Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.     

Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.     

Guanylate cyclase activity of human lymphocytes from peripheral blood, thymus, and tonsils. A comparative study

The subcellular repartition and the distinctive properties of guanylate cyclase (EC 4.6.1.2) vary according to the lymphocyte population studied and according to the presence of detergent. Guanylate cyclase of non-adherent peripheral lymphocytes and of thymus lymphocytes is recovered by more than 90% in the soluble fraction of the homogenate. Kinetics according to the substrate (5'-GTP-Mn2+) is Michaelian, the Ca2+ ion acts as an activator, especially in the case of blood lymphocytes, and the detergent has no effect on the enzyme activity. On the other hand, the guanylate cyclase of tonsil lymphocytes reside in the particulate fraction. It has non-Michaelian kinetics for the substrate, a strong stimulating effect of detergent, and an inhibitory effect of Ca2+. A comparison of the enzymatic activities of unseparated and of non-adherent tonsil lymphocytes obtained from the same donor points to a correlation between their T and B properties: predominant soluble activity in the T population and particulate guanyl cyclase activity in the B subset.     

Identification of calmodulin-sensitive and calmodulin-insensitive adenylate cyclase in rat kidney

Adenylate cyclase from rat kidney membranes solubilized with Lubrol-PX, was resolved into calmodulin-insensitive and calmodulin-sensitive forms using DEAE-Sephacel and calmodulin-Sepharose affinity chromatography. The major fraction, 90% of the activity recovered, did not bind to the calmodulin-Sepharose in the presence of Ca2+, and was insensitive to activation by calmodulin. The calmodulin-sensitive enzyme, approximately 10% of the recovered activity, bound to the affinity column and was eluted with buffer containing 2 mM EGTA. In the presence of free Ca2+, calmodulin increased the specific activity of the calmodulin-sensitive adenylate cyclase from 15.2 to 60.4 pmol/mg protein-1 min-1. Maximum stimulation occurred at 0.035-0.076 mM Ca2+. The apparent Ka for calmodulin was 8 nM. The calmodulin-mediated increase in activity was inhibited by trifluoperazine, but not by its analog trifluoperazine-5-oxide. In contrast, trifluoperazine did not inhibit the calmodulin-insensitive activity. The GTP analog, guanyl-5'-yl imidodiphosphate, did not activate either fraction. Furthermore, activation by calmodulin did not require the presence of a guanyl nucleotide. The present finding of a calmodulin-sensitive form of adenylate cyclase in kidney raises the possibility that a calmodulin-mediated mechanism is involved in the formation of cAMP in this organ.     

Evidence for reversability of age-related decrease in human lymphocyte adenylate cyclase activity

Age-related loss of adenylate cyclase responsiveness to guanyl nucleotide was demonstrated in lymphocytes freshly isolated from human subjects. Enzyme activity of cells from young (<40 years) and elderly (>65 years) subjects were markedly sensitive to inhibition by non-ionic detergents. When enzyme activity in the presence of guanyl nucleotide and low concentrations of Triton X-100 was determined in a mixture of cells from the young and aged donors, the activity was 40±17 percent (mean ± S.D.) greater than anticipated from the activity of the cells of the two age groups assayed separately. The detergent range which facilitated the enhanced enzyme activity was too low to extract the catalytic subunit of adenylate cyclase from the cells. These results further suggest that in man, changes distal to receptors contribute to diminished responsiveness of lymphocyte adenylate cyclase as a function of age. In addition, these age-related changes may be partially reversible by reconstitution with factors from cells from younger subjects.     

Coupling of glucagon receptor to adenylyl cyclase. Requirement of a receptor-related guanyl nucleotide binding site for coupling of receptor to the enzyme.

Effects of glucagon and guanyl nucleotides on the rat liver plasma membrane adenylyl cyclase were studied. It was established that: 1) glucagon stimulates the fully guanyl-5'-yl imidodiphosphate (GMP-P(NH)P)-activated enzyme between 20 and 70%, provided a guanyl nucleotide is present in the assay; 2) glucagon has no effect on adenylyl cyclase activity in membranes activated fully by GMP-P(NH)P and then washed free of nucleotides. It is concluded that occupancy of the guanyl nucleotide binding site that activates the catalytic moiety of the system is not sufficient to promote hormone-receptor coupling to adenylyl cyclase and that occupancy of a second site by guanyl nucleotides is essential to effect stimulation of adenylyl cyclase by the glucagon-receptor complex. The data presented raise the question whether the guanyl nucleotide site that promotes coupling is distinct from the guanyl nucleotide site that modulates binding of glucagon to receptor and whether the occupancy of the guanyl nucleotide site associated with the catalytic moiety is necessary for coupling.     

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product.

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.     

Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells.

