Simultaneous electron microscopic detection of colloidal gold labeled ligand and adenylate cyclase

Summary Concanavalin-A-colloidal gold (Con-A-G) complex and adenylate cyclase activity were detected simultaneously in electron microscopic preparations of human fibroblast cultures, by a combined histochemical technique. The colloidal gold particles appeared as round bodies which could be readily differentiated from the amorphous product of the adenylate cyclase enzyme reaction. The combined technique makes possible the simultaneous visualization of the bound ligand (i.e. of its binding site), and of the enzyme activated by the lignad. Treatment of the cells with Con-A accounted for a considerable increase in intracellular adenylate cyclase activity. The activity increase was disproportionally greater than the amount of bound ligand, and it also appeared in localizations showing no indication of ligand binding. Treatment of the fibroblasts with Con-A was followed by internalization of the lignand and the enzyme inside at least seemingly segregated vesicles.     

Calmodulin-mediated adenylate cyclase from mammalian sperm

Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.     

Isolation, purification and characterization of regulatory properties of adenylate cyclase from rabbit heart]

Rabbit heart membranes possessing the adenylate cyclase activity were isolated and purified by extraction with high ionic strength solutions and centrifugation in the sucrose density gradient. It was shown that the membranes are characterized by a high percentage of cholesterol (molar ratio cholesterol/phospholipids is 0.24) and an increased activity of Na, K-ATPase, which suggests the localization of adenylate cyclase in the sarcolemma. During centrifugation in the sucrose density gradient the activities of andenylate cyclase and Na,K-ATPase are not separated. Treatment of heart sarcolemma with a 0.3% solution of lubrol WX results in 10--20% solubilization of adenylate cyclase. Purification of the enzyme in the membrane fraction is accompanied by a decrease in the activity of phosphodiesterase; however, about 2% of the heart diesterase total activity cannot be removed from the sarcolemma even after its treatment with 0.3% lubrol WX. Epinephrine and NaF activate adenylate cyclase without changing the pH dependence of the enzyme. The alpha-adrenergic antagonist phentolamine has no effect on the adenylate cyclase activation by catecholamines, glucagon and histamine; the beta-adrenergic antagonist alprenolol competitively inhibits the effects of isoproterenol, epinephrine and norepinephrine, having no effect on the enzyme activation by glucagon and histamine. There is no competition between epinephrine, glucagon and histamine for the binding site of the hormone; however, there may occur a competition between the hormone receptors for the binding to the enzyme. A combined action of several hormones on the membranes results in the averaging of their individual activating effects. When the hormones were added one after another, the extent of adenylate cyclase activation corresponded to that induced by the first hormone; the activation was insensitive to the effect of the second hormone added. It is assumed that the outer membrane of myocardium cells contains a adenylate cyclase and three types of receptors, each being capable to interact with the same form of enzyme. The activity of adenylate cyclase is determined by the type of the receptor, to which it is bound and by the amount of the enzyme-receptor complex.     

Effect of modification of synaptosomal membrane glycoproteins on adenylate cyclase activity

The effect of the modification of synaptosomal membrane glycoproteins on the activity of adenylate cyclase was studied. It was found that the binding of concanavalin A to unmodified guinea pig cerebral cortex synaptosomal membrane did not change adenylate cyclase activity. Concanavalin A binding to synaptosomal membrane of hypoxic brain cortex resulted in no decrease of enzyme activity. The level of protein-bound sialic acid in these synaptosomal fractions was 20% lower than in the control. Treatment of synaptosomal membranes with neuraminidase resulted in a decrease of sialic acid content by about 70%, but it had no significant effect on adenylate cyclase activity. The modification with concanvalin A of sugar end groups exposed by neuraminidase treatment resulted in significant decrease of both basal and fluoride-stimulated adenylate cyclase activity. These results seem to indicate that some component of the adenylate cyclase complex of brain synaptosomal membranes is closely interacting with a carbohydrate-containing macromolecule on the cell surface.This work was supported by, the Polish Academy of Sciences within the project 10.4.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

The role of phospholipids in TSH stimulation of adenylate cyclase in thyroid plasma membranes

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.     

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.     

Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity.     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Inhibition of adenylate cyclase by polyadenylate

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

Activation of adenylate cyclase in cultured fibroblasts by trypsin

Adenylate cyclase activity measured in membranes of cultured normal rat kidney (NRK) fibroblasts was markedly increased by prior treatment of the intact cells with trypsin. Cell population density influenced the extent of activation observed. Trypsin treatment of sparse cells significantly enhanced adenylate cyclase activity, whereas similar treatment of confluent cells caused only a slight increase in adenylate cyclase activity. The degree of activation noted after trypsin treatment also varied depending on the adenylate cyclase function measured. Activity determined in the presence of GTP alone showed the greatest increase after trypsin treatment. Similar enhancement of adenylate cyclase activity of a washed cell membrane preparation was achieved by the addition of low concentrations of trypsin directly to the adenylate cyclase reaction mixture. The membranes of confluent NRK fibroblasts initially exhibited higher adenylate cyclase activity than did membranes of sparse cells. The present results suggest that this change in adenylate cyclase activity at cell confluence is not due to an increase in the amount of adenylate cyclase in the cell membrane but rather to a change in membrane components that regulate its activity. Proteolytic activation of adenylate cyclase appears to result from degradation of cell membrane proteins that modulate the activity of this enzyme.     

