Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.     

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.     

Stimulation of partially purified adenylate cyclase from bull sperm by bicarbonate

Solubilized and partially purified adenylate cyclase from bull sperm was found to be specifically activated (up to 6-fold) by sodium bicarbonate (NaHCO3) and to a lesser extent by NaNO3. Other sodium salts were either ineffective (e.g. NaCOOH) or inhibitory (e.g. NaHSO3, NaHSO4 and Na2B4O7). Stimulation by NaHCO3 was dose-dependent in the range of 0-40 mM and was greater when enzyme activity was assayed in the presence of magnesium as compared with manganese ions. Bicarbonate seems to affect maximal enzyme velocity (Vmax) and has no effect on the Km of adenylate cyclase for Mn-ATP. Stimulation of adenylate cyclase by NaHCO3 coincided with the elution pattern of the enzyme as recorded following chromatography on DEAE-cellulose or gel filtration on BioGel P-100. These results suggest that in the course of stimulation of sperm adenylate cyclase, bicarbonate is likely to interact directly with the enzyme. Furthermore, this intrinsic and unique property of sperm adenylate cyclase may explain results reported by others on the stimulation of cAMP production by bicarbonate in intact and broken sperm preparations and suggest a biochemical basis for enhanced sperm motility associated with high bicarbonate concentrations.     

Role of heme in the regulation of human and rat platelet soluble guanylate cyclases.

The chromatography of soluble human and rat platelet guanylate cyclases (105000 g supernatants) on DEAE-cellulose in 50 mM Tris HCl buffer, containing 0.22 M NaCl, has yielded virtually identical elution profiles, each with two protein peaks (I and II). Only peak II was found to have guanylate cyclase activity. Experiments with human platelets showed that inactive protein peak I inhibited the activity of guanylate cyclase preparation (peak II) and restored the already lost ability of the enzyme to be activated by sodium nitroprusside. In experiments with rat platelets, inactive fraction I had no effect on guanylate cyclase activity (peak II), and the enzyme was not activated by sodium nitroprusside either before or after DEAE-cellulose. 105000g supernatant of human platelets had an absorbance maximum at 415 nm (Soret band), which disappeared from the spectrum of the active fraction (II) but was found in the spectrum of the inactive (inhibitory) fraction I. Experiments with rat platelets demonstrated the absence of Soret band in the corresponding spectra. It was concluded that, contrary to the generally accepted notion, heme is not a prosthetic group of the soluble rat platelet guanylate cyclase.     

Insulin release from mouse islets. Effect of glucose and hormones on adenylate cyclase

The adenylate cyclase system of normal mouse islets was characterized. The pH optimum of the system was 7.6. The enzyme preparation contained particulate phosphodiesterase activity. This could be removed by treatment with 0.4% (v/v) Triton X-100 or inhibited by 8mm-theophylline in the presence of 2mm-cyclic AMP (adenosine 3':5'-cyclic monophosphate). ATP at 0.32mm produced one-half maximal enzyme activity. The enzyme was stimulated in the presence of F(-) and strongly inhibited by Ca(2+). The isolated enzyme retained hormonal sensitivity and was stimulated by glucagon, pancreozymin and secretin at physiological concentrations. Glucose at 17mm, 8mm and 2mm had no direct effect on the activity of the enzyme; neither did galactose at the same concentrations. Groups of islets incubated in 17mm- or 2mm-glucose for 5 or 15min and then homogenized and assayed for adenylate cyclase activity showed no differences in adenylate cyclase activity. The results suggest that the mechanism of glucose-mediated insulin release is not via the adenylate cyclase system. Hormones, however, could mediate insulin secretion via their effects on the adenylate cyclase system.     

Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclases

Previous studies using calmodulin-Sepharose affinity chromatography have suggested that bovine brain may contain a mixture of calmodulin-sensitive and -insensitive adenylate cyclase activities (Wescott, K. R., La Porte, D. C., and Storm, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 82, 3086-3090). In this study, mice were immunized with a purified preparation of the calmodulin-sensitive adenylate cyclase from bovine brain, and a polyclonal antiserum was obtained which was specific to the calmodulin-sensitive form of the enzyme. The antiserum was not inhibitory and precipitated enzyme activity from a homogeneous preparation of the calmodulin-sensitive adenylate cyclase catalytic subunit. Furthermore, the antiserum did not interact with calmodulin-insensitive adenylate cyclase which was resolved from the calmodulin-sensitive form of the enzyme by calmodulin-Sepharose affinity chromatography. Since the only polypeptide specifically precipitated by the antiserum had an Mr of 135,000, which was identical to the Mr of the catalytic subunit of the enzyme, it is concluded that the antiserum interacted directly and specifically with the catalytic subunit of the calmodulin-sensitive isozyme of adenylate cyclase. Detergent-solubilized membranes from several rat tissues were examined for the presence of calmodulin-sensitive adenylate cyclase using anti-calmodulin-sensitive adenylate cyclase antiserum. Approximately 40-60% of the total adenylate cyclase activity of rat brain and kidney were immunoprecipitated by the antiserum, whereas liver and testes contained no detectable calmodulin-sensitive adenylate cyclase. Approximately 15% of the total adenylate cyclase activity in rat heart and lung was the calmodulin-sensitive form. These data indicate that the calmodulin-sensitive and insensitive adenylate cyclases from bovine brain are immunologically distinct and support the proposal that there may be two or more distinct adenylate cyclase isozymes in brain.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Adenylate cyclase system of bovine adrenal plasma membranes.

The adenylate cyclase system present in a preparation enriched in plasma membranes derived from bovine adrenal cortex was investigated in considerable detail. This system is stimulated by adrenocorticotropic hormone (ACTH), by biologically active analogs of this hormone, and by fluoride ion. The preparation contains sodium-potassium- and magnesium-dependent ATPases that are markedly inhibited by 50 mM sodium fluoride. Incorporation of a pyruvate phosphokinase ATP generating system into the adenylate cyclase assay medium provided constant substrate levels. In the presence of the ATP generating system, the rate of cyclic AMP formation (basal, fluoride, and ACTH-activated) was proportional to enzyme concentration and was linear with time. Proportionality with respect to enzyme concentration as concerned the hormone-activated adenylate cyclase was achieved only when the ratio of hormone to enzyme protein was kept constant. The temperature optimum of the adenylate cyclase, basal or activated, was approximately 30 degrees. Michaelis-Menten kinetics were observed when the ratio of Mg2+ to ATP was approximately 6:1. Both calcium and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid completely inhibited the adenylate cyclase system at concentrations of 5 and 0.5 mM, respectively. GTP was inhibitory at concentrations of 10-2 M but had little effect at lower concentrations. Freezing in liquid nitrogen and storage at -60 degrees exerted little effect on the fluoride-stimulated enzyme but lowered hormone stimulated activity. Preincubation in the presence of ACTH afforded a high degree of stabilization of the enzyme system while preincubation with a biologically inactive analog afforded no protection.     

Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the bacterium responsible for whooping cough, releases a soluble, calmodulin-sensitive adenylate cyclase into its culture medium. B. pertussis mutants deficient in this enzyme are avirulent, indicating that the adenylate cyclase contributes to the pathogenesis of the disease. It has been proposed that B. pertussis adenylate cyclase may enter animal cells and increase intracellular adenosine cyclic 3',5'-phosphate (cAMP) levels. We have purified the enzyme extensively from culture medium using anion-exchange chromatography in the presence and absence of calmodulin and gel filtration chromatography. The enzyme was purified 1600-fold to a specific activity of 608 mumol of cAMP min-1 mg-1 and was free of islet activating protein. The molecular weight of the enzyme was 43 400 in the absence of calmodulin and 54 200 in the presence of calmodulin. The Km of the bacterial enzyme for adenosine 5'-triphosphate was 2.0 mM, whereas the Km of the calmodulin-sensitive adenylate cyclase from bovine brain was 0.07 mM. Although the enzyme was not purified to homogeneity, its turnover number of 27 000 min-1 is the highest documented for any adenylate cyclase preparation.     

Inhibition of a parathyroid hormone-stimulated renal adenylate cyclase by cyclic AMP.

Plasma membranes were isolated from bovine renal cortex. This particulate, adenylate cyclase-containing fraction was stimulated to produce cyclic AMP by parathyroid hormone and fluoride. When the time-course of adenylate cyclase activity was investigated, it was found that while PTH-stimulated cyclic AMP production comes to a halt in about 15 minutes after the initiation of the reaction, fluoride-stimulated activity continues unabated for at least an hour. Experiments to determine the cause of this showed that the cyclase enzyme is not degraded under our experimental conditions, but is inhibited by a soluble, unbound product of the reaction which requires ATP for its synthesis. In our experiments degradation of parathyroid hormone was relatively slow and could not account for the rapid inhibition of PTH-stimulated cyclase activity. Of the various agents tested, cyclic AMP was found capable of inhibiting PTH-stimulated cyclic AMP production by our purified membrane preparation. Half-maximal inhibition was observed at around 10(-6) M concentrations of the nucleotide. Pyrophosphate, adenosine, 5'-AMP and ADP had no effects. The significance of these results in relation to the regulation of adenylate cyclase activity is discussed.     

