Component Of Adenylate Cyclase Research Articles

Observations on Cyclic Nucleotides in Human Semen

In agreement with earlier studies, we have found that the human seminal plasma yields considerably higher values for cyclic nucleotides when compared with spermatozoa. However, we have also shown that these values vary markedly both between different individuals and in specimens from the same individual at different times. Levels recorded for seminal plasma were 25 to 18, 937 nM of cyclic AMP and 0.54 to 3.57 nM of cyclic GMP. These levels were unrelated to those of either fructose (from the seminal vesicles) or zinc and citric acid (from the prostate). We have demonstrated also that in most specimens of seminal plasma the level of cyclic AMP remained stable during a 1‐hour incubation at 37 C or during prolonged storage at −40 C. Washed suspensions contained 13.2 to 27.2 pmol cyclic AMP/100 million spermatozoa. Adenylate cyclase activity of human semen was confined to the spermatozoa; it was several times higher in the presence of Mn2+ than Mg2+ and was unresponsive to sodium fluoride. The latter findings are consistent with enzyme activity derived from a dissociated catalytic component of adenylate cyclase.

Purification of the regulatory component of adenylate cyclase.

The regulatory component (G/F) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rabbit liver plasma membranes has been purified essentially to homogeneity. The purification was accomplished by three chromatographic procedures in sodium cholate-containing solutions, followed by three steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 mumol x min-1 x mg-1 (reconstituted adenylate cyclase activity). Purified G/F reconstitutes guanine nucleotide-, fluoride-, and hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant of S49 murine lymphoma cells. G/F also recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain three polypeptides with approximate molecular weights of 52,000, 45,000, and 35,000. The active G/F protein behaves as a multisubunit complex of these polypeptides. Treatment of G/F with [32P]NAD+ and cholera toxin covalently labels the molecular weight 52,000 and 45,000 polypeptides with 32P.

Hybridization of adenylate cyclase components by membrane fusion and the effect of selective digestion by trypsin.

Hybridization of adenylate cyclase components by membrane fusion and the effect of selective digestion by trypsin.

Characteristics of the guanine nucleotide regulatory component of adenylate cyclase in human erythrocyte membranes

This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable β-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.

The regulatory component of adenylate cyclase from uncoupled S49 lymphoma cells differs in charge from the wild type protein.

The uncoupled (UNC) variant of the S49 lymphoma possesses the components known to be required for a hormone-sensitive adenylate cyclase system, including receptors for hormones, a guanine nucleotide-binding regulatory protein (G/F), and a catalyst; however, hormones are ineffective in stimulating enzymatic activity in these cells. Two proteins (Mr = 45,000 and 55,000) are labeled with [32P]NAD when wild type or UNC membranes are incubated with the labeled nucleotide and cholera toxin (Johnson, G.L., Kaslow, H.R., and Bourne, H.R. (1978) J. Biol. Chem. 253, 7120-7123). The 45,000-dalton component appears to be a constituent of G/F. Two dimensional electrophoresis of the labeled proteins reveals that both are more acidic when derived from UNC cells.

A regulatory component of adenylate cyclase from human erythrocyte membranes.

A regulatory component of adenylate cyclase from human erythrocyte membranes.

Hydrodynamic properties of the regulatory component of adenylate cyclase.

Hydrodynamic parameters of the regulatory component of adenylate cyclase, G/F, have been estimated by gel filtration and sucrose density gradient centrifugation. In solutions containing Lubrol 12A9, the protein has an apparent molecular weight of 130,000. G/F from various sources and resolved from the catalytic moiety of the enzyme by different techniques behaves similarly. Consistent with our previous proposal that this protein is the site of action of both guanine nucleotides and fluoride, treatment with these activating ligands causes a reduction in both the sedimentation coefficient and the Stokes radius of G/F. These changes suggest a loss of mass of approximately 40,000 daltons. Nevertheless, this alteration is fully reversible when ligands are removed, even if the liganded protein is first fractionated by gel filtration or sucrose density gradient centrifugation.

Cholera toxin activation of adenylate cyclase. Roles of nucleoside triphosphates and a macromolecular factor in the ADP ribosylation of the GTP-dependent regulatory component.

