Allosteric modification of adenylate deaminase activity: appearance of adenosine deaminase activity as an effect of potassium ions]

The activation of purified adenylate deaminase from the duck myocardium by K+ is accompanied by modification of the substrate specificity and by the appearance of the capacity to deaminate adenosine and adenine. Adenosine deaminase activity originates at the concentration of K+ of 0.15 M that possesses the most stimulating effect on adenylate deaminase activity; with the increase of potassium ions concentration adenosine deaminating activity is enhanced as well, with a parallel reduction of Hill's constant. The PH-dependence, mode of inhibition by phosphate ions and the effect of alkaline metals suggests that adenosine deamination is carried out by natural adenylate deaminase active centres when their conformation is changed under the activator action.     

Hormonal control of the adenyl cyclase activity of adipose cell membranes prepared from badger, rabbit, fox and rat adipose tissues]

1. We have shown differences in hormonal regulation of adenylate cyclase activity in fat cell ghosts prepared from rat, rabbit, fox and badger adipose tissue, under the influence of catecholamines, ACTH and insulin. a) In the rat, catecholamines induced a large stimulation (+315%) of adenylate cyclase. b) In the rabbit, ACTH was the most effective hormone. c) In the fox and the badger, only catecholamines could stimulate adenylate cyclase. d) In both rat and rabbit, insulin did not reduce spontaneous enzymatic activity. Moreover, the activation of adenylate cyclase by ACTH in the rabbit was not altered by insulin, while in the rat, this hormone slightly decreased epinephrine stimulation. 2. Hormonal regulation of adenylate cyclase correlated with the lipolytic response.     

Biochemical evidence for PGI2 and PGE2 receptors in the rabbit renal preglomerular microvasculature

It has been proposed that a portion of the biologic actions of vasodilator prostaglandins occurs via an interaction with specific adenylate cyclase-linked receptors. This hypothesis was explored further in the renal microvasculature by examining the effects of PGI2, PGE1, and PGE2 on rabbit preglomerular microvascular adenylate cyclase. A membrane preparation derived from freshly isolated rabbit renal preglomerular microvessels was used in these studies. NaF, forskolin, or 5'-guanylyl imidodiphosphate were found to be effective in increasing adenylate cyclase activity in the absence of exogenous guanosine-5'-triphosphate. A dose-dependent stimulation of adenylate cyclase was also observed with guanosine-5'-triphosphate. PGE1, PGE2, and PGI2 produced a dose-dependent stimulation of adenylate cyclase activity only in the presence of guanosine-5'-triphosphate suggesting that this nucleotide is essential for prostaglandin-induced stimulation of the enzyme. PGI2 exhibited a time-dependent increase in adenylate cyclase activity and this increased activity reached a plateau at 20-25 min. When PGE1 and PGE2 were added together, no additive effect on adenylate cyclase stimulation was noted whereas PGI2 and PGE2 when added together produced an additive stimulatory effect. When viewed together, these data suggest the presence of separate PGI2 and PGE adenylate cyclase-linked receptors in rabbit renal preglomerular microvessels. These findings also suggest that in the renal microvasculature, cyclic AMP may be a second messenger mediating the vasodilatory effects of both PGI2 and PGE2.     

Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme.

In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5'-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5'-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.     

Protein kinase C interferes with Ni-mediated inhibition of human platelet adenylate cyclase

Addition of phorbol ester-activated, partially purified protein kinase C to membranes of human platelets had no effect on forskolin stimulation of the adenylate cyclase and increased stimulation by prostaglandin E1 only at high GTP concentrations by preventing inhibition by GTP. Hormonal inhibition of the platelet adenylate cyclase by epinephrine was eliminated or largely impaired. At low GTP concentrations, epinephrine even caused a small increase in cyclase activity. The data suggest that activated protein kinase C interferes with GTP- and hormone-induced adenylate cyclase inhibition probably by phosphorylating the inhibitory guanine nucleotide-binding regulatory component Ni.     

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.     

