The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity. Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed. These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.     

Eukaryotic-like adenylyl cyclases in Mycobacterium tuberculosis H37Rv: cloning and characterization.

Screening the Mycobacterium tuberculosis H37Rv genomic library for complementation of catabolic defect for cAMP-dependent expression of maltose operon produced the adenylyl cyclase gene (Mtb cya, (1997)) annotated later as Rv1625c (Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, et al. (1998) Nature 393, 537-544). The deduced amino acid (aa) sequence (443 aa) encoded by Mtb cya contains a single hydrophobic domain of six transmembrane helices (152 aa) in the amino-terminal half of the protein. Flanking this domain are an arginine-rich (17%) amino-terminal cytoplasmic tail (46 aa) and a carboxyl-terminal cytoplasmic domain (245 aa) with extensive homology to the catalytic core of eukaryotic adenylyl cyclases. Site-directed mutagenesis of Arg(43) and Arg(44) to alanine/glycine showed a loss of adenylyl cyclase activity, whereas mutagenesis to lysine restored the activity. Hence it is proposed that the formation of the catalytic site in Mtb adenylyl cyclase requires an interaction between Arg(43) and Arg(44) residues in the distal cytoplasmic tail and the carboxyl-terminal cytoplasmic domain. Mtb adenylyl cyclase activity at the physiological concentration of ATP (1 mm) was 475 nmol of cAMP/min/mg of membrane protein in the presence of Mn(2+) but only 10 nmol of cAMP/min/mg of membrane protein in the presence of Mg(2+). The physiological significance of the activation of Mtb adenylyl cyclase by Mn(2+) is discussed in view of the presence of manganese transporter protein in mycobacteria and macrophages wherein mycobacteria reside.     

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides

The adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is localized to the plasma membrane of the cell. The enzyme utilizes Mn2+/ATP as substrate and free Mn2+ ions as an effector. Unlike the baker yeast Saccharomyces cerevisiae, S. pombe adenylyl cyclase does not utilize Mg2+/ATP as substrate and the activity is not stimulated by guanyl nucleotides. The optimal pH for the S. pombe adenylyl cyclase activity is 6.0. The activity dependence on ATP is cooperative with a Hill coefficient of 1.68 +/- 0.14.     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.     

Manganese and manganese-ATP interactions with bovine sperm adenylate cyclase

The adenylate cyclase of mammalian spermatozoa shares some of the properties of the isolated catalytic component from somatic cell adenylate cyclases. One of these properties is the large apparent stimulation by Mn2+. We have used the direct linear plot according to Eisenthal and Cornish-Bowden to explore whether this apparent stimulation is due to direct stimulation by Mn2+ or due to complexation of free ATP, a postulated inhibitor of cyclase activity. We have observed the activity of the particulate adenylate cyclase from bovine caudal epididymal spermatozoa as a function of calculated equilibrium values for the concentrations of Mn2+, free ATP, and the enzyme's substrate, the manganese-ATP complex. Direct linear plots for activity and substrate concentration over the apparent inhibitory concentration range of free ATP give the pattern expected for a hyperbolic substrate response. By contrast, direct linear plots in which Mn2+ concentration varies over its apparent stimulatory range show that as Mn2+ concentration increases, activities are higher than would be predicted for a hyperbolic substrate response. We conclude that for particulate bovine sperm adenylate cyclase, free ATP is not strongly inhibitory, and Mn2+ is a positive effector, reaching half-maximal stimulation at 0.2 mM. The unique nature of the sperm adenylate cyclase and its possible regulation by Mn2+ under physiological conditions is discussed.     

Characterization of an adenylyl cyclase activity in particulate preparations from epimastigote forms of Trypanosoma cruzi.

Particulate preparations from epimastigote forms of Trypanosoma cruzi contain an adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) which could be stored at --20 degree C and resisted 5 cycles of freezing and thawing over 10 days without significant loss of activity. The enzyme reaction strictly required Mn2+, had a pH optimum of 7.7 and was not inhibited or stimulated by NaF. Particles prepared in the presence of 10 mM Mn2+ or Mg2+ were 3--4 times more active than particles prepared in the absence of these cations. However, Mg2+ could not substitute for Mn2+ during enzyme assay nor did it enhance activity in the presence of saturating concentrations of Mn2+. The binary complex Mn - ATP2- was shown to be the true substrate for the adenylyl cyclase and free ATP was highly inhibitory. Plots of enzyme activity against equimolar concentrations of ATP - Mn gave sigmoid curves with n values in Hill plots ranging between 1.5 and 2.0. Excess Mn2+ activated the cyclase catalyzed reaction at low but not at high concentrations of ATP - Mn. In the presence of an excess of 1 mM Mn2+, which transforms 97% of the added ATP to productive Mn - ATP2- complex, the substrate saturation curve assumed a Michaelian pattern with an apparent Km =0.2 mM.     

