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.     

Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site

6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.     

Rat brain adenylate cyclase. Further studies on its stimulation by a Ca2+-binding protein.

Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol-PX, a nonionic detergent, requires a Ca²⁺-binding protein activator for full activity (Cheung et al., 1975, Biochem. Biophys. Res. Commun.66, 1055–1062). We now show that particulate rat brain adenylate cyclase also required the activator for maximum activity. A brain particulate fraction was extracted with a hypertonic NaCl solution containing [ethyl-enebis(oxyethylenenitrilo)] tetraacetic acid. This procedure removed preferentially the activator, making adenylate Cyclase activator deficient and, consequently, dependent on an exogenous activator for maximum activity. The activator increased the V of adenylate cyclase without affecting its apparent K_m for ATP. In the presence of the activator, the enzyme was more stable against thermal inactivation, suggesting that the activator probably induced a conformational change to the enzyme. F^? and 5′-guanylylimidodi-phosphate [GMP-p(NH)p] greatly stimulated brain adenylate cyclase. Adenylate cyclase activity obtained in the presence of the activator and F^? was comparable to the summed activities of the two agents assayed separately, indicating that their effects were additive. Similarly, the effects of the activator and GMP-p(NH)p were additive. These results suggest that the action of the activator is independent of the other two ligands. Since the activator is present in excess over adenylate cyclase, the cellular flux of Ca²⁺ is believed to be important in modulating the enzyme activity. The role of the Ca²⁺/ activator is discussed with respect to cyclic AMP metabolism in brain.     

Comparative titration of arginyl residues in purified D-β-hydroxybutyrate apodehydrogenase and in the reconstituted phospholipid-enzyme complex

In order to titrate and understand the role of arginyl residues of D-β-hydroxybutyrate dehydrogenase, arginyl specific reagents: butanedione, 1,2-cyclohexanedione and phenylglyoxal were incubated with three different forms of the enzyme; native enzyme (inner mitochondrial membrane bound), purified apoenzyme (phospholipid -free) and phospholipid-enzyme complex (reconstituted active form).After complete inactivation of the enzyme by [¹⁴C]-phenylglyoxal, the number of modified arginyl residues was different: one with the lipid-free apoenzyme and three with the phospholipid-enzyme complex, suggesting a conformational change of the enzyme triggered by the presence of phospholipids.After exhaustive chemical modification either of the apoenzyme or of the phospholipid-enzyme complex with [¹⁴C]-phenylglyoxal, four arginyl residues were titrated indicating that these residues are located in the hydrophilic part of the enzyme, not interacting with phospholipids.Reconstituted enzyme inactivated by butanedione could no longer bind a pseudosubstrate (succinate) which indicates that an arginyl residue is involved in the enzyme-substrate complex formation.The values of second order rate constants of D-β-hydroxybutyrate dehydrogenase inactivation by butanedione and 1,2-cyclohexanedione were unchanged with the three enzyme forms, suggesting that phospholipids are not involved in the substrate binding mechanism.     

Biochemical characterization of histamine-sensitive adenylate cyclase in mammalian brain.

Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (K_a, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the K_m (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg²⁺ stimulated enzymatic activity; the kinetic properties of this activation by Mg²⁺ suggest the existence of a Mg²⁺ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg²⁺. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg²⁺, low concentrations of Ca²⁺ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca²⁺. Other divalent cations, including Zn²⁺, Cu²⁺, and Cd²⁺, were also inhibitory. Of the divalent cations tested, only Co²⁺ and Mn²⁺ could replace Mg²⁺ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F^?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed.     

The thermal lability of adenylate cyclase: Mechanisms of stabilization

The thermal inactivation of adenylate cyclase was investigated in human lymphocytes and in the N-protein deficient cyc-S49 mouse lymphoma cell line. The enzyme is rapidly inactivated at 37C with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 5.5 and 4.5 min respectively in human and cyc^? membranes. Thermal inactivation is prevented by at least two mechanisms. The first mechanism involves ATP which stabilizes adenylate cyclase in a concentration dependent manner similar to the K_m of ATP for cAMP formation. However, the inhibition of inactivation does not require Mg⁺⁺ while the enzyme catalysis of ATP to cAMP does. The second mechanism involves substances which activate the enzyme. The human lymphocyte enzyme is equally stabilized by either NaF, GppNHp, or forskolkin. In contrast, the cyc^? enzyme is fully stabilized by forskolin but only partially stabilized by NaF. When human erythrocyte N-protein extract is added to cyc^? membranes, NaF fully stabilizes the enzyme. These data suggest that an activated N-protein is instrumental in stabilizing adenylate cyclase and that there is some N-protein component in cyc^? membranes through which NaF may be exerting its stabilizing action.     

Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Preparative isoelectric focusing and some properties of solubilized adenylate cyclase from rat brain

A new procedure for the purification of rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) is presented. The enzyme solubilized in Lubrol PX was purified either by molecular sieving or by hydrophobic chromatography, followed by a preparative isoelectric focusing step. For this purpose, a new isoelectric focusing technique was developed which allows a good resolution of adenylate cyclase in a short period of time. When resolved by this procedure, the enzyme migrated as a single molecular species with a pI of 6.3. When isoelectric focusing was performed in the presence of EGTA, two distinct peaks of activity could be detected at pI 6.1 and 7.3. This suggests that adenylate cyclase consists of two subunits held together by divalent ions. It is shown that the purified adenylate cyclase has a smaller sedimentation coefficient and is less hydrophobic than the native one. We conclude that the adenylate cyclase containing complex was at least partially disaggregated by this procedure.     

The adenylate cyclase activity of isolated hepatocytes actions of glucagon and sodium fluoride

Isolated hepatocytes converted exogenous [α-³²P]ATP to cyclic [³²P]AMP at high rates. This system was used for kinetic studies of the effects of glucagon, fluoride, free magnesium and free ATP^4? on adenylate cyclase. In the absence or presence of glucagon, free Mg²⁺ activated adenylate cyclase by decreasing the K_m for MgATP^2? without changing V. Free ATP^4? was not a potent inhibitor of adenylate cyclase and the only effect of glucagon was to increase V.Fluoride also increased the V of adenylate cyclase, but, in contrast to the results obtained with glucagon, the effect increased as the concentration of free Mg²⁺ increased. One explanation of the effect of fluoride, consistent with the idea that free Mg²⁺ activates adenylate cyclase and free ATP is not an inhibitor, is that fluoride increases the affinity of the enzyme for Mg²⁺. Weak inhibition of adenylate cyclase by ATP^4? in the presence of fluoride cannot be excluded.     

Identification of a common domain in calmodulin-activated eukaryotic and bacterial adenylate cyclases

Bordetella pertussis and Bacillus anthracis, two taxonomically distinct bacteria, secrete adenylate cyclase toxins that are activated by the eukaryotic protein calmodulin. The two enzymes contain a well-conserved stretch of 24 amino acid residues [Escuyer et al. (1988) Gene 71, 293-298]. Antibodies have been obtained against two synthetic heptadecapeptides, covering part of the conserved sequences. The anti-peptide antibodies specifically reacted in Western blots with the rat brain adenylate cyclase as well as with the two bacterial enzymes. Anti-rat brain adenylate cyclase serum contained antibodies that were retained by the immobilized peptides, and the affinity-purified antibodies yielded the same recognition pattern of the eukaryotic enzyme as did the unfractionated serum. These results indicate that the eukaryotic adenylate cyclase contains an epitope closely related to that specified by the conserved bacterial sequence. The synthetic peptides and the bacterial adenylate cyclases appeared to compete for ATP (KD of the ATP-peptide complex ca. 0.2 mM), suggesting that the conserved sequence may be part of the substrate binding site in these two enzymes.     

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.     

Protein activator of cyclic 3':5'-nucleotide phosphodiesterase of bovine or rat brain also activates its adenylate cyclase.

Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol PX contained an activator which was separated from the enzyme by an anionic exchange resin column. Dissociation of the activator from adenylate cyclase rendered the enzyme less active, and reconstituting with an exogenous activator restored full enzyme activity. A pure protein activator of cyclic 3′:5′-nucleotide phosphodiesterase (EC 3.1.4.17) isolated from bovine brain also stimulated this adenylate cyclase. Stimulation of adenylate cyclase by the activator required Ca⁺⁺, the effect being immediate and reversible. Although the activator was specific, it lacked tissue specificity; an activator isolated from bovine brain cross-activated effectively adenylate cyclase from rat, and vice versa. These findings indicate that brain adenylate cyclase required an activator for activity and that this activator is functionally identical to the protein activator of phosphodiesterase (J.B.C. 249: 4943–4954, 1974).     

Characterization of an octopamine-sensitive adenylate cyclase from insect brain (Mamestra configurata Wlk.).

An adenylate cyclase present in the brain of the moth Mamestra configurata Wlk. that is stimulated selectively by low (micromolar) concentrations of octopamine has been characterized with respect to several properties. The optimum pH, optimum ATP:Mg2+ ratio, the concentration of ATP required for half-maximal and maximal reaction velocity, metal ion specificity, effect of NaF, and effects of GTP and 5'-guanylylimidodiphosphate were in general similar to those of catecholamine-sensitive adenylate cyclases from various regions of mammalian brain. However, ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA), a calcium chelator, stimulated both basal and octopamine-sensitive enzyme activity in the insect brain, whereas in mammalian brain EGTA is usually observed to inhibit basal activity but not catecholamine-stimulated activity. Adenylate cyclase activity of the 47,000 g particulate fraction of the insect brain was almost undetectable in the absence of added GTP. Addition of saturating concentrations (100 micrometer) of GTP to the particles restored about 30% of the basal and octopamine-sensitive enzyme activity present in the homogenate. Addition of 100,000 g supernatant to the particles doubled both basal and octopamine-sensitive enzyme activity in the presence of saturating concentrations of GTP, indicating that in addition to GTP, a cytosolic factor(s) is necessary for enhanced adenylate cyclase activity.     

