Epinephrine-stimulated Adenylate Cyclase Activity Research Articles

Modulation by docosahexaenoic acid of the epinephrine-stimulated adenylate cyclase activity of the bovine retina.

This work shows that unsaturated fatty acids enhance the epinephrine-stimulated adenylate cyclase activity in bovine retina. The modulating effect on the epinephrine-stimulated formation of cyclic AMP seems to be linked to the degree of unsaturation of the fatty acid. Treatment of the intact retina with docosahexaenoic acid in the concentration range 0.5 X 10(-6)-1 X 10(-3) M does not affect the enzyme activity measured in the absence of the hormone but markedly increases the cyclase activity when the tissue is incubated in the presence of 0.1 mM epinephrine. Docosahexaenoic acid enhances the maximal response to epinephrine without affecting the apparent ED50 value for this effector. Docosahexaenoic acid at 0.5 mM also increases the hormone-stimulated adenylate cyclase activity in retinal cell-free homogenate, whereas it has no effect on the epinephrine-sensitive enzyme solubilized from the membrane fraction with 1% Triton X-305. When docosahexaenoic acid-preincubated intact retina and cell-free homogenate are incubated in the presence of defatted albumin, both the observed activating effect of the fatty acid on the epinephrine-stimulated adenylate cyclase activity and the enhancement of the enzyme response to the hormone significantly diminish.

Monoclonal antibodies to the beta-adrenergic receptor: modulation of catecholamine-sensitive adenylate cyclase by the antibody.

We have developed two types of hybridomas producing monoclonal antibodies to the turkey erythrocyte beta 1-adrenergic receptor in order to study the beta-adrenergic-cAMP system of epidermis. Splenic cells from BALB/c mice immunized with partially purified turkey erythrocyte beta 1-receptors were fused with mouse myeloma cell line SP2/0-Ag14. Five hybridomas of 17 positive cells producing antibodies which could precipitate soluble turkey erythrocyte beta 1-receptors were cloned by the limiting dilution method. The antibodies cross-reacted with beta 1- and beta 2-adrenergic receptors and stained epidermal basal cells with immunocytochemical techniques. Neither type of antibody interfered with the antagonist binding, i.e., all antibodies bound to sites other than the ligand binding site on the surface. One type of antibody inhibited epinephrine-stimulated adenylate cyclase activity in our "leaky" epidermal cell system. The data suggest that the antibody interferes with the coupling of the receptor to the regulatory protein.

Depressed epinephrine-stimulated adenylate cyclase activity in diabetic rat heart

Depressed epinephrine-stimulated adenylate cyclase activity in diabetic rat heart

Relationship of the inhibition of epinephrine-stimulated adenylate cyclase in turkey erythrocytes by adenosine to its “R-” and “P-site”-mediated effects

Adenosine and its analogs inhibited epinephrine-stimulated adenylate cyclase activity in turkey erythrocyte membranes in a manner suggesting the presence of both “R-” and “P-site” effects. The existence of the “R-site” receptor-mediated effect implies that, even when the degree of coupling of the adenosine receptor to the enzyme is low, the mode of coupling is permanent.

Enhanced receptor-cyclase coupling and augmented catecholamine-stimulated lipolysis in exercising rats.

To test the hypothesis that alterations of adipocyte beta-adrenergic receptors provide a molecular mechanism for enhanced catecholamine-stimulated lipolysis in physically trained animals, we studied adipocytes derived from rats subjected to 14 wk of swimming and from sedentary controls. Peak glycerol release and peak adenylate cyclase activity in response to epinephrine were increased in swimmers to 255% (P less than 0.01) and 156% (P less than 0.01) of control values, respectively, but neither basal glycerol release, basal cyclase activity, NaF-stimulated cyclase activity, beta-receptor number, nor receptor affinity for [3H]dihydroalprenolol were altered. Epinephrine-stimulated adenylate cyclase activity remained increased in adipocytes from swimmers in the presence of theophylline or adenosine. In the absence of exogenous guanine nucleotide, we observed no differences in the dissociation constants for either the high-affinity (KD = 0.025 microM) or the low-affinity (KL = 11 microM) classes of binding sites for (-)-epinephrine, but the proportion of high-affinity sites was greater in membrane preparations from swimmers than from controls (74 vs. 42%; P less than 0.01). We conclude that receptor-cyclase coupling is enhanced in adipocytes from exercising rats, perhaps due to an improved ability of adrenergic agonists to form the guanine nucleotide reversible high-affinity agonist-receptor complex.