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.     

Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.     

Regulation of the Ca2+-inhibitable adenylyl cyclase type VI by capacitative Ca2+ entry requires localization in cholesterol-rich domains

The endogenous Ca(2+)-inhibitable adenylyl cyclase type VI of C6-2B glioma cells is regulated only by capacitative Ca(2+) entry and not by a substantial elevation of [Ca(2+)](i) from either intracellular stores or via ionophore-mediated Ca(2+) entry (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). The present studies explored the role of cholesterol-rich domains in maintaining this functional association. The cholesterol-binding agent, filipin, profoundly inhibited adenylyl cyclase activity. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin did not affect forskolin-stimulated adenylyl cyclase activity and did not affect capacitative Ca(2+) entry. However, cholesterol depletion completely ablated the regulation of adenylyl cyclase by capacitative Ca(2+) entry. Repletion of cholesterol restored the sensitivity of adenylyl cyclase to capacitative Ca(2+) entry. Adenylyl cyclase catalytic activity and immunoreactivity were extracted into buoyant caveolar fractions with Triton X-100. The presence of adenylyl cyclase in such structures was eliminated by depletion of plasma membrane cholesterol. Altogether, these data lead us to conclude that adenylyl cyclase must occur in cholesterol-rich domains to be susceptible to regulation by capacitative Ca(2+) entry. These findings are the first indication of regulatory significance for the localization of adenylyl cyclase in caveolae.     

Signaling pathways for modulation of mouse sperm motility by adenosine and catecholamine agonists

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.     

Inhibition of adenylyl cyclase activity in brain membrane fractions by arachidonic acid and related unsaturated fatty acids

Pretreatment of mouse brain membranes with arachidonic acid (AA) and related unsaturated fatty acids at 30 degrees C for 10 min decreased basal activity and isoproterenol/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and forskolin-stimulated activities of adenylyl cyclase to a level less than 5% of control. The presence of the carboxyl group on the fatty acids was essential for the inhibition, because no such inhibition was found with ethyl arachidonate or AA attached to diacylglycerols and phospholipids. The AA-mediated inhibition was observed when the activity was measured in the presence of Mn2+ or forskolin and was insensitive to pertussis toxin or guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), indicating a mechanism independent of GTP-binding proteins. In addition, the fact that stimulators of the adenylyl cyclase catalytic unit, ATP, GTP gamma S and forskolin, when present during pretreatment, attenuate the inhibitory effect of AA may suggest that the catalytic unit is a target of AA. Bovine serum albumin suppressed the inhibition when present in the mixtures for pretreatment, but could not restore the adenylyl cyclase activity that had been reduced by AA, indicating an irreversible inhibition by AA. The effect of AA was found to be additive to P-site-mediated inhibition. The present study suggests the existence of another mechanism of regulation of adenylyl cyclase by unsaturated fatty acids.     

Activation of Adenylyl Cyclase by Preincubation of Rat Cerebral-Cortical Membranes Under Phosphorylating Conditions: Role of ATP, GTP, and Divalent Cations

Abstract: The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl₂ resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation-dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg²⁺ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral-cortical membranes with adenosine-5′-0-(3-thiophosphate) (ATPγS), but this activation was not reversed by prolonged preincubation times. The activation by ATPγS was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATPγS to guanosine-5′-0-(3-thiophosphate) (GTPγS), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 μ.M GTP and 10 mM MgCl₂ also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of ³⁵S from [³⁵S]ATPγS into a protein component with apparent M_r of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATPγS. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [³⁵S]ATPγS. Endogenous activity utilizing [γ-³²P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane-bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent M_r of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP-preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation.     

Adenylyl cyclase and G-proteins in Phytomonas

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.     

Discriminating between capacitative and arachidonate-activated Ca(2+) entry pathways in HEK293 cells.

We have recently questioned whether the capacitative or store-operated model for receptor-activated Ca(2+) entry can account for the influx of Ca(2+) seen at low agonist concentrations, such a those typically producing [Ca(2+)](i) oscillations. Instead, we have identified an arachidonic acid-regulated, noncapacitative Ca(2+) entry mechanism that appears to be specifically responsible for the receptor-activated entry of Ca(2+) under these conditions. However, it is unclear whether these two systems reflect the activity of distinct entry pathways or simply different mechanisms of regulating a common pathway. We therefore used the known selectivity of the Ca(2+)-stimulated type VIII adenylyl cyclase for Ca(2+) entry occurring via the capacitative pathway (Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444) to attempt to discriminate between these two entry mechanisms in HEK293 cells. Consistent with the earlier reports, we found that thapsigargin induced an approximate 3-fold increase in adenylyl cyclase activity that was unrelated to global changes in [Ca(2+)](i) or to the release of Ca(2+) from internal stores but was specifically dependent on the induced capacitative entry of Ca(2+). In marked contrast, the arachidonate-induced entry of Ca(2+) completely failed to affect adenylyl cyclase activity despite producing a substantially greater rate of entry than that induced by thapsigargin. These data demonstrate that the arachidonate-activated entry of Ca(2+) occurs via an entirely distinct influx pathway.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.     