Presence of an essential arginyl residue in D-β-hydroxybutyrate dehydrogenase from mitochondrial inner membrane

D-β-hydroxybutyrate dehydrogenase, a lipid requiring enzyme, is rapidly and completely inactivated by phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione. Inactivation, which occurs at the millimolar range, depends on the nature of buffer, borate ions are required to get enzyme inactivation by 2,3-butanedione. Most of the inactivation follows a pseudo first order kinetics, the stoichiometry being of one to one. Presence of NAD⁺ or methylmalonate (a substrate-like compound) prior addition of inhibitor does not affect inactivation, while methylmalonate in presence of NAD⁺ strongly protects against inactivation. Chemical modification of the enzyme does not affect K_D of NAD while K_M of β-hydroxybutyrate and Ki of methylmalonate (protecting agent) increase. In view of the high specificity of these inhibitors for arginyl residues of proteins, these results are in favour of the presence of at least one arginyl residue essential for enzyme activity and located in, or near the substrate binding site.     

Osteosarcoma cytosol factor promotes parathyroid hormone stimulation of adenylate cyclase independent of GTP

The adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1)-stimulating factor from rat osteosarcoma cytosol was purified 600-fold by ion-exchange chromatography. The factor has an apparent M_r of 20 000, is cold-labile, but retains activity at ?20°C in 10% glycerol.The factor enhanced parathyroid hormone stimulation of adenylate cyclase and restored hormone responsiveness to membranes washed with 0.5 M NaCl. These ‘GTP-like’ effects were not inhibited by 100 μM GDP-β-S, which completely abolished the GTP enhancement of both basal and hormone-stimulated adenylate cyclase.Adenylate cyclase activity in the presence of the stimulating factor was linear with time, and showed hyperbolic dependence on factor concentration. The factor also linearized (in double reciprocal plots) the downward-concave Mg²⁺-dependence of adenylate cyclase, increasing the apparent affinity of the enzyme for Mg²⁺.The presence of the factor in two clonal osteosarcoma cell lines correlated with parathyroid hormone-stimulatable adenylate cyclase. Factor stimulation was absent while GTP stimulation was retained in the hormone-nonresponsive clone. Factor and hormone sensitivity were restored by in vivo passage. This factor thus may represent a guanyl nucleotide-independent path for cellular regulation of hormone response.     

Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.     

Adenylate cyclase system of human skeletal muscle: Subcellular distribution and general properties

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

Phosphorylated adenosine derivatives as low-affinity adenosine-receptor agonists. Methodological implications for the adenylate cyclase assay.

In cellular systems provided with activatory (Ra-site) receptors for adenosine, such as rat cerebral microvessels and rat liver plasma membranes, the adenosine-receptor antagonist 8-phenyltheophylline (10 microM) significantly decreased adenylate cyclase activity if ATP was the substrate and only if GTP was present. With dATP as substrate, adenylate cyclase activities in both preparations remained unaffected by 8-phenyltheophylline. In rat cerebral-cortical membranes, with inhibitory (Ri-site) receptors for adenosine, 8-phenyltheophylline significantly enhanced adenylate cyclase activity only in the presence of GTP and if ATP was the substrate. In rat cardiac ventricular membranes, which are devoid of any adenylate cyclase-coupled adenosine receptor, the methylxanthine had no GTP-dependent effect, irrespective of the substrate used. All assay systems contained sufficiently high amounts of adenosine deaminase (2.5 units/ml), since no endogenous adenosine, formed from ATP, was found chromatographically. In order to demonstrate a direct influence of phosphorylated adenosine derivatives on adenylate cyclase activity, we investigated AMP in a dATP assay system. AMP was verified chromatographically to remain reasonably stable under the adenylate cyclase assay conditions. In the microvessels, AMP increased enzyme activity in the range 0.03-1.0 mM, an effect competitively antagonized by 8-phenyltheophylline. In the cortical membranes, 0.1 mM-AMP inhibited adenylate cyclase, which was partially reversed by the methylxanthine. The presence of GTP was again necessary for all observations. In the ventricular membranes, AMP had no effect. Since the efficacy of adenosine-receptor agonists and, probably, that of other hormones on adenylate cyclase activity can be more efficiently measured with dATP as the enzyme substrate, this nucleotide seems preferable for adenylate cyclase measurements in systems susceptible to modulation by adenosine.     

MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.