Cholera toxin-catalyzed ADP ribosylation of the membrane-bound GTP-binding protein that regulates adenylate cyclase activity requires certain components of the cytosol. In broken cells it is prevented by depletion of endogenous nucleotides and is restored by the provision of GTP (1 to 3 PM half-maximum) or ITP (30 PM) although not by ATP, CTP, TTP, or UTP. An hydrolysis-resistant GTP analog, guanyl-5’-yl imidodiphosphate (Gpp(NH)p, 1 to 3 p ~ ) , also supports the toxin-catalyzed reaction, but adenyl-5’-yl imidodiphosphate is only weakly effective. The ability of nucleoside triphosphates to support toxin-catalyzed ADP ribosylation or the toxin-catalyzed activation of adenylate cyclase correlate well. Even with excess GTP or Gpp(NH)p, the rate of ADP ribosylation and adenylate cyclase activation depends upon the presence of a macromolecular component of the erythrocyte cytosol. If GTP is employed, ADP ribosylation requires the simultaneous presence of this nucleotide, cytosolic macromolecules, NAD’, toxin, and membranes. However, membranes may be predisposed to respond to the toxin by preincubating them with Gpp(NH)p and cytosolic macromolecules. Preincubated membranes, washed free of unbound nucleotide, can then be ADP ribosylated by incubation with cholera toxin and NAD+ without additional cofactors. Both Gpp(NH)p and the macromolecular fraction of the cytosol are required for such preactivation. It is complete in 15 min at 37°C and is about three times slower at 25°C. It requires magnesium, is accelerated by isoproterenol, and is inhibited by GTP or sodium fluoride. The conditions for preactivation resemble those for the specific binding of Gpp(NH)p to the regulatory component of adenylate cyclase and consequent quasi-permanent activation of the cyclase, except that direct activation of the cyclase does not depend upon a cytosolic macromolecule. We conclude that the substrate for cholera toxin is a complex of the GTP-binding protein with GTP, in some way modified by a macromolecule of the cytosol.

Transient complexes. A structural model for the activation of adenylate cyclase by hormone receptors (guanine nucleotides/irradiation inactivation)

1. The irradiation-inactivation procedure was used to study changes in the state of association of the protein components of adenylate cyclase in intact rat liver plasma membranes by measurement of alterations in the target size determined from the catalytic activity of the enzyme. 2. A decrease in target size at 30 degrees C in response to p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate) or GTP was demonstrated, which we take to reflect the dissociation of a regulatory subunit. The effect of GTP is potentiated by glucagon. This effect is not observed at 0 degrees C. 3. An increase in target size was observed in response to glucagon in the absence of guanine nucleotides, which we take to reflect the association of glucagon receptor with adenylate cyclase. 4. We propose a model for the activation of adenylate cyclase by glucagon in which the binding of the hormone to its receptor causes an initial association of the receptor with the catalytic unit of the enzyme and a regulatory subunit to form a ternary complex. The subsequent activation of the adenylate cyclase results from the dissociation of the ternary complex to leave a free catalytic unit in the activated state. This dissociation requires the binding of a guanine nucleotide to the regulatory subunit. 5. The effects of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides were examined and are discussed in relation to the irradiation-activation data. 6. The effectiveness of hormones, guanine nucleotides and combinations of hormone and guanine nucleotides as activators of adenylate cyclase in both rat liver and rat fat-cell plasma membranes was studied and the results are discussed in relation to the model proposed, which is also considered in relation to the observations published by other workers.

Reconstitution of dopamine-sensitive adenylate cyclase from dissociated components in canine caudate nucleus

The catalytic component of adenylate cyclase and [ 3H ]dopamine binding protein were solubilized with 2% Lubrol PX in the presence of NaF from the synaptic membranes of canine caudate nucleus and were separated into distinct fractions by gel exclusion chromatography on a Sephadex G-200 column. The dissociated adenylate cyclase was no longer responsive to dopamine but was considerably stimulated by 10 m m NaF. Dissociated [ 3H ]-dopamine binding protein possessed the apparent dissociation constant of 3.2 μ m for dopamine, almost identical to that of the particulate preparations. The affinities of [ 3H ]-dopamine binding protein to catecholamines and neuroleptics were also very similar to those of particulate preparations. After the adenylate cyclase and [ 3H ]dopamine binding protein were preincubated together at 4 °C for 30 min, the cyclase activity displayed a dose-dependent increase by dopamine with the K a of 1.6 μ m, the concentration of dopamine to stimulate half-maximally. Stimulation of the reconstituted adenylate cyclase by dopamine was maximally 2.7-fold and was strongly inhibited by neuroleptics such as chlorpromazine and haloperidol. These results suggest that [ 3H ]dopamine binding protein is identical to the regulatory subunit of dopamine-sensitive adenylate cyclase in the synaptic membranes of canine caudate nucleus.

A regulatory component of adenylate cyclase is located on the inner surface of human erythrocyte membranes

Summary Human erythrocyte membranes contain the guanine nucleotide regulatory component of adenylate cyclase termed N, which can be assayed by in vitro complementation of adenylate cyclase in membranes of the cyc − variant of the S49 lymphoma cell. We used inside-out (IO) and rightside-out (RO) membrane vesicles to determine the membrane orientation of N: Trypsin inactivates N in IO but not in RO vesicles. Treatment with cholera toxin and NAD + activates N in IO but not in RO vesicles. These experiments provide strong evidence that N is located on the inner surface of the human erythrocyte membrane.