A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin

Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [α-³²P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K_M), the v₀^max value of ATP turnover, and the IC₅₀ values for three inhibitors of EF by this newly developed fluorescent method.     

Studies of the guanylate cyclase of the social amoeba Dictyostelium discoideum

Observations on the properties of the guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) of the social amoeba Dictyostelium discoideum are reported. On the basis of similarities in kinetic and fractionation properties, it is shown that the activity from vegetative cells and the sixfold higher activity from starved cells appear to be due to the same enzyme. Most of the activity is found to be soluble, and by gel exclusion chromatography a molecular weight of 250,000 has been estimated for this form. As the enzyme shows considerably more activity with Mn+2 than Mg+2, the Km for Mn+2 activation was determined (700 microM), and compared to the levels of total cell Mn+2 (10 microM) and Mg+2 (3mM). These data suggest that Mg+2 is probably the physiological cofactor. A previous report [J. M. Mato, (1979) Biochem. Biophys. Res. Commun. 88, 569-574] that the enzyme is activated about twofold by ATP was confirmed; but contrary to that report, activation by the ATP analog 5'-adenylyl-imidodiphosphate was also obtained. Since this analog does not donate its phosphate in kinase reactions, it is likely that ATP activates the guanylate cyclase by direct binding rather than by phosphorylation. The known in vivo agonist of the guanylate cyclase, cAMP, did not activate the enzyme in vitro, either alone or in various combinations with calcium, calmodulin, ATP, and phospholipids.     

Studies of the thermal inactivation of cardiac adenylyl cyclase: evidence for a conformational change in the reaction mechanism

Membrane bound cardiac adenylyl cyclase was shown to undergo a spontaneous and irreversible thermal inactivation with a t1/2 of approximately 10 min. The loss of activity could not be explained by the action of endogenous proteases. Repeated freeze-thaw of membrane preparations resulted in a much increased rate of thermal inactivation (t1/2 = approx. 2 min). ATP, adenylimidodiphosphate, ADP, and PPi protected the enzyme from thermal inactivation with dissociation constants (Kd) of 193, 5.04, 84.4, and 6.3 microM, respectively. 5'-AMP and cyclic AMP were ineffective as protectors at concentrations as high as 3 mM. Activators of adenylyl cyclase such as Mn2+, forskolin, 5-guanylylimidodiphosphate, and NaF and 9 mM Mg2+ protected against thermal inactivation with Kd of 16.8 microM, 8.81 microM, 0.23 microM and 1.04 mM, respectively. Mg2+ alone was without effect. Thermal inactivation was first order under all conditions tested. Arrhenius plots of the rate constants for inactivation vs temperature were linear. The increased stability of ligand bound adenylyl cyclase was shown to be associated with an increased free energy of activation (delta G 0). These data provide evidence for the existence of two distinct conformations of cardiac adenylyl cyclase based on different susceptibilities to thermal inactivation. These enzyme conformations, termed E1 and E2, may be important reaction intermediates. The thermal stability of E1 was highly influenced by the enzyme's membrane lipid environment. The formation of E2 from E1 was enhanced by interaction with substrate, PPi, activators of adenylyl cyclase, and by interaction with dissociated stimulatory guanine nucleotide binding protein-alpha beta gamma heterotrimers.     

Zinc inhibition of adenylyl cyclase correlates with conformational changes in the enzyme

We have previously demonstrated that Zn(2+) inhibits hormone and forskolin stimulation of cAMP synthesis in intact N18TG2 cells, corresponding plasma membranes, and of recombinant adenylyl cyclase isoforms. If, however, the enzyme is pre-activated by hormone or forskolin, Zn(2+) inhibition is attenuated [J. Biol. Chem. 277 (2002) 11859]. We have extended our analyses of this inhibition to investigations of soluble adenylyl cyclase, composed of the CI and CII domains of the full-length protein. The properties of Zn(2+) inhibition of the soluble enzyme parallel that of the full-length protein, including the fact that inhibition is not competitive with Mg(2+). By monitoring intrinsic and extrinsic fluorescence, we demonstrate changes in enzyme conformers in response to the addition of varied effectors. The data suggest a possible mechanism by which Zn(2+) inhibits adenylyl cyclase activity.