Guanylate cyclase from the rat renal medulla. Physical properties and comparison with adenylate cyclase.

Guanylate cyclase from the rat renal medulla is found in both the soluble and particulate fractions of the cell. Sucrose density gradient centrifugation and gel filtration in H2O and D2O indicate that the enzyme from the soluble cell fraction has the following properties: S20w, 6.3 S; Stokes radius, 54 A; partial specific volume, 0.75 ml/g; mass, 154,000 daltons; f/fo, 1.4; axial ratio (prolate ellipsoid), 7. The addition of 0.1% Lubrol PX to this fraction activates the enzyme and changes thartial specific volume, 0.74 ml/g; mass, 148,000 daltons; f/fo, 1.6; axial ratio (prolate ellipsoid), 11. These findings show that detergent activates the enzyme by changing its conformation and not simply by dispersing nonsedimentable membrane fragments. The dimensions of this guanylate cyclase in detergent are very similar to those of detergent-solubilized adenylate cyclase from the same tissue (Neer, E.J. (1974) J. Biol. Chem. 249, 6527-6531). Guanylate cyclase can be solubilized from the particulate cell fraction with 1% Lubrol PX but has properties quite different from those of the guanylate cyclase in the soluble cell fraction. It is a large aggregate with a value of S20,w of about 10 S, Stokes radius of 65 A, and a mass of approximately 300,000 daltons. However, the peaks of guanylate cyclase activity in column effluents and sucrose density gradients are very broad indicating a mixture of different size proteins. The conditions used to solubilize guanylate cyclase from the particulate fraction also solubilize adenylate cyclase, and the two activities can be separated on the same sucrose gradient. Studies of this sort require a rapid, accurate guanylate cyclase assay. We have developed an assay for guanylate cyclase activity which meets these criteria by adapting the competitive protein binding assay for guanosine cyclic 3':5' monophosphate originally described by Murad et al. (Murad, F., Manganiello, V., and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 736-739).     

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.     

Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells.

The membrane-bound adenylase cyclase (ATP pyrosphosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Milipore filters with pores 0.22 micron in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5'-guanylyl-imidodiphosphate at a concentration of 1 . 10(-6)M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5'-guanylyl-imidodiphosphate.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. I. Inhibition by heparin of gonadotrophin-stimulated ovarian adenylate cyclase.

Heparin inhibits (I50 = 2 microgram/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5'-(beta,gamma-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (I50 = 6 microgram/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Heparin (3 microgram/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

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

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

Regulation of Dictyostelium discoideum adenylate cyclase by manganese and adenosine analogs

Adenylate cyclase is the critical enzyme in the chemotactic signal relay mechanism of the slime mold amoeba, Dictyostelium discoideum. However, few studies examining the regulation of this enzyme have been performed in vitro due to the instability of enzyme activity in crude lysates. For studies presented in this communication, a membrane preparation has been isolated that exhibits a high specific activity adenylate cyclase that is stable during storage at -70 degrees C and under assay conditions at 27 degrees C. The enzyme was activated by micromolar concentrations of MnCl2. GTP and its non-hydrolyzable analog, guanosine 5'-(beta, gamma-imino)triphosphate, inhibited the enzyme non-competitively in the presence of either Mg2+ or Mn2+. However, this inhibition was more pronounced in the presence of Mn2+. Since guanylate cyclase activity in the D. discoideum membranes was less than 10% of the adenylate cyclase activity, there could not be a significant contribution by guanylate cyclase toward the production of cyclic AMP. Experiments indicate that D. discoideum adenylate cyclase was also regulated by adenosine analogs. The enzyme was inhibited by 2',5'-dideoxyadenosine and 2'-deoxyadenosine and inhibition was augmented by the presence of Mn2+. However, the inhibition was not entirely consistent with that which would be expected for the P-site of eukaryotic systems because some purine-modified adenosine analogs also inhibited the enzyme. Guanine nucleotides had no effect on the inhibition by either purine-modified or ribose-modified adenosine analogs. The binding of cyclic AMP to its receptor on the D. discoideum membranes was not affected by either MnCl2 or adenosine analogs.     

