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.     

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.     

Mechanism of glucagon activation of adenylate cyclase in the presence of Mn2+

For a variety of ligand states, adenylate cyclase activity in the presence of Mn2+ was greater than with Mg2+. Trypsin treatment of intact hepatocytes, under conditions which destroy cell surface glucagon receptors, led to a first order loss of glucagon-stimulated adenylate cyclase activity in isolated membranes assayed in the presence of Mn2+ whether or not GTP (100 microM) was present in the assays. Arrhenius plots of basal activity exhibited a break at around 22 degrees C, those with NaF were linear and those with glucagon +/- GTP (100 microM) were biphasic with a break at around 28 degrees C. It is suggested that Mn2+ perturbs the coupling interaction between the glucagon receptor and catalytic unit of adenylate cyclase at the level of the guanine nucleotide regulatory protein. This appears to take the form of Mn2+ preventing GTP from initiating glucagon's activation of adenylate cyclase through a collision coupling mechanism.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

Characterization of a calcium-modulated adenylate cyclase from abalone spermatozoa

Abalone spermatozoa contain a particulate adenylate cyclase that displays maximal catalytic activity when Mn2+ is present as a metal cofactor in excess of ATP. Unlike other sperm adenylate cyclases, the abalone enzyme displays a high Mg2+-supported catalytic activity (Mg2+/Mn2+ activity ratio = 0.8). Kinetics analyses demonstrate that the enzyme contains both a MgATP catalytic site and a separate Mg2+ regulatory site. Mg2+-supported enzyme activity, however, is not stimulated by guanine nucleotides, NaF, cholera toxin, forskolin, or a variety of hormones. The enzyme from unfractionated sperm homogenates is inhibited by added Ca2+ in a concentration-dependent manner, when EGTA is not present in the assay. Methylxanthines, such as 1-methyl-3-isobutylxanthine and theophylline, also inhibit enzyme activity in a concentration-dependent manner through a noncompetitive mechanism. On the other hand, when intact cells are preincubated with Ca2+ prior to breakage and assayed for enzyme activity, Ca2+ stimulates enzyme activity at low concentrations. Enzyme activity of intact sperm preincubated with methylxanthines, in either the absence or presence of added Ca2+, is also stimulated. This effect is expressed via an effect on the velocity of the enzyme. A-23187 has similar stimulatory effects on the enzyme under these conditions. These data provide further support for the role of Ca2+ conductance in modulating sperm adenylate cyclase activity. The abalone sperm enzyme also appears to have regulatory properties that are unique among other sperm types.     

Prostacyclin stimulation of adenylate cyclase activity in human thyroid membranes

Effect of prostacyclin (PGI2) on adenylate cyclase activity in human thyroid membranes was examined. PGI2 caused a dose- and time-dependent production of cyclic AMP (cAMP) with high potency. When GTP was added in concentrations up to 100 uM, the activation of adenylate cyclase by PGI2 was increased. In the assay medium containing 3 mM ATP, 10 uM GTP and nucleotide regenerating system, the replacement of Mg2+ by increasing concentrations of Mn2+ caused a progressive loss of PGI2 as well as TSH-stimulated adenylate cyclase activities, while high concentrations of Mg2+ (12 or 18 mM) slightly suppressed the activity stimulated by either PGI2 or TSH. Both agents had an additive effect on the stimulation of adenylate cyclase activity in the presence of either 6 mM Mg2+ or 6 mM Mn2+. Gamma-globulin fraction containing non-stimulatory TSH receptor antibody which was prepared from a patient with chronic thyroiditis, suppressed only TSH- but not PGI2-stimulation of the adenylate cyclase activity. These results suggest that PGI2 can stimulate the adenylate cyclase activity in human thyroid tissue, and that PGI2-stimulation may be mediated by the different system from TSH-dependent one.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Withdrawal of GTP-dependent inhibition

12-O-Tetradecanoylphorbol-13-acetate (TPA) enhances the apparent maximal velocity of adenylate cyclase in S49 lymphoma cells, an effect that seems not to result from an increased rate of activation of the catalytic subunit by the stimulatory GTP-binding protein (Gs) (Bell, J. D., Buxton, I. L. O., and Brunton, L. L. (1985) J. Biol. Chem. 260, 2625-2628). In membranes from wild type S49 cells, this enhancing effect of TPA is largely GTP-dependent; TPA enhances forskolin-stimulated adenylate cyclase activity by 35% in the presence of guanine nucleotide but only slightly (approximately 10%) in its absence. TPA causes comparable results in membranes from the cyc- variant that lacks the GTP-binding subunit of Gs. Blockade of the activity of the inhibitory GTP-binding protein (Gi) by high concentrations of Mg2+ (100 mM) or Mn2+ (3 mM) abolishes the effect of TPA to enhance adenylate cyclase activity in wild type membranes. The potentiation by TPA of cAMP accumulation in intact cells is greater than and not additive with the similar effect of pertussis toxin (an agent known to abolish hormonal inhibition of adenylate cyclase). Kinetic experiments indicate that TPA decreases the rate of activation of Gi by guanine nucleotide. We conclude that the resultant withdrawal of tonic inhibition of adenylate cyclase is one mechanism by which phorbol esters enhance guanine nucleotide-dependent cAMP synthesis.     

Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5'-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.     

Inhibition of adenylate cyclase by the 2',3'-dialdehyde of adenosine triphosphate

The periodate-oxidized analog of ATP, 2',3'-dialATP, competitively inhibited bovine brain and rat liver adenylate cyclase. The apparent Ki for inhibition of brain adenylate cyclase by 2',3'-dialATP was 196 microM in the presence of Mg2+ and 37 microM in the presence of Mn2+. The Ki values for inhibition of rat liver adenylate cyclase by 2',3'-dialATP were 48 and 30 microM in the presence of Mg2+; and Mn2+, respectively. Adenylate cyclase activity was irreversibly inactivated by 2'3'-dialATP in the presence of NaCNBH3 and the kinetics for loss in enzyme activity were pseudo-first order. Both ATP and Tris protected adenylate cyclase from irreversible inhibition by 2',3'-dialATP and NaCNBH3. It is proposed that 2',3'-dialATP forms a Schiff's base with an amino group at the active site of the enzyme and that Na-CNBH3 reduction of this Schiff's base causes irreversible modification of the catalytic subunit. The Km for 2',3'-dialATP inactivation, the maximal rate constant of inactivation, and protection of the enzyme by ATP were not affected by the presence or absence of free Mg2+. These data indicate that a divalent cation is not required for binding of 2',3'-dialATP to the active site of adenylate cyclase.     

Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts

Alterations in receptor-independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn2+, forskolin, and Gpp(NH)p. Analyses were performed 3, 6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn2+ decreases with the age of the culture; the apparent activation constant for Mg2+ does not. Bimodal activation by Mn2+, i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg2+, forskolin activation occurs with low-and high-affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low- and high-affinity activation only in the presence of high Mg2+. In contrast, the Vmax for forskolin increases with increasing Mg2+ in all culture ages. Although Gpp(NH)p-dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg2+, low Mg2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 microM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera- and pertussis toxin-dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.     

Insulin exerts actions through a distinct species of guanine nucleotide regulatory protein: inhibition of adenylate cyclase.

Insulin failed to exert an effect on the basal and glucagon- and guanosine 5'-[beta, gamma-imido]-triphosphate-stimulated adenylate cyclase activities of hepatocyte membranes. In the presence of high GTP (0.1 mM) concentrations, however, insulin was shown to inhibit adenylate cyclase activity. This effect was dose-dependent, exhibiting an EC50 (median effective concentration) of 3 microM for GTP. Elevated glucagon concentrations blocked the inhibitory effect of insulin in a dose-dependent fashion, with an EC50 of 1 nM. The insulin inhibition was dose-dependent (EC50 = 90 pM). The inhibitory effects of insulin were abolished using membranes from either glucagon-desensitized hepatocytes or cholera-toxin-treated hepatocytes. If either Mn2+ replaced Mg2+ in adenylate cyclase assays or Na+ was removed from the assay mixtures then insulin failed to exert any inhibitory effect. It is suggested that insulin exerts its action on adenylate cyclase through an inhibitory guanine nucleotide protein. This is integrated with the proposal [Heyworth, Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & Houslay (1983) Biochem. J. in the press] that insulin mediates a variety of cellular effects through a specific guanine nucleotide regulatory protein and associated protein kinase(s).     

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+. These properties indicated the catalytic unit was not sensitive to regulation by the GS regulatory protein. Guanine nucleotide inhibited catalysis, however, and was a competitive inhibitor of the ATP substrate (Ki approximately 50 microM). Since inhibition affected Km but not Vmax, the catalytic unit also seemed insensitive to regulation by the Gi regulatory protein, which does not act like a competitive inhibitor in other enzyme systems. The catalytic unit was also phospholipid sensitive. Only phosphatidic acid (Pho-A) had a direct effect on catalysis and was a potent inhibitor. Its effects were antagonized by the concomitant addition of phosphatidylcholine (Pho-C) but not by phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol. Acyl chain composition had a marked effect on Pho-C binding when this was determined by antagonism of Pho-A-dependent inhibition. These properties suggest the catalytic unit has both polar head group and acyl chain requirements for phospholipid binding.     

