Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

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

Postmortem Stability of Dopamine-Sensitive Adenylate Cyclase, Guanylate Cyclase, ATPase, and GTPase in Rat Striatum

The stability of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase was measured in homogenates of rat striatal tissue frozen from 0 to 24 h postmortem. ATPase, GTPase, and Mg2+-dependent guanylate cyclase activities showed no significant change over this period. Mn2+-dependent guanylate cyclase activity was stable for 10 h postmortem. Basal and dopamine-stimulated adenylate cyclase activity decreased markedly during the first 5 h. However, when measured in washed membrane preparations, these adenylate cyclase activities remained stable for at least 10 h. Therefore, the postmortem loss of a soluble activator, such as GTP, may decrease the adenylate cyclase activity in homogenates. These results are not consistent with an earlier suggestion that there is a postmortem degradation of the enzyme itself. Other kinetic parameters of dopamine-sensitive adenylate cyclase can also be measured independently of postmortem changes. Thus, it is possible to investigate kinetic parameters of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in human brain obtained postmortem.     

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.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis.

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.     

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.     

Parathyroid hormone increases sodium/calcium exchange activity in renal cells and the blunting of the response in aging

Na+-dependent Ca2+ efflux was demonstrated in cells isolated from the rat renal cortex, suggestive of the presence of a Na+/Ca2+ exchange carrier in the cells. Parathyroid hormone, when incubated with the cells in vitro, increased Na+-dependent Ca2+ efflux about 60%. The effect of the hormone was specific for biologically active parathyroid hormone analogs and could be mimicked by cyclic nucleotides and forskolin. The effects of parathyroid hormone concentration on Ca2+ efflux and cyclic AMP formation were similar. These findings would be consistent with the view that the cyclic nucleotide might act as the intracellular messenger to increase Na+/Ca2+ exchange activity. Cells isolated from parathyroidectomized rats had decreased Na+-dependent Ca2+ efflux. When these cells were treated in vitro with parathyroid hormone, Na+-dependent Ca2+ efflux was enhanced to the same rate as found with cells from sham-operated animals. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent (24 months) rats. Basal Na+-dependent Ca2+ efflux and Na+-independent Ca2+ efflux were not altered in the aged animal. Parathyroid-stimulated adenylate cyclase was also decreased in aging. In contrast, forskolin-stimulated Na+-dependent Ca2+ efflux and adenylate cyclase did not change with senescence. These findings would be compatible with a mechanism of desensitization that occurred at the level of the receptor or hormone-receptor coupling to adenylate cyclase. These results may be of physiological significance in understanding calcium homeostasis and the imbalances in mineral metabolism associated with old age.     

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.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000.Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg²⁺ as well as with Mn²⁺. Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase.These experiments show that adenylate and guanylate cyclase activities are physically separable.     

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.     

Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.     

Phorbol esters induce two distinct changes in GH3 pituitary cell adenylate cyclase activity

Phorbol esters alter cyclic AMP levels in a number of tissues, including the anterior pituitary. We report that membrane preparations from GH3 cells exposed to phorbol esters exhibit decreased vasoactive intestinal peptide (VIP)-stimulated and enhanced forskolin-stimulated adenylate cyclase activity. The responsiveness of adenylate cyclase activity to NaF, guanylyl-imidodiphosphate, and Mn2+ was also reduced by phorbol ester treatment. The ability of somatostatin to inhibit forskolin-stimulated adenylate cyclase activity was reduced while phorbol ester exposure had no apparent effect on somatostatin inhibition of VIP-stimulated adenylate cyclase activity. We suggest that protein kinase C alters at least two distinct components of the adenylate cyclase system. One modification disrupts hormone receptor-Gs interaction (lowering VIP efficacy) and the second perturbation augments the activity of the adenylate cyclase catalytic subunit.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

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.     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

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

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

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.     

A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose- and acidification-induced cAMP signalling and not the basal cAMP level

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.     

Microtubule-associated adenylate cyclase

Twice-cycled bovine brain or rat brain microtubule protein contains an adenylate cyclase activity that passes 0.2 micron filters, is activated 2-7-fold by 30 microM forskolin, shows modest stimulation by fluoride (especially in the presence of added AI3+), but is virtually insensitive to added guanine nucleotides. The activity is insensitive to various hormones or Ca2+/calmodulin. The adenylate cyclase is active with both Mg2+ and Mn2+ but activity is less in the presence of Mg2+ than with Mn2+. The cyclase is inhibited by agonists of the adenosine P site. It is proposed that the catalytic unit of adenylate cyclase and probably small quantities of the guanine nucleotide regulatory protein, Ns, are cycled along with microtubules.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase.

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.