Proof of adenylate cyclase activity in the coronary artery of cattle]

In two fractions obtained from the bovine A. coronaria adenylate cyclase activity was identified and characterized. The adenylate cyclase activity of the 75,000 X g sediment shows a pH optimum at 7.4. The temperature dependence of this adenylate cyclase activity is linear when represented in the Arrhenius plot, and an Arrhenius activation energy of 13.2 kcal Mol-1 can be calculated for the enzyme reaction. The Km-value of the enzyme to ATP is 6 +/- 0.6 - 10(-4) M. The adenylate cyclase activity of the 75,000 X g sediment can be stimulated by NaF. 5'AMP and adenosine inhibit the adenylate cyclase activity of the 75,000 X g sediment. With regard to the enzyme activity, Mn++ and Co++ replace Mg++, but not Ca++. The monovalentcations Na+ and K+ do not influence the adenylate cyclase activity. In a particulate fraction containing plasma membranes, adenylate cyclase activity was also identified. This adenylate cyclase activity can be stimulated by catecholamines, noradrenaline, and isoproterenol. This stimulation can, however, only be proved for the enzyme in the coronaries of 9-week-old and 2-year-old animals. The adenylate cyclase activity from the coronaries of adult animals is not affected by catecholamines. These findings are discussed with regard to hypertension frequently found in adult animals.     

Galanin inhibits adenylate cyclase of rat brain membranes.

The ubiquitous neuropeptide, galanin, strongly inhibits adenylate cyclase in rat brain membranes. While basal enzyme activity was not altered, galanin from 10(-11) M to 5 x 10(-7) M decreased forskolin- and VIP-stimulated adenylate cyclase with a half-maximal effect being elicited by 0.7 nM neuropeptide and a maximal 80% inhibition of the enzyme activity. The galanin fragments (2-29) and (1-15) dose-dependently inhibited the forskolin-stimulated adenylate cyclase, while the fragments (3-29) and (10-29) were found inactive. These results indicate that the regulatory action of galanin in the central nervous system involves the coupling of galanin receptors to the inhibition of the adenylate cyclase system.     

Modulation of quail oviduct adenylate cyclase activity by estradiol and progesterone

Oviduct adenylate cyclase activity of the quail was measured by radiochemical analysis following different hormonal treatments. A single injection of estradiol benzoate (EB) to immature female quails resulted in a prereplicative surge of adenylate cyclase activity. A second surge of enzyme activity was observed during the proliferative phase induced by EB. Estradiol-17 alpha, estrone, estriol and testosterone were ineffective. Tamoxifen completely inhibits the growth-promoting effect of EB and the second surge of adenylate cyclase activity but does not inhibit the prereplicative increase of enzyme activity. This prereplicative increase of adenylate cyclase activity was also observed, even in the absence of increased plasma estradiol, when estradiol-17 beta (E2) was perfused through the hepatic portal vein. Moreover, E2 had no effect on enzyme activity when added directly to the oviduct homogenate preparation, at concentrations ranging from 10(-9) to 10(-7) M. In response to progesterone injection, oviduct adenylate cyclase activity followed a different pattern, beginning its increase after 3 h and remaining elevated up to 24 h. The activation by estradiol was independent of the presence of guanylylimidodiphosphate. Moreover, the enzyme was more sensitive to forskolin at submaximal concentration in estradiol treated birds than in control. These results demonstrate that transient activation of adenylate cyclase at the early stages of the action of estradiol does not occur through the classic nuclear receptor-gene activation pathway or a membrane receptor mediated process, but involves an indirect pathway, yet to be defined.     

Crayfish abdominal muscle adenylate cyclase. Studies on the stimulation by a Ca2+-binding protein.

A plasma-membrane preparation of crayfish muscle showed an adenylate cyclase activity which is inhibited to about 80% of its original activity by 100 microM-EGTA. Measurements of the enzyme activity in the presence of 100 microM-EGTA and various concentrations of Ca2+ revealed an increase in enzyme activity of about 400%, indicating an adenylate cyclase which is dependent on Ca2+ for activity. Fluphenazine (1 mM), a blocker of the Ca2+-binding protein calmodulin, decreased enzyme activity to zero. The enzyme can be re-activated by the addition of certain concentrations of calmodulin to the assay medium. This suggests that crayfish muscle adenylate cyclase is dependent on Ca2+ and calmodulin for activity.