In vivo effect of human growth hormone on hepatic adenylate cyclase activity.

Significant increases in basal, and glucagon and fluoride stimulated adenylate cyclase activity were observed in liver plasma membranes of hypophysectomized rats compared to normal adult and weanling rats. The fluoride stimulated adenylate cyclase activity was 2-3 fold greater in the membranes from hypophysectomized animals while the glucagon stimulated activity was 5-7 fold greater, and the basal activity was approximately double that of membranes from normal adult animals. Administration of growth hormone to hypophysectomized rats by an intramuscular or intravenous route decreased adenylate cyclase activity to levels equivalent to those in normal adult rats. Estradiol and thyroxine replacement did not alter the adenylate cyclase activity of the membranes from hypophysectomized animals. The fluoride or epinephrine stimulated adenylate cyclase activity of rat diaphragm homogenates was not affected by hypophysectomy.

β-adrenegic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0–8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the β-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na +K +(Mg 2+)-ATPase activity and a 6–8-fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2–3-fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological β 2 type response with isoproterenol (1.8 · 10 −8 M) > epinephrine (1.1 · 10 −7 M) > norinephrine (3.2 · 10 −6 M). In contrast, binding studies employing (−)-[ 3H] dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (−)-[ 3H]dihydroalprenolol by catecholamine agonists. Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of M r 80 000 and 86 000, whereas in the adult only a single M r 133 000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.

Ovarian gonadotropin-releasing hormone receptors. I. Properties and inhibition of luteal cell function.

Gonadotropin-releasing hormone (GnRH) and its agonist analogs exert paradoxical antifertility effects in several species by causing regression of ovarian function. In collagenase-dispersed rat luteal cells, the agonist analog [D-Ser(tBu)6]des-Gly10-GnRH-N-ethylamide (GnRHa) caused inhibition of hCG-stimulated progesterone production. Epinephrine- stimulated progesterone production in isolated luteal cells was also partially inhibited by the GnRH analog. On the other hand, there was no effect of the analog on the steroid doseresponse curve to dibutyryl cAMP. The effect of the GnRH analog on the steroidogenic response to both hormones was to shift the dose-response curve to the right without changing the maximum production of progesterone. Natural sequence GnRH also inhibited steroid production, with a half-maximal effect occurring at 5.10-9 M in agreement with the binding affinity of GnRH for its gonadal receptors (Ka = 5 × 108 M-1). GnRHa also caused a shift to the right in the dose-response curve for cAMP production stimulated by hCG. However, no direct inhibitory effects of GnRH and its agonist analogs could be detected on hCG- or epinephrine-stimulated adenylate cyclase activity measured in ovarian homogenates. GnRH did not affect the binding affinity or capacity of rat ovarian receptors for hCG. Specific GnRH receptors in luteinized rat ovaries were identified with [l25I]GnRHa and found to have high affinities for the analog (Ka = 5 × 109 M-1). A potent antagonist analog ([D-pGlu1,D-Phe2,DTrp36]GnRH) was also bound with high affinity (Ka = 3 times; 109 M-1) by the ovarian receptors. GTP, guanosine 5′-(β,γ-imido) triphosphate, and ATP as well as prostaglandin F2a and epinephrine did not alter binding of the analog to GnRH receptors. These studies demonstrate that the ovary contains specific, high affinity receptors for GnRH, through which GnRH and its gonist analogs can inhibit steroidogenic responses to hormonal stimulation. Since there was no inhibition of dibutyryl cAMPstimulated progesterone production and no direct action on hCG binding, it is suggested that GnRH interferes with the mechanism of hormonal stimulation of cAMP production.