Protease inhibitors block hormonal activation of adenylate cyclase

To investigate the mechanism of serine protease stimulation of rat ovarian adenylate cyclase, a variety of synthetic protease inhibitors were used. These inhibitors blocked trypsin, chymotrypsin and hCG stimulation of adenylate cyclase in nearly the same manner. The inhibition of hormone stimulated adnylate cyclase could not be explained by a loss of [¹²⁵I]hCG binding. Cholera toxin and epinephrine stimulation of adenylate cyclase were similarly inhibited, whereas basal and fluoride-stimulated activities were only affected by higher doses of the inhibitors. The results suggest that adenylate cyclase in the ovary may be regulated by membrane protease activity.     

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

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.     

Electron microscopic demonstration of adenylate cyclase in rabbit small intestine enterocytes doubly stimulated by cholera toxin and sodium fluoride]

Cytochemical investigations showed adenylate cyclase in the rabbit small intestine enterocytes to be activated both with cholera toxin and sodium fluoride. Following double stimulation of adenylate cyclase in the intestinal enterocytes by the mentioned two substances maximal critical levels of cAMP were attained resulting in self-inhibition of adenylate cyclase; in this case only a low adenylate cyclase activity, if any, could be demonstrated by electron microscopy.     

Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone.

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.     

Stimulation of adenylate cyclase by adenosine and other agonists in mesenteric artery smooth muscle cells in culture

An adenosine-sensitive adenylate cyclase has been characterized in cultured mesenteric artery smooth muscle cells. N-Ethylcarboxamide-adenosine (NECA), N-Methylcarboxamide-adenosine (MECA), L-N6-phenylisopropyladenosine (PIA) and 2-chloroadenosine (2-cl-Ado) all stimulated adenylate cyclase in a concentration dependent manner. NECA was the most potent analog (EC50, 1 microM), whereas PIA (EC50, 15 microM), 2-Cl-Ado (EC50, 15 microM) and MECA (EC50, 24 microM), were less potent and had efficacies relative to NECA of 0.61, 0.61 and 0.65, respectively. Adenosine showed a biphasic effect: stimulation at lower concentrations and inhibition at higher concentrations, whereas 2' deoxyadenosine only inhibited adenylate cyclase activity. The stimulatory effect of NECA on adenylate cyclase was dependent on metal ion concentration and was blocked by 3-isobutyl-l-methylxanthine (IBMX) and 8-phenyltheophylline (8-PT). Adenylate cyclase from these cultured cells was also stimulated by other agonists such as epinephrine, norepinephrine, prostaglandins, dopamine, NaF and forskolin. The stimulation of adenylate cyclase by isoproterenol, epinephrine and norepinephrine was blocked by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol and flupentixol all inhibited dopamine-stimulated adenylate cyclase activity. In addition, the stimulation by an optimal concentration of PIA was additive or almost additive with maximal stimulation caused by catecholamines and prostaglandins. These data indicate the presence of adenosine (Stimulatory "Ra"), catecholamine and prostaglandin receptors in mesenteric artery smooth muscle cells and suggest that these agents may exert their physiological actions through their interaction with their respective receptors coupled to adenylate cyclase.     

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.     

Activation of adenylate cyclase from rat striatum and tuberculum olfactorium by adenosine

Adenosine caused a dose-dependent stimulation of adenylate cyclase in homogenates from rat striatum and tuberculum olfactorium (200 and 300% stimulation by 100 muM adenosine). The effect of adenosine was not antagonized by haloperidol. Subcellular fractionation suggested that adenosine stimulates a different adenylate cyclase than dopamine. Basal adenylate cyclase activity in freshly prepared homogenates was reduced by dialysis and by the addition of adenosine deaminase. Basal adenylate cyclase activity was enchanced by papaverine and dipyridamole, but reduced by theophylline and isobutylmethylxanthine. The results are compatible with the opinion that endogenous adenosine is capable of activating adenylate cyclase in these areas of the rat brain.     