The membrane-bound adenylase cyclase (ATP pyrosphosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Milipore filters with pores 0.22 micron in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5'-guanylyl-imidodiphosphate at a concentration of 1 . 10(-6)M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5'-guanylyl-imidodiphosphate.     

Stimulation of human fat cell adenylate cyclase by GDP and guanosine 5'-O-(2-thiodiphosphate)

GDP regulation of basal and receptor-mediated catecholamine-sensitive human fat cell adenylate cyclase was studied using purified plasma membrane preparations and assay conditions selected to minimize conversion of GDP to GTP. Under ordinary assay conditions (low NaCl concentration) and with App(NH)p as substrate to prevent GDP conversion to GTP, basal enzyme activity was stimulated up to 2-fold by GDP (0.1 mM) while addition of epinephrine (0.1 mM) eliminated stimulation by GDP and reduced basal adenylate cyclase activity. With ATP as substrate, the enzyme was not responsive to hormone in the absence of guanyl nucleotides and GDP augmentation of basal activity was small (0-1.5-fold) while stimulatory effects of epinephrine and isoproterenol were minimally but definitely exhibited (1.5-fold over basal). Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analog resistant to phosphorylation and hydrolysis and an antagonist of GTP, stimulated enzyme activity more than did GDP but did not promote epinephrine action. Rather, inhibition of GDP beta S-stimulated adenylate cyclase activity was seen with both epinephrine and isoproterenol and also with GTP. In the presence of NaCl (200 mM), which alone produced 2-3-fold increase in basal enzyme activity, GDP (0.1 mM) and GDP beta S (50 microM) produced 8- and 15-fold increases of activity, respectively. Addition of UDP, to prevent possible conversion of GDP to GTP, had no effect on NaCl-enhanced activation by GDP. The results indicate that the human fat cell adenylate cyclase system is unique in responding to GDP and its analog GDP beta S by stimulation in the absence of hormone but suggest that as in other systems catecholamine-mediated stimulation is normally dependent on GTP. Salts (Na+) appear to stimulate the enzyme by facilitating the interaction of the guanyl nucleotide regulatory protein (N8) with the catalytic unit.     

Effect of guanyl nucleotides and hyperbaric oxygenation on the adenylate cyclase activity of the heart in rabbits with myocardial hypertrophy

The authors studied the role of guanyl nucleotides and hyperbaric oxygenation (HBO) in desensitization and resensitization of adenylate cyclase of the heart during hypertrophy which was induced by aorta stenosing. The basal activity of the enzyme and the rate of its activation with adrenaline and guanyl nucleotides were discovered to be reduced. In the presence of guanyl nucleotides, HBO gave rise to the recovery of enzyme sensitivity to the hormone. The data obtained indicate that during myocardial hypertrophy, guanyl nucleotides participate both in densensitization of adenylate cyclase to hormonal exposure and in the enzyme resensitization during HBO therapy.     

GUANYL CYCLASE IN CHICK EMBRYO BRAIN CELL CULTURES: EVIDENCE OF NEURONAL LOCALIZATION

Abstract— Guanyl cyclase activity was studied in dissociated chick embryo brain cell cultures presenting different ratios of neuronal to glial elements. The cultures containing neurons in substantial numbers always had higher guanyl cyclase activities than those consisting mainly of glial cells. No guanyl cyclase activity could be found in cultures made up of pure glial or meningeal cells. These results provide further evidence for our conclusion based on subcellular fractionation studies (G oridis & M organ , 1973), that brain guanyl cyclase might be overwhelmingly concentrated in neurons. Guanyl cyclase activity of chick embryo cerebral hemispheres increased sixfold between day 12 and day 16 after fertilization; an increase, though of much smaller magnitude, was also seen in cultured cells of the same age.     

Bordetella pertussis invasive adenylate cyclase. Partial resolution and properties of its cellular penetration

The existence of an invasive adenylate cyclase in dialyzed urea extracts of the bacterium Bordetella pertussis has been suggested recently. Gel filtration of B. pertussis dialyzed urea extract shows that the invasive enzyme constitutes only a small portion of the total adenylate cyclase activity found in the extract. Its size is different than the size of the two peaks of adenylate cyclase activity identified in the extract. Ca2+ is absolutely required for the penetration of the invasive enzyme, it also controls the rate of intracellular cAMP accumulation in human lymphocytes exposed to dialyzed extract. These characteristics may be attributed to the increase in the size of the invasive enzyme as found by gel filtration chromatography of the extract in the absence of Ca2+. Removal of nonpenetrating adenylate cyclase that adheres to lymphocytes permits a direct assay of the intracellular enzyme. The time course of intracellular accumulation of adenylate cyclase activity is similar to the time course of intracellular accumulation of cAMP, suggesting that the invasive enzyme is rapidly deactivated, but not degraded, since it can be detected upon cell disruption. No appreciable amount of the enzyme is introduced when cells are incubated with extract at 4 degrees C for 120 min, then washed and incubated further at 37 degrees C. Concanavalin A inhibits cAMP accumulation irrespective of the time of its addition, and EGTA prevents penetration of the invasive enzyme even if added 20 min after addition of extract. These findings are different from those observed in other bacterial toxins thought to be internalized by receptor-mediated endocytosis. However, the cellular penetration of B. pertussis adenylate cyclase is cell-selective. It does not occur in human erythrocytes. In addition to human lymphocytes, S49 cyc- murine lymphoma, turkey erythrocytes, and rat oocytes accumulate cAMP in response to B. pertussis extract.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