Determinants of the stimulation of fat cell adenylate cyclase by high concentrations of sodium and magnesium salts. Implications for the role of magnesium in regulation of enzyme activity

Stimulation of adenylate cyclase by high concentrations of Mg 2+ has been attributed to either occupation of a divalent cation site on the enzyme or Mg 2+ binding of inhibitory species of uncomplexed ATP. However, the stimulation of adenylate cyclase by other salts led us to reexamine the alleged specificity of Mg 2+. The stimulatory effects of high concentrations of magnesium and sodium salts on adenylate cyclase were compared in rat fat cell membranes (‘ghosts’). Under standard assay conditions of 5 mM MgCl 2 and 1 mM ATP, added MgCl 2 (45 mM), NaCl (90 mM), MgSO 4 (45 mM), and Na 2SO 4 (45 mM) stimulated basal activity in a temperature-dependent manner. At 30°C all four salts produced 2–3-fold stimulation with linear time course to 10 min. At 37°C time courses were nonlinear, and although MgCl 2 and NaCl were stimulatory over 10 min, sulfate salts of both cations had little or no effect. When 10 −4 M GTP was added, however, all four salts became stimulatory at 37°C with linear time courses. In the presence of GTP, MgCl 2 and NaCl were equally effective at equimolar concentrations (100 mM) of Cl −. MgCl 2 and NaCl were stimulatory (in the absence or presence of GTP) over the same range of Cl − concentrations up to 200 mM. In contrast to the results with GTP, magnesium salts enhanced stimulation by the GTP analog 5′-guanylyl-imidodiphosphate (GMP-P(NH)P) much more than did sodium salts. MgCl 2 and MgSO 4 each approximately tripled GMP-P(NH)P stimulation, while NaCl and Na 2SO 4 had only minimal stimulatory effect. None of the salts increased 10 −4 M epinephrine-stimulated adenylate cyclase activity in the absence or presence of GTP. Our results show that the effects of ‘high Mg’ are, under certain conditions, approximated by high concentrations of another cation (Na) and are in part determined by the accompanying anion. Stimulation by high MgCl 2, therefore, appears to be a generalized salt effect rather than a specific effect of Mg 2+. In addition, the degree of stimulation of adenylate cyclase of fat cell ghosts by salts, including those of Mg 2+, is dependent on temperature and the presence or absence of guanine nucleotide and hormone.

Beta-Adrenergic receptors and catecholamine-sensitive adenylate cyclase of the human placenta.

Beta-Adrnergic receptor and beta-adrenergic sensitive adenylate cyclase were demonstrated in membrane fractions of human placenta. Placental membranes from normal term pregnancies bound the beta-adrenergic antagonist (-)[3H]dihydroalprenolol to a single saturable class of sites (Kd = 2.31 +/- 0.23 nM; n = 9; maximal capacity, 112 +/- 9 fmol/mg). Competition for binding was stereoselective for (-)isomers of propranolol, and beta-adrenergic agonists displayed competition for the placental receptor in the order (-)isoproterenol greater than (-)epinephrine greater than (-)norepinephrine, typical of a beta 2 type receptor. Beta-Adrenergic receptor was present in placental tissue as early as 10 weeks gestational age, and binding capacity decreased slightly with advancing gestation. [3H]Dihydroalprenolol binding was coupled to epinephrine-stimulated adenylate cyclase activity throughout gestation. The subcellular distribution of both beta-adrenergic receptors and epinephrine-stimulated adenylate cyclase suggest their localization primarily in nonbrush border membrane fractions, presumably from plasma membranes more closely related to the fetal rather than to the maternal circulation. Epinephrine-sensitive adenylate cyclase was not present in purified brush border preparations which were directly exposed to maternal blood in the intervillous space.

Adenylate cyclase from human decidua parietalis

Adenylate cyclase (EC 4.6.1.1) activity was demonstrated in membrane fractions of human decidua parietalis. Adenylate cyclase activity was associated with membrane fractions of decidua but not chorion or amnion. Prostaglandins PGE1 and PGE2 but neither PGD2 nor PGF2α stimulated this activity at gestational ages 18 to 40 weeks. Fluoride and guanosine triphosphate stimulated decidual adenylate cyclase; however, epinephrine-stimulated adenylate cyclase activity was demonstrable only in the presence of guanosine triphosphate. The possible roles of adenylate cyclase activity in the decidua are discussed.