Proof of adenylate cyclase activity in the coronary artery of cattle]

In two fractions obtained from the bovine A. coronaria adenylate cyclase activity was identified and characterized. The adenylate cyclase activity of the 75,000 X g sediment shows a pH optimum at 7.4. The temperature dependence of this adenylate cyclase activity is linear when represented in the Arrhenius plot, and an Arrhenius activation energy of 13.2 kcal Mol-1 can be calculated for the enzyme reaction. The Km-value of the enzyme to ATP is 6 +/- 0.6 - 10(-4) M. The adenylate cyclase activity of the 75,000 X g sediment can be stimulated by NaF. 5'AMP and adenosine inhibit the adenylate cyclase activity of the 75,000 X g sediment. With regard to the enzyme activity, Mn++ and Co++ replace Mg++, but not Ca++. The monovalentcations Na+ and K+ do not influence the adenylate cyclase activity. In a particulate fraction containing plasma membranes, adenylate cyclase activity was also identified. This adenylate cyclase activity can be stimulated by catecholamines, noradrenaline, and isoproterenol. This stimulation can, however, only be proved for the enzyme in the coronaries of 9-week-old and 2-year-old animals. The adenylate cyclase activity from the coronaries of adult animals is not affected by catecholamines. These findings are discussed with regard to hypertension frequently found in adult animals.     

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.     

Properties of Muscarinic-Stimulated Adenylate Cyclase Activity in Rat Olfactory Bulb

The muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was characterized, with the aim of elucidating the nature of the molecular mechanism involved. Carbachol (CCh) stimulated the enzyme activity in either crude or purified cell membrane preparations and increased cyclic AMP accumulation in miniprisms of olfactory bulb. The CCh stimulation of adenylate cyclase activity displayed a fast onset and was rapidly reversed by addition of atropine. The stimulation was associated with an increase in the apparent Vmax of the enzyme, with no change in the Km for Mg-ATP. The affinity of the enzyme for Mg2+ was enhanced by CCh. The muscarinic effect required GTP at concentrations higher than those needed for enzyme stimulation with either l-isoproterenol or vasoactive intestinal peptide. Moreover, contrary to the beta-adrenergic stimulation, the muscarinic effect disappeared when guanosine 5'-O-(3'-thiotriphosphate) was substituted for GTP. In vivo treatment of olfactory bulbs with pertussis toxin completely prevented the muscarinic stimulation of adenylate cyclase, whereas cholera toxin was without effect. These results indicate that in rat olfactory bulb muscarinic receptors increase adenylate cyclase activity by interacting with a pertussis toxin-sensitive GTP-binding protein different from the stimulatory GTP-binding protein.     

Inhibition of E. coli adenylate cyclase activity by inorganic orthophosphate is dependent on IIIglc of the phosphoenolpyruvate:glycose phosphotransferase system

The relationship of adenylate cyclase, inorganic orthophosphate and the proteins of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was studied. A strain deleted for the genes for Enzyme I and IIIglc of the PTS was transformed with plasmids expressing either Enzyme I and HPr, IIIglc or all three proteins. The fully reconstituted strain showed a Pi-dependent stimulation of adenylate cyclase activity; in contrast, the strain expressing only IIIglc showed a Pi-dependent inhibition of adenylate cyclase activity.     

Monoclonal antibody approach to the relationship between immunological structure and biological activity of thyrotropin

In order to locate the domains involved in the biological activity of TSH and to get some insight in the relationship between immunological and biological properties of TSH, 24 monoclonal antibodies (mAb) to 11 different antigenic regions of hTSH were tested for both binding to hTSH and inhibition of hTSH stimulation of adenylate cyclase in human thyroid membranes. These mAb were also investigated for binding to bovine TSH (bTSH), and interference with bTSH binding to the receptor and stimulation of adenylate cyclase. Radioiodinated human TSH (hTSH) was incubated with increasing concentrations of mAb. Maximum hTSH binding by the various mAb ranged from 15-75% and was not related to the apparent affinity of the mAb for hTSH. Maximum inhibition by the mAb of hTSH stimulation of adenylate cyclase ranged from 3-92%. As compared to the antigenic map of hTSH, it was observed that mAb reacting with the same antigenic regions might display varying inhibition of hTSH. Nevertheless, it was clearly shown that the most potent inhibitors of hTSH stimulatory activity interacted with epitopes located on the alpha- and beta-subunits or expressed only by holo hTSH. Only 11 of the 24 mAb cross-reacted significantly with bTSH. Seven exhibited the same inhibition of hTSH and bTSH stimulatory activity; the four remaining mAb rather than to inhibit adenylate cyclase stimulation as observed with hTSH, did not interfere or even increased adenylate cyclase stimulation by bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of phospholipids in TSH stimulation of adenylate cyclase in thyroid plasma membranes

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.