The invasive adenylate cyclase of Bordetella pertussis. Intracellular localization and kinetics of penetration into various cells.

The penetration of Bordetella pertussis adenylate cyclase into various mammalian cells exhibits similar kinetics; the accumulation of both intracellular cyclase activity and cyclic AMP is rapid, reaching constant levels after 15-60 min of incubation. The kinetics of enzyme penetration into turkey erythrocytes is different; cyclase activity and cyclic AMP accumulate linearly and do not reach constant levels even after 6 h of incubation. In the preceding paper [Friedman, Farfel & Hanski (1987) Biochem. J. 243, 145-151] we have suggested that the constant level of intracellular cyclase activity reflects a steady state formed by continuous penetration and intracellular inactivation of the enzyme. In contrast with other mammalian cells, no inactivation of cyclase is observed in turkey erythrocytes. These results further support the notion that there is continuous penetration and deactivation of the invasive enzyme in mammalian cells. A 5-6-fold increase in specific activity of the invasive cyclase is detected in a pellet fraction of human lymphocytes in which a similar increase in specific activity of the plasma-membrane marker 5'-nucleotidase is observed. A similar increase in the invasive-cyclase specific activity is detected in a membrane fraction of human erythrocytes. Cyclase activity in a membrane-enriched fraction of human lymphocytes reached a constant level after 20 min of cell exposure to the enzyme. Similar time courses were observed for accumulation of cyclase activity and cyclic AMP in whole lymphocytes [Friedman, Farfel & Hanski (1987) Biochem, J. 243, 145-151]. We suggest therefore that cyclic AMP generation by the invasive enzyme as well as the intracellular inactivation process occur while it is associated with a membrane fraction identical, or closely associated, with the plasma membrane.     

Studies on the assay and activities of guanyl and adenyl cyclase of rat liver

In applying recently developed methods for measuring adenyl and guanyl cyclase activities, we found that some modifications produced much better cyclic nucleotide recovery, lower assay backgrounds, and greater reliability than previously reported. The reliability and specificity of the assay methods were confirmed by substrate and product analysis. Kinetic analysis of rat liver guanyl and adenyl cyclase was subsequently performed to investigate regulatory properties of both enzymes. The Michaelis-Menton constant of guanyl cyclase activity of a 30,000g supernatant fraction of rat liver for guanosine 5′-triphosphate (GTP) was 0.04 mm. This enzyme was competitively inhibited by adenosine 5′-triphosphate (ATP) (K_i = 0.011 mM). Guanyl cyclase was activated in vitro by secretin but unaffected by carbamylcholine, hist-amine, methoxamirie, serotonin, glucagon, and pancreozymin. Liver homogenate adenyl cyclase had a Michaelis-Menten constant for ATP of 0.2 mm. This enzyme was activated by secretin, pancreozymin, glucagon, sodium fluoride, and isoproterenol. GTP (0.005 mm) enhanced the activation by both isoproterenol and glucagon. Methoxamine had no effect on adenyl cyclase activity in the presence or absence of GTP. These results suggest that both guanyl cyclase and adenyl cyclase may be mediators of hormone action in the liver.     

GTP-dependent anion-sensitive adenylate cyclase in snail ganglia potentiation of neurotransmitter effects

Snail ganglia possess an anion-sensitive adenylate cyclase. This enzyme was stimulated 100% by chloride in a strictly GTP-dependent manner. The apparent affinity of chloride for adenylate cyclase was 2 X 10(-4) M. Halogens were found to be the most active anions. Some inorganic anions such as SO4(2-) and H2PO4- were inactive, as were all the organic anions tested. Stimulation was not cumulative for any maximal concentration of the active anions except fluoride. Chloride potentiated the effect of fluoride, indicating that the anion effect is not fluoride-like. Another striking result is that chloride enhanced adenylate cyclase sensitivity to the neurotransmitters serotonin and dopamine. The absence of chloride stimulation when Mg2+ was replaced by Mn2+ further indicates a role of the GTP-binding protein (the G/F unit). Chloride could reversibly stimulate the adenylate cyclase activity already maximally stimulated by guanyl 5'-imidodiphosphate. We therefore suggest that, in snail ganglia, chloride raises the activity of the G/F unit-catalytic unit complex at some stage after its formation. The same specific anion-sensitive adenylate cyclase was also found in some of the rat tissues tested.     

