calcitonin, parathyroid hormone, or glucagon blocked only 12 to 18% of the binding. The dose-response curves for inhibition of binding of iodinated calcitonin by unlabeled calcitonin and the activation of adenylate cyclase by the hormone each showed 50% maximal effect at a concentration between 4.5 and 8 muM porcine calcitonin and maximal effect at a concentration between 33 and 66 muM porcine calcitonin.     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.