Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate

A general feature of membrane-bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane-bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane-bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane-bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by P_i or PP_i, and (4) and apparent loss of the Me²⁺-reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane-bound form.     

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.     

Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm. Inhibition by adenosine

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by alpha-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 mumol cyclic AMP formed mg protein-1 min-1 at 30 degrees C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30 degrees C, 20 degrees C and 4 degrees C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4 degrees C. The purified enzyme exhibited a Km value similar to that of the native enzyme (Km = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that adenosine acts on the catalytic component itself and the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10,818-10,826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.     

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.     

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.     

Isolation and characterization of the adenylate cyclase structural gene of Neurospora crassa.

A single gene (nac) encoding an adenylate cyclase was cloned from the genomic DNA library of Neurospora crassa, using the DNA fragment encoding the catalytic domain of adenylate cyclase of Saccharomyces cerevisiae as a probe. The open reading frame of this gene (6900 base pairs) was interrupted three time by introns. The protein encoded consists of 2300 amino acids and has adenylate cyclase activity. N. crassa adenylate cyclase has a high degree of homology with the catalytic domains of yeast and bovine brain adenylate cyclases.     

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 hormone-sensitive adenylate cyclase activity with resolved components of the enzyme.

Adenylate cyclase can be resolved into at least two proteins, a thermolabile, N-ethylmaleimide-sensitive component and a second protein (or proteins) that is more stable to either of these treatments. Neither component by itself catalyzes the formation of cyclic AMP using MgATP as substrate. However, mixture of the two reconstitutes MgATP-dependent fluoride- and guanyl-5'-yl imidodiphosphate (Gpp(NH)p)-stimulatable adenylate cyclase activity. The more stable component can be resolved from the first in various tissues or cultured cells by treatment of membrnes or detergent extracts with heat or N-ethylmaleimide. The two proteins have also been resolved genetically in two clonal cell lines that are deficient in adenylate cyclase activity. An adenylate cyclase-deficient variant of the S49 lymphoma cell (AC-) contains only the thermolabile activity, while the activity of the more stable protein is found in a complementary hepatoma cell line (HC-1). In addition, AC-S49 cell plasma membranes contain MnATP-dependent adenylate cyclase activity. The protein that catalyzes this reaction appears to be the same as that which can combine with the thermostable component to reconstitute Mg2+-dependent enzyme activity because both activities co-fractionate by gel exclusion chromatography and sucrose density gradient centrifugation, both activities have identical denaturation kinetics at 30 degrees C, and both activities are stabilized at 30 degrees C and labilized at 0 degree C by various nucleotides and divalent cations with similar specificity. It is thus hypothesized that the thermolabile factor is the catalytic subunit of the physiological adenylate cyclase and that the Mn2+-dependent activity is a nonphysiological expression of the catalytic protein. The thermostable moiety of the enzyme, which is proposed to serve a regulatory function, appears to consist of two functional components, based upon differential thermal lability of its ability to reconstitute hormone-, NaF-, or Gpp(NH)p-stimulated adenylate cyclase activity. These components have not, however, been physically separated. The thermolabile and thermostable components can interact in detergent solution or in a suitable membrane. Mixing of the detergent-solubilized regulatory component with AC-membranes that contain only the catalytic protein and beta-adrenergic receptors reconstitutes catecholamine-stimulatable adenylate cyclase activity; however, addition of the catalytic protein to membranes that contain receptor and the regulatory component yields MgATP-dependent enzymatic activity that is unresponsive to hormone.     

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.     

Effects of noradrenaline on the activation and the stability of brain adenylate cyclase.

At constant 1 mM-ATP, the Mg2+-saturation curves for adenylate cyclase (EC 4.6.1.1) particulate preparations obtained from corpus striatum and cortex tissues of rat brain show that addition of 0.1 mM-noradrenaline increases the apparent Vmax. for Mg2+ by 300% in corpus striatum particles, and by 280% in cortex particles. At 10 mM-MgCl2, the addition of 0.1 mM-noradrenaline increased by 800% the adenylate cyclase activity of corpus striatum particles. At all Mg2+ concentrations, the addition of 0.3 mM-CaCl2 suppressed the noradrenaline-induced stimulation of adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase activity of cortex particles. The addition of noradrenaline during a 3 h preincubation of particle preparations of brain cortex at 38 degrees C decreased by more than 4-fold the half-life of the decay of adenylate cyclase activity. The addition of MgATP protected against noradrenaline-induced inactivation.     