Membrane Receptor Function and the Loss of Glucagon-Stimulated Adenylate Cyclase Activity in Hepatomas*

Plasma membranes were prepared from homogenates of two well differentiated hepatomas (Morris rat 7787 and Dalton mouse 9815), two poorly differentiated hepatomas (Morris rat 7288-C and Dalton mouse 129), and normal liver. Adenylate cyclase activity and [125I]iodoglucagon binding were measured in the plasma membrane preparations over a wide range of glucagon concentrations. Nether glucagon-stimulated adenylate cyclase activity nor [125I]iodoglucagon binding could be detected in the poorly differentiated hepatomas. Fluoride and epinephrine stimulated adenylate cyclase activity in all hepatomas. Maximum activity of glucagon-stimulated adenylate cyclase and maximum binding of glucagon in the wall differentiated hepatomas were less than those of normal liver. Plasma membranes from liver and hepatomas were solubilized with Lubrol-PX and, after reducing the concentration of detergent, were incubated with [125I]iodoglucagon and then chromatographed on a column of Bio-Gel A 1,5 m. Two peaks containing both protein and [125I]iodoglucagon were found for normal liver but not for the poorly differentiated hepatomas. Fractions from the Bio-Gel column containing the greatest concentration of protein were also subjected to a binding microassay. Material from the poorly differentiated tumors did not bind glucagon in this system, whereas the solubilized normal liver membranes bound up to 1.4 pmol [125I]iodoglucagon/mg protein. This indicates that there is no detectable glucagon receptor in these undifferentiated tumors.

Inhibition of basal and hormone-stimulated adenylate cyclase in adipocyte ghosts by the prostaglandin endoperoxide prostaglandin H2

The prostaglandin endoperoxide PGH2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid), at a concentration of 2.8 x 10(-5) M inhibited basal adenylate cyclase activity 11% and epinephrine-stimulated activity 30 to 35%. PGH2 inhibited epinephrine-stimulated enzyme activity in the presence of 10 mM theophylline, 2.5 mM adenosine 3':5'-monophosphate (cAMP), or in the absence of inhibitors or substrates of the cAMP phosphodiesterase. When the cAMP phosphodiesterase was assayed directly using 62 nM and 1.1 muM cAMP, PGH2 did not affect the 100,000 x g particulate cAMP phosphodiesterase from fat cells. The inhibition of adenylate cyclase by PGH2 was readily reversible. A 6-min preincubation of ghost membranes with PGH2, followed by washing, did not alter subsequent epinephrine-stimulated adenylate cyclase activity. During epinephrine stimulation, the PGH2 inhibition was apparent on initial rates of cAMP synthesis, and the addition of PGH2 to the enzyme system at any point during an assay markedly reduced the rate of cAMP synthesis. Between 2.8 x 10(-7) M and 2.8 x 10(-5) M, PGH2 inhibited epinephrine-stimulated enzyme activity in a concentration-dependent manner. The stimulation of adenylate cyclase by thyroid-stimulating hormone, glucagon, and adrenocorticotropic hormone as well as by epinephrine was antagonized by PGH2, suggesting that PGH2 may be an endogenous feedback regulator of hormone-stimulated lipolysis in adipose tissue.

Stimulation and Desensitization of Macrophage Adenylate Cyclase by Prostaglandins and Catecholamines

Abstract Preparations of peritoneal macrophages were obtained by selecting from a peritoneal exudate of guinea pigs those cells which adhere to glass. By preincubating the macrophages in a hypotonic pH 8.0 buffer, homogenization was achieved under gentle conditions. The adenylate cyclase activity of homogenates was stimulated by catecholamines and by the prostaglandins E1 and E2 (PGE1 and PGE2). Using inhibitors and other criteria the catecholamine receptor was classified as a β-adrenergic receptor. Since propranolol inhibits the catecholamine stimulation but not the prostaglandin stimulation of adenylate cyclase, it was concluded that these two classes of hormones interact with separate receptor sites. It is still unclear whether they affect the same or different catalytic sites. Two types of experiments yielded information about the effects on adenylate cyclase activity of exposure of intact cells to hormones. Intact macrophages were incubated as monolayers with epinephrine or PGE2 for 2 hours, after which the hormone was removed and the cells homogenized. Preincubation of macrophages with PGE2 greatly reduced the subsequent response of adenylate cyclase to PGE2. The basal, epinephrine-stimulated, and NaF-stimulated adenylate cyclase activities were unaffected. Similarly, treatment of cells with epinephrine left the basal, PGE2-stimulated and NaF-stimulated activities unaffected but markedly reduced the epinephrine-stimulated adenylate cyclase activity. This desensitization, i.e. loss of response to hormone due to treatment with hormone, was found to be dose-dependent over the range of 10-7 to 10-4 m PGE2. Incubation of macrophage monolayers in a serum-containing medium leads to an increase in adenylate cyclase activity (basal) after 24 hours. Inclusion of PGE2 in these cultures caused the basal adenylate cyclase activity to be markedly decreased relative to control cultures incubated without PGE2. This decrease was dose-dependent over the range of 10-7 to 10-4 m PGE2 and was not observed with prostaglandin F1α.