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides

The adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is localized to the plasma membrane of the cell. The enzyme utilizes Mn2+/ATP as substrate and free Mn2+ ions as an effector. Unlike the baker yeast Saccharomyces cerevisiae, S. pombe adenylyl cyclase does not utilize Mg2+/ATP as substrate and the activity is not stimulated by guanyl nucleotides. The optimal pH for the S. pombe adenylyl cyclase activity is 6.0. The activity dependence on ATP is cooperative with a Hill coefficient of 1.68 +/- 0.14.     

Mechanism of molybdate activation of adenylate cyclase

Molybdate activation of rat liver plasma membrane adenylate cyclase has been examined and compared with the effect of glucagon, Gpp(NG)p and fluoride. Glucagon does not stimulate the detergent solubilized enzyme, though molybdate, fluoride, and Gpp(NH)p are effective in this regard. The stimulatory effects of either fluoride or molybdate are additive with those of GTP and do not require guanyl nucleotide to evoke their activation. Neither fluoride nor molybdate can substitute for GTP when glucagon is the activator of rat liver adenylate cyclase. The stimulatory effects of either ion on adenylate cyclase are additive with that produced by glucagon. Activation of adenylate cyclase by either molybdate or fluoride occurs by a mechanism distinct from that of glucagon or guanyl nucleotide. The data presented here suggest that fluoride and molybdate may act via a similar mechanism of action. Neither ion displays a lag in activation of adenylate cyclase. The pH profiles of fluoride and molybdate-stimulated adenylate cyclase activity are similar, and distinct from guanyl nucleotide-stimulated activity. Cholera toxin treatment of adenylate cyclase blocks fluoride and molybdate stimulation of the enzyme to the same extent, while enhancing the activation obtained with GTP and hormones.     

The epinephrine-sensitive adenylate cyclase of rat liver plasma membranes. Role of guanyl nucleotides.

The epinephrine sensitivity in vitro of the adenylate cyclase system in liver plasma membranes from adrenalectomized rats was increased by the addition of 1 to 100 muM GTP or GDP in the incubation medium. Basal and glucagon-stimulated cyclase activities were also enhanced by GTP and GDP. These effects occurred even in the absence of an ATP-regenerating system. They were mimicked by 5'-guanyl diphosphonate and a series of guanyl derivatives, indicating that the structural requirement for the GTP action is not very stringent. Guanyl nucleotides did not increase the affinity of the adenylate cyclase system for the activating hormones, nor did they protect the enzyme activity against denaturation. Their synergic effect was due to an enhancement of the affinity of the enzyme for the substrate MgATP and also to an increase of the maximal velocity of the reaction. It is proposed that the guanyl nucleotides act directly and primarily upon the catalytic component of the cyclase system, independently of their effects on the binding of the activating hormones to liver plasma membrane. Since the activating effects of epinephrine and glucagon are similar in the presence of GTP, but not in its absence, it is suggested that the lower efficiency of epinephrine under normal conditions is not due to intrinsic membrane characteristics, but rather, to superimposed extraneous modulations.     

Activation of solubilized liver membrane adenylate cyclase by guanyl nucleotides.

Liver plasma membranes of hypophysectomized rats were purified, treated with 0.1 m Lubrol-PX and centrifuged at 165,000g for 1 h. The detergent solubilized 50% of the membrane protein; adenylate cyclase activity was present in the supernatant fraction. Optimal substrate concentration of the soluble enzyme was 0.32 mm ATP. Basal activity of 25 preparations of the solubilized enzyme ranged from 124 to 39 pmol cyclic AMP/mg protein/10 min. The solubilized enzyme retained the same sensitivity to activation by guanyl nucleotides as was present in the membrane preparation from which it was derived. Relative sensitivity of the solubilized enzyme with 0.1 mm nucleotides or -side was GDP > GTP > GMP > guanosine; GMP-PNP = GMP-PCP > ITP > GTP. GTP, GMP-PCP, GMP-PNP and other nucleotides were hydrolyzed by phosphohydrolases present in liver membranes that were solubilized with Lubrol-PX along with adenylate cyclase. The presence of the ATP regenerating system in the adenylate cyclase assay also aided in maintaining guanyl nucleotide concentrations. The degree of adenylate cyclase activation by guanyl nucleotides was not related to the sparing effects of nucleotides on substrate ATP hydrolysis. These findings demonstrate that activation of adenylate cyclase by nucleotides is a consequence of a nucleotide-enzyme interaction that is independent of membrane integrity.