Specific Mg 2+ and adenosine sites involved in a bireactant mechanism for adenylate cyclase inhibition and their probable localization on this enzyme's catalytic component

The specificity of adenosine sites involved in adenylate cyclase inhibition (P sites) is identical on membrane-bound and on solubilized enzyme. Kinetic analysis indicates that in addition to a low affinity Mg 2+ site involved in adenylate cyclase stimulation (Km = 10 mM), there is a high affinity Mg 2+ site (Km = 2.10 −4M) involved together with P sites in a bireactant mechanism for triggering adenylate cyclase inhibition. Guanyl nucleotide-binding protein does not seem to be implicated in this inhibition. We were not able to separate the catalytic component of adenylate cyclase from P sites, either on a sucrose density gradient or in gel filtration experiments. It is suggested that P sites are located on the catalytic component of the enzyme.

Fluoride and guanosine nucleotide activated adenylate cyclase from [formula omitted][formula omitted]: Reconstitution after inactivation

Detergent solubilized extracts from the liver fluke, Fasciola hepatica , contain adenylate cyclase activity which can be persistently activated by NaF or guanosine-5′-(3-0-thio) triphosphate. Treatment with heat or N-ethylmaleimide inactivated the cyclase. Activity can be partially reconstituted by mixing inactivated preparations with solubilized enzyme that has not been activated. The results provide a means to assay for adenylate cyclase components that are necessary for activation by NaF or the guanine nucleotides and may help in further resolution and purification of the components necessary for cyclase activity.

Guanine nucleotide‐controlled interactions between components of adenylate cyclase

Guanine nucleotide‐controlled interactions between components of adenylate cyclase

Effects of cholera toxin and guanosine 5′-[βγ-imido]triphosphate on β-adrenergic-receptor affinity

A comparison was made of the effects of cholera toxin and p[NH]ppG on the binding affinity of beta-adrenergic receptors in toad erythrocyte membranes. This was determined by studying the ability of isoproterenol and propranolol to compete for the receptor with (-)-[3H]dihydroalprenolol. p[NH]ppG decreased the receptor affinity for the agonist isoproterenol (i.e. a 'right' shift in the displacement-concentration curve), but was without effect on the affinity for the antagonist propranolol. Toad erythrocyte membranes after treatment with cholera toxin exhibited increased receptor affinity for isoproterenol (i.e. a 'left' shift in the displacement curve), but did not affect the affinity for propranolol. p[NH[ppG was able to exert its right shift even in cholera-toxin treated membranes. The ability of cholera toxin to alter beta-adrenergic-receptor affinity is interpreted as further evidence that the toxin affects the nucleotide-regulatory component of adenylate cyclase. The regulatory component affected may be the catecholamine-sensitive guanosine triphosphatase.

Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase.

Cholera toxin, using [32P]NAD+ as substrate, specifically radiolabels at least two proteins in plasma membranes of wild type S49 mouse lymphoma cells. The toxin-specific substrates are detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as bands corresponding to molecular weights of 45,000 and a doublet of 52,000 to 53,000. Membranes of two other cell types exhibit similar patterns of radiolabeled bands specifically produced by incubation with cholera toxin: the "uncoupled" variant S49 cell, which possesses adenylate cyclase activity unresponsive to hormones, and the HTC4 rat hepatoma cell, which lacks detectable catalytic adenylate cyclase activity but contains components of the cyclase system necessary for regulation by guanyl nucleotides and NaF. Little or no toxin-specific radiolabeling is observed in membranes of a fourth cell type, the adenylate cyclase activity-deficient S49 variant, which functionally lacks components of the cyclase system involved in cholera toxin action and regulation by guanyl nucleotides and NaF. The toxin-specific labeling pattern is not observed in membranes prepared from wild type S49 cells previously treated with cholera toxin in culture. One or both of the toxin substrates thus appears to be involved in regulation of adenylate cyclase by guanyl nucleotides and fluoride ion.

Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase.

The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process tha produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.