Adenylate cyclase activity in immature rabbit lung.

Extract: Adenylate cyclase activity of lung homogenates from fetal and neonatal rabbits is considerably higher than that found in adult animals. This activity does not change significantly between 21 and 31 days postconception, but maturational changes are observed in responses of adenylate cyclase activity to incubation with epinephrine, glucagon, and NaF. Epinephrine stimulated adenylate cyclase activity at all ages studied, 21, 24, 27, 30 and 31 days postconception. NaF depressed adenylate cyclase activity at 21 and 24 days but thereafter stimulated it at 27, 30, and 31 days. Glucagon stimulated adenylate cyclase activity only after delivery. We conclude that hormonally mediated responses in formation of adenosine 3′, 5′-monophosphate (cyclic AMP) occur in the lung of the fetal animal. Speculation: Adenylate cyclase may be important in regulating pulmonary phospholipid biosynthesis in fetal animals. Activity of adenylate cyclase can be augmented by epinephrine in fetal lung homogenates and may mediate growth of the lung or premature formation of pulmonary surface active phospholipids.

Influence of lithium ions on epinephrine-stimulated adenylate cyclase activity in cultured glial tumor cells

1. 1. Adenylate cyclase associated with a particulate fraction of rat glial tumor tumor cells (clone C 6) exhibited optimum activity at Mg 2+: ATP ratios near 2. In the presence of excess Mg 2+, the enzyme exhibited a K m for ATP of 0.14 + 0.04 mM . 2. 2. Li + enhanced the effect of epinephrine on adenylate cyclase activity in glial cell particles up to 2-fold but only at Mg 2+: ATP ratios greater than 1. Li + increased the V of adenylate cyclase but did not alter the apparent K m for ATP. The influence of Li + on epinephrine-stimulated enzyme activity also was observed in intact cells. 3. 3. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) enhanced adenylate cyclase activity in the presence of various combinations of epinephrine and Li + apparently by chelating Ca 2+. 4. 4. The influences of Li + and Ca 2+ on adenylate cyclase activity associated with glial cells was different from that reported for brain cortical slices and homogenates, suggesting that other cell types also contribute to adenylate cyclase activity in brain.

Effect of insulin and epinephrine on the carbohydrate metabolism and adenylate cyclase activity of rhesus fetal muscle.

Extract: Carbohydrate metabolism of skeletal muscle from rhesus fetuses at 58% of gestation (95 days) is sensitive to epinephrine in vitro. Epinephrine increased lactate production and decreased glucose uptake, 14C-lactate production, glycogen content, and 14C-glycogen formation as well as 14CO2 production. These responses to epinephrine are similar to those in adult muscle. However, in some cases the magnitude of these responses appears lower in fetal muscle. The content of cyclic 14C-adenosine 3‘,5’-monophosphate (cyclic 14C-AMP) was about 2.5-fold higher in the 100-day fetal muscle than in the adult. Epinephrine stimulated adenylate cyclase activity almost threefold in fetal and fourfold in adult muscle. When incubated with muscle from 85-day fetal monkeys, insulin increased glucose uptake, lactate and lactate-14C production, and 14CO2 production; the greatest effect was found in the increased incorporation of labeled glucose into glycogen. Both synthetase I and phosphorylase a activities were present at 78–80 days of fetal age. Our data show that as early as about 58% of term the carbohydrate metabolism of fetal rhesus muscle in vitro is sensitive to epinephrine and that the hormone probably acts through the adenylate cyclase and the “second messenger” system of cyclic AMP as it does in adult muscle.Speculation: Our data offer indirect evidence that insulin and epinephrine mediate glycogen metabolism via cyclic AMP in rhesus fetal muscle as early as 85 days of gestational age (52% of term) and that these hormones operate through similar enzyme systems in fetal and adult muscle. It is possible that glycogen synthetase and phosphorylase, or other enzymes mediating the cyclic AMP response, are not identical in fetal and adult muscle; fetal isoenzyme patterns for muscle lactate dehydrogenase differ markedly from those of the adult. However, it is difficult to reconcile the existence of major differences in the enzyme milieu in fetal and adult muscle with the similar overall actions of epinephrine and insulin on glycogenesis and glycogenolysis demonstrated in our experiments.