Modulation of guanine nucleotide affinity does not affect the first order rate constant of activation of adenylate cyclase in native membranes

A new method was developed to follow the rate of activation of adenylate cyclase in rat brain membranes by rapid freezing and N-ethylmaleimide treatment at 0 degrees C. This method was used to investigate the relationship between the rate of activation of adenylate cyclase by p(NH)ppG and GTP gamma S and their apparent affinities. These studies established the following. 1) The kinetics of activation by p(NH)ppG and GTP gamma S were indistinguishable although the apparent affinity of p(NH)ppG was 20-fold lower than the affinity of GTP gamma S. Activation was first order, kobs varying approximately 1.5-fold (average t 1/2 = 3.5 min, 30 degrees C) between 20-90% occupancy by either guanine nucleotide. 2) Final levels of activity were strictly dependent on the concentration of the nucleotides in a saturable manner. 3) Mg2+ increased the apparent affinity of either guanine nucleotide by 10-20-fold between 0.1 microM and 3 mM free Mg2+ in the presence of 2 mM EDTA but did not enhance the rate or maximal extent of activation. 4) The effects of Mg2+ were expressed through two independent classes of sites with affinities in the nanomolar and micromolar range. 5) A Mg2+ X guanine nucleotide complex was not the substrate for activation. The affinity of Mg2+ for nucleotides was determined as 6.25 mM GTP gamma S, 0.930 mM GTP, 0.156 mM p(NH)ppG. 6) Full activation by p(NH)ppG was completely reversible but activation by GTP gamma S was only partially reversible. These results suggest that: activation of adenylate cyclase in native membranes does not require Mg2+ or irreversible binding of the guanine nucleotide and there are two independent pathways for formation of active adenylate cyclase. A minimal mechanism for activation is discussed in light of current models.     

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 microM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, Ns, by fluoride and the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 microM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, Ni. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg2+ concentration and was reversed into a stimulation by Mn2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the Ni protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.     

Regulation of adenylate cyclase by adenosine and other agonists in rat myocardial sarcolemma

The presence of adenosine receptors coupled to adenylate cyclase in rat heart sarcolemma is demonstrated in these studies. Heart sarcolemma was isolated by the hypotonic shock-Lithium bromide treatment method. This preparation contained negligible amounts (2-4%) of contamination by other subcellular organelles such as mitochondria, sarcoplasmic reticulum, and myofibrils as verified by electron microscopic examination. In addition this preparation was also devoid of endothelial cells, since angiotensin-converting enzyme activity was not detected in this preparation. N-Ethylcarboxamide adenosine (NECA), L-N6-phenylisopropyladenosine (PIA), and adenosine N'-oxide (Ado N'-oxide) were all able to stimulate adenylate cyclase in heart sarcolemma, but not in crude homogenate, with an apparent Ka of 3-7 microM. The activation of adenylate cyclase by NECA was dependent on the concentrations of metal ions such as Mg2+ or Mn2+. The maximal stimulation was observed at lower concentrations of the metal ions (0.2-0.5 mM). At 5 mM Mg2+ or Mn2+, the stimulation by NECA was completely abolished. The stimulatory effect of NECA on adenylate cyclase was also dependent on guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine. In addition, 2'-deoxyadenosine showed an inhibitory effect on adenylate cyclase. The myocardial adenylate cyclase was also stimulated by beta-adrenergic agonists, dopamine and glucagon, and inhibited by cholinergic agonists such as carbachol and oxotremorine. The stimulation of adenylate cyclase by NECA was found to be additive with maximal stimulation obtained by epinephrine. These data suggest that rat heart sarcolemma contains adenosine (Ra), beta-adrenergic, dopaminergic, glucagon, and cholinergic receptors, and the stimulation of adenylate cyclase by epinephrine and adenosine occurs by distinctly different mechanism or adenosine and epinephrine stimulate different cyclase populations.     

Lipids associated with bovine kidney glomerular basement membranes.

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.     

Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.     

Characterization of a calmodulin-stimulated adenylate cyclase from abalone spermatozoa

Abalone sperm adenylate cyclase activity is particulate in nature and displays a high Mg2+-supported activity (Mg2+/Mn2+ = 0.8) as compared to other sperm adenylate cyclases. Approximately 90% of the enzyme activity in crude homogenates is inhibited by EGTA in a concentration-dependent manner which is overcome by added micromolar free Ca2+. The EGTA-inhibited Ca2+-stimulated enzyme activity is also inhibited by phenothiazines. Added calmodulin, however, has no effect on enzyme activity prepared from crude homogenates. Preparation of a twice EGTA-extracted 48,000 X g pellet fraction yields a particulate enzyme activity that can be stimulated 10-65% by added calmodulin in the presence of micromolar free Ca2+. Detergent extraction (1% Lubrol PX) of the EGTA-washed 48,000 X g pellet solubilizes 2-5% of the total particulate adenylate cyclase activity, and this solubilized enzyme is activated up to 125% by calmodulin. The ability of the different enzyme preparations to be stimulated by calmodulin is inversely proportional to the endogenous calmodulin concentration. Calmodulin stimulation of the Lubrol PX-solubilized enzyme is specific to this Ca2+-binding protein and is mediated as an effect on the velocity of the enzyme. This stimulation is completely Ca2+ dependent and is fully reversible. These data suggest that the control of sperm cAMP synthesis by changes in Ca2+ conductance may be mediated via this Ca2+-binding protein.     

Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes.

Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control.     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.