β-Adrenergic receptor-sensitive adenosine cyclic 3′,5′-monophosphate accumulation in homogenates of the rat corpus striatum. A comparison with the dopamine receptor-coupled adenylate cyclase

The conversion of newly formed [ 3H]adenosine triphosphate (ATP) to [ 3H]adenosine cyclic 3′,5′-monophosphate (cAMP) was studied in osmotically shocked, crude synaptosomal fractions of the rat corpus striatum. Of the β-hydroxylated catecholamines tested, the potency of isoproterenol (Ec 50 about 0.01 μM) was greater than that of norepinephrine (Ec 50 about 1.0 μM). Stereoselectivity was displayed with the (−)isomer of isoproterenol being more potent that its (+)isomer. Of the non-β-hydroxylated catecholamines studied, N-isopropyldopamine demonstrated greater potency than dopamine, whereas apomorphine was inactive. No “additive stimulatory effect” was observed when dopamine was combined with a maximum effective concentration of isoproterenol. The β-adrenergic antagonist propranolol, completely blocked both the dopamine- and norepinephrine-induced increases in cAMP formation. Properties characteristic of a β-type system were, likewise, exhibited by homogenates of both the cerebral cortex and hindbrain. Furthermore, the conversion to [ 3H]cAMP was increased only slightly (about 16 per cent) by exposure to sodium fluoride, and the stimulatory response to isoproterenol was not altered significantly by high concentrations of ATP but was lost upon sonication of the tissues. In contrast, by assaying adenylate cyclase activity with a high saturating concentration of exogenous [ 3H]ATP, striatal and cerebral cortical homogenates exhibited responses characteristic of a specific dopamine receptor-coupled adenylate cyclase. Sonication of tissues did not alter the stimulatory effect of dopamine, and sodium fluoride produced about a 2-fold stimulation of the adenylate cyclase activity. Thus, findings in osmotically shocked, crude synaptosomal fractions of the corpus striatum suggest that the paniculate component of adenylate cyclase, which utilizes exogenous ATP as substrate, exhibits properties characteristic of a dopamine receptor-coupled adenylate cyclase. In contrast, the membrane-enclosed, particulate component of adenylate cyclase, which utilizes endogenously synthesized ATP as substrate, conforms to criteria identified with a β-adrenoreceptor-linked adenylate cyclase.

Neuronal localization of the enhanced adenylate cyclase responsiveness to catecholamines in the rat cerebral cortex following reserpine injections

Rats were injected daily with reserpine (2.5 mg/kg) for four days. The animals were sacrificed 4 hr subsequent to the last injection. Studies were performed as a means to determine alterations in catecholamine sensitivity of adenylate cyclase in the cerebral cortex following reserpine. In incubated tissue slices from reserpinized animals a greater accumulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in response to norepinephrine and isoproterenol was seen. In control animals both α-(phentolamine) and β-blocking agents (propranolol) inhibited norepinephrine-induced cyclic nucleotide accumulation, while in the reserpine-injected rats only propranolol was effective. The accumulation of cyclic AMP elicited by isoproterenol was inhibited by propranolol alone whether or not reserpine was administered. In cortical homogenates adenylate cyclase responsiveness to norepinephrine, isoproterenol and dopamine was enhanced in animals pretreated with reserpine. An identical observation was found in isolated neuronal fractions, although increased sensitivity of the enzyme to dopamine was now absent. Neither a- nor β-blocking agents were effective in broken cellular preparations. These data are interpreted to suggest that following depletion of catecholamines by reserpine, the β-receptor component of adenylate cyclase in the cortical neurones becomes hypersensitive to stimulation by catecholamines.

Influence of tricyclic antidepressants on the adenylate cyclase-phosphodiesterase system in the rat cortex

The actions of ten tricyclic antidepressant drugs were evaluated with respect to inhibition of the adenylate cyclase-cyclic adenosine 3': 5'-monophosphate (cyclic AMP)-phosphodiesterase system in various preparations of rat cerebral cortex. At highest concentrations (10 −4M) all drugs exhibited antagonism of the stimulation of cyclic AMP accumulation by norepinephrine in incubated tissue slice (whole cell) preparations. In these experiments iprindole and desipramine (10 −4–10 −8M) were the most potent agents, while lesser degrees of inhibition were seen with amitriptyline, chlorimipramine, nortriptyline, protriptyline, imipramine, opipramol, 2-hydroxyimipramine and 2-hydroxydesipramine in approximately that order. On the other hand, in broken cellular preparations from isolated neuronal and glial-enriched fractions, extremely high concentrations (1–10 mM) of the tricyclic antidepressants were necessary to inhibit by 50% the basal, fluoride-sensitive (catalytic site) and norepinephrine-sensitive (receptor) components of adenylate cyclase. In similar studies with isolated neuronal and glial fractions somewhat lower concentrations (0.1–1 mM) of tricyclic antidepressants were required to inhibit by 50% the total phosphodiesterase activity. The data indicate a basic problem relating to drug experimentation on cyclic nucleotide systems. Results obtained from intact cellular investigations i.e. tissue slices may not always be extrapolated to similar findings with enzyme preparations derived from broken cells.