Epinephrine binding and the selective restoration of adenylate cyclase activity in fat-fed rats

Fat feeding results in a progressive loss of epineph-rine- and glucagon-stimulated adenylate cyclase activity in adipocyte plasma membrane sacs (ghosts). Basal and NaF-stimulated adenylate cyclase activities in fat-fed animals are not significantly different from those in preparations obtained from chow-fed rats. The high fat diet increases the mean adipocyte diameter rapidly, but increased cell size, at least in the case of epinephrine stimulation, is not responsible for the decreased hormone-stimulated adenylate cyclase activity. Diet shifts to high carbohydrate or high protein regimens result in the restoration of the epinephrine-stimulated, but not the glucagon-stimulated, activity without a significant reduction in mean cell diameter. Both hormone-resistant adipocyte ghosts from fat-fed animals and ghosts obtained from hormone-sensitive adipocytes bind the same amount of [(3)H]epinephrine per milligram of membrane protein. These data indicate that the fat diet inhibits epinephrine-stimulated adenylate cyclase activity at a point between the hormone receptor and the catalytic unit of adenylate cyclase.

Insulin-like activity of concanavalin A and wheat germ agglutinin--direct interactions with insulin receptors.

Concanavalin A and wheat germ agglutinin are as effective as insulin in enhancing the rate of glucose transport and in inhibiting epinephrine-stimulated lipolysis in isolated adipocytes. These lectins, also like insulin, inhibit basal as well as epinephrine-stimulated adenylate cyclase activity of membranes obtained from homogenates of fat cells. Low concentrations of wheat germ agglutinin enhance the specific binding of insulin to receptors of fat cells and liver membranes. Higher concentrations of this plant lectin, as well as of concanavalin A, competitively displace the binding of insulin to receptors in these tissues. These effects are equally apparent in insulin-binding proteins solubilized from membranes, indicating that the plant lectins interact directly with insulin receptors. All of the effects observed with the plant lectins are reversed by simple sugars that bind specifically to these plant proteins. Agarose derivatives of the plant lectins effectively adsorb solubilized insulin-binding proteins, and these can be eluted with buffers containing specific simple sugars. The possible implications of these findings to certain biological properties (mitogenicity) of these lectins and to the mechanism of action of other growth-promoting substances are considered.

α,β-Methylene-adenosine 5′-Triphosphate: A COMPETITIVE INHIBITOR OF ADENYLATE CYCLASE IN FAT AND LIVER CELL MEMBRANES

Abstract The ATP analog, α,β-methylene-adenosine 5'-triphosphate (Ap(CH2)pp), competitively inhibits the basal as well as the glucagon- and NaF-stimulated activity of adenylate cyclase in isolated liver membrane preparations. The dissociation constant (Ki) of the inhibitor, 0.5 mm, is independent of the concentration of glucagon or NaF and is somewhat lower than the apparent Km for ATP. The phosphonic acid analog of ATP also inhibits competitively (Ki, 1.2 mm) the epinephrine-stimulated adenylate cyclase activity of membranes prepared from homogenates of isolated fat cells. The enzyme activity stimulated by NaF in these membranes is also inhibited by Ap(CH2)pp. The ADP analog, α,β-methylene-adenosine 5'-diphosphate does not significantly inhibit the activity of adenylate cyclase of liver cell membranes.