Chymotrypsin selectively decreases forskolin stimulation of adenylate cyclase

We studied the effects of chymotrypsin on turkey erythrocyte membrane adenylate cyclase activity. Proteolysis with chymotrypsin led to a concentration- and time-dependent increase in activation of adenylate cyclase by isoproterenol + guanine nucleotides, and fluoride, and to a decrease in activation by forskolin. Maximal effects (up to 10-fold increases in fluoride- and isoproterenol + guanine nucleotide-stimulated activity, and up to 100% inhibition of forskolin-stimulated activity) occurred under similar conditions (10-20 micrograms/ml chymotrypsin for 10-15 min at 30 degrees C). Augmentation of isoproterenol + guanosine-3'-O-thiotriphosphate (GTP-gamma-S)-stimulated activity by chymotrypsin occurred only if proteolysis preceded stimulation with isoproterenol + GTP-gamma-S. Addition of isoproterenol + GTP-gamma-S to membranes before proteolysis, however, did not prevent chymotrypsin from augmenting subsequent stimulation by these agents. In contrast, addition of forskolin during proteolysis with chymotrypsin prevented the time- and concentration-dependent decline in forskolin stimulation observed with chymotrypsin. Proteolysis decreased the magnitude of stimulation at any concentration of forskolin, but did not alter the concentration dependence of forskolin stimulation (apparent half-maximum = 3 microM). The data are consistent with the existence of a chymotrypsin-sensitive site essential for forskolin stimulation of adenylate cyclase. In view of the simultaneous effect of chymotrypsin to augment fluoride- and isoproterenol + guanine nucleotide-stimulated activities, it is highly unlikely that the site is on the stimulatory guanine nucleotide binding protein. Since forskolin is thought to act directly on the catalytic unit of adenylate cyclase, and since forskolin can protect against the effect of proteolysis with chymotrypsin, the site involved may be on the catalytic unit itself.     

Adenylate cyclase in silkworm. Properties of the enzyme in pupal fat body.

Adenylate cyclase was assayed in a sonicated preparation of silkworm pupal fat body. The adenylate cyclase was found mostly in the particulate fraction. The activity depended upon either Mg2+ or Mn2+, and the degree of stimulation by Mn2+ was 2 times greater than that by Mg2+ compared at the saturating concentrations. In the presence of Mg2+, the enzyme was inhibited by both EGTA and high concentrations of Ca2+, showing biphasical response to Ca2+. The enzyme was stimulated several-fold by NaF. The enzyme exhibited typical Michaelis-Menten kinetics and Km values were 0.13 mM for MgATP and 0.086 mM for MnATP.     

Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.     

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.     

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 beta-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.     

Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.     

Mechanism of action of forskolin on adenylate cyclase: effect on bovine sperm complemented with erythrocyte membranes

The mechanism of action of forskolin stimulation of adenylate cyclase was investigated by examining its effects on the enzyme's Mg2+ activated catalytic unit (C) from bovine sperm, both preceding and following complementation with human erythrocyte membranes as a source of guanine nucleotide regulatory protein (N). Prior to complementation, sperm C was not activated by either NaF (10 mM) or 5'-guanylyl-beta-gamma-imidodiphosphate (Gpp(NH)p, 10 microM), suggesting that functional N was not present in this preparation. Forskolin (100 microM) was also without effect on C. Following complementation of the sperm membranes with those of erythrocytes, Mg2+-dependent sensitivity to forskolin, NaF, and Gpp(NH)p was imparted to C. Our findings are incompatible with the current hypothesis that forskolin stimulates adenylate cyclase by direct activation of C. Rather, the data suggest that the activation process occurs through an effect on N or by augmentation of the interaction between the components of the adenylate cyclase complex.     

Mechanisms of heart adenylate cyclase activation by epinephrine and fluoride ions

The effects of epinephrine and NaF on the membrane preparations of adenylate cyclase from rabbit heart were studied. After preincubation with epinephrine or NaF at 37 degrees C and subsequent washing of the membranes at 4 degrees C from the effectors, adenylate cyclase passes into the activated state and loses its sensitivity to epinephrine and NaF. The effect may be "reversed" by preincubation of the membranes at 37 degrees C. The addition of ATP to the preincubation media does not affect the regulatory and catalytic properties of the enzyme. It is assumed that adenylate cyclase regulation by hormones and fluoride ions may occur without hypothetical processes of phosphorylation and dephosphorylation of the enzyme. The effect of preincubation is probably due to the temperature-dependent association and dissociation of the enzyme-receptor complex in the membrane. Epinephrine and NaF partially protect the cyclase against trypsin-induced inactivation, which is indicative of structural or conformational changes of the adenylate cyclase complex during its interaction with activators.     

Inhibition of adenylate cyclase by polyadenylate

The effects of ribo- and deoxyribonucleic acids on the activity of detergent-dispersed adenylate cyclases from rat and bovine brain were examined. Mn2+ (10 mM)-activated adenylate cyclase was inhibited by micromolar concentrations of poly(A) (IC50 congruent to 0.45 microM). This inhibition was directly due to poly(A) and was not mediated by: (a) protein contamination of the poly(A) preparation, (b) metal chelation, (c) formation of an acid-soluble inhibitor of adenylate cyclase, (d) effects on the specific activity of [alpha-32P]ATP, (e) competition with MnATP for binding to adenylate cyclase, or (f) diversion of substrate to an alternate polymerase reaction. Inhibition of adenylate cyclase by poly(A) was on the enzyme's catalytic unit, as purified preparations of the enzyme from bovine brain were inhibited by poly(A). This inhibition by poly(A) was not likely mediated via the enzyme's "P"-site, through which activated forms of the enzyme are selectively inhibited by specific adenosine phosphates. In contrast with inhibition by the "P"-site agonist 3' AMP, inhibition of adenylate cyclase by poly(A) was slow in onset and was not reversible by dilution and showed a different metal-dependence. Inhibition of adenylate cyclase was relatively specific for poly(A) as poly(U) caused less than 50% inhibition and deoxyribonucleic acids had no effect. The potency and specificity of the inhibition of adenylate cyclase by poly(A) imply a biochemically interesting interaction that is possibly also of physiological significance.     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.