Vasopressin-sensitive adenylate cyclase reversibility of hormonal activation

The reversibility of adenylate cyclase activation induced by vasopressin was studied by reducing the concentration of active peptide in contact with kidney medullo-papillary membranes. Reversibility of hormonal activation was only partial. The use of antagonists failed to demonstrate the reversibility of an adenylate cyclase activation induced by high affinity agonists. When antagonist was added after the agonist to membranes, a non-competitive inhibitio was apparent. Active peptide was also eliminated from the incubation medium by treatment with agents capable of reducing the disulfide bridge of the hormonal molecule. Direct effects of reducers on adenylate cyclase activity were measured on enzyme activation induced by peptides lacking a disulfide bridge. There was no apparent correlation between the abilities of different reducers to inactivate free peptide in solution and their abilities to promote the reversibility of hormone-induced enzyme activation. Upon the addition of dithiothreitol, enzyme activity could be lowered to baseal value and adenylate cyclase was again fully stimulatable. However, when dithiothreitol addition to stimulated enzyme was combined with a 60-fold dilutionof the incubation medium, no reversibility of hormonal activation occurred. These results illustrate that the processes involved in adenylate cyclase activation are only partially reversible.     

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

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

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

Heparin inhibits (I₅₀ = 2 μg/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′-(β,γ-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by hepatin (I₅₀ = 6 μg/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. Herapin (3 μg/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 herapin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

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.     

Vasopressin-sensitive adenylate cyclase. Reversibility of hormonal activation

The reversibility of adenylate cyclase activation induced by vasopressin was studied by reducing the concentration of active peptide in contact with kidney medullo-papillary membranes. Reversibility of hormonal activation was only partial. The use of antagonists failed to demonstrate the reversibility of an adenylate cyclase activation induced by high affinity agonists. When antagonist was added after the agonist to membranes, a non-competitive inhibition was apparent. Active peptide was also eliminated from the incubation medium by treatment with agents capable of reducing the disulfide bridge of the hormonal molecule. Direct effects of reducers on adenylate cyclase activity were measured on enzyme activation induced by peptides lacking a disulfide bridge. There was no apparent correlation between the abilities of different reducers to inactivate free peptide in solution and their abilities to promote the reversibility of hormone-induced enzyme activation. Upon the addition of dithiothreitol, enzyme activity could be lowered to basal value and adenylate cyclase was again fully stimulatable. However, when dithiothreitol addition to stiumlated enzyme was combined with a 60-fold dilution of the incubation medium, no reversibility of hormonal activation occurred. These results illustrate that the processes involved in adenylate cyclase activation are only partially reversible.     

Modulation byn-alkanols of rat cardiac adenylate cyclase activity

Summary n-Alkanols (from methanol to decanol) have a biphasic effect on rat cardiac adenylate cyclase either basal or stimulated by GTP, GppNHp, NaF or hormones (isoproterenol, glucagon, secretin) in the presence of GTP. At high concentration, all the enzyme activities are inhibited. At low concentration, adenylate cyclase activity is either unchanged or potentiated depending on both the stimulus and the alkanols involved. Potentiation is due to an increase of maximum velocity with no change in the activation constant of the enzyme. Basal activity is unchanged as well as the isoproterenol-and glucagon-stimulated enzyme. The secretin-stimulated enzyme is potentiated. It is the guanyl nucleotide regulatory protein-mediated stimulation of adenylate cyclase which is mainly affected. An attempt was made to relate these effects on adenylate cyclase with physical parameters of the alkanols (partition coefficient). From the data obtained as a function of the alkanol chain-length and of temperature on the adenylate cyclase stimulated by GTP, GppNHp, NaF and permanently activated, it is concluded that the increase in efficacy observed in the presence of alkanol is due to an interaction with the protein moeity particularly with the guanyl nucleotide regulatory protein.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

Concanavalin A binding to brain particulate preparations and its effect on adenylate cyclase.

[³H] -Concanavalin A binding to brain particulate preparations measured by a filtration technique was found to show a characteristic regional specificity with the caudate-putamen area exhibiting the greatest density of concanavalin A (con A) binding sites. The synaptic membranes were shown to be the most highly enriched of the subcellular fractions examined in terms of lectin-binding glycoproteins. Con A was also shown to inhibit the basal adenylate cyclase activity of cerebral, cerebellar, and caudate-putamen particulate preparations in a concentration-dependent manner. This lectin sensitivity of the adenylate cyclase is apparently an intrinsic property of the enzyme complex since a detergent dispersed preparation of the cerebral cortical enzyme was equally inhibited by con A. It is proposed that one of the membrane con A binding sites in brain tissue is a component of the adenylate cyclase system.