Inhibition of fat cell membrane adenylate cyclase by 2'-deoxyadenosine-3'-monophosphate

A naturally occurring compound, 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP), is a potent inhibitor (K_i about 10 μM) of the adenylate cuclase activity of various rat tissues, toad erythrocytes, and mammalian cells in culture. This inhibition is reversible and appears to be noncompetitive with respect to the substrate, ATP. In addition, in the presence of the inhibitor, adenylate cyclase exhibits negative cooperativity with respect to MgCl₂. Cyclase activity stimulated by preincubation with NaF is as sensitive to inhibition by 2'-deoxy-3'-AMP as is control activity. However, preincubation of adenylate cyclase with 5'-guanylylimidodiphosphate (GPPNHP) and isoproterenol reduces its sensitivity to inhibition by the deoxynucleotide. Furthermore, adenylate cyclase exhibits negative cooperativity with respect to inhibition by 2'-deoxy-3'-AMP in the presence but not in the absence of isoproterenol. Finally, the deoxynucleotide inhibitor lowers the observed maximal isoproterenol-stimulated cyclase activity and simultaneously increases the concentration of catecholamine required for half-maximal activation. The possible role of 2'-deoxy-3'-AMP as a regulator of adenylate cyclase and intracellular hormonal responsiveness is discussed.     

Inhibition of cyclic amp phosphodiesterase by 5'-deoxy-5'-S-isobutylthioadenosine at biologically active concentrations of drug

5'-Deoxy-5'-S-isobutylthioadenosine (SIBA), a synthetic analogue of S-adenosylhomocysteine, has been reported by others to inhibit a number of biological processes and these effects of SIBA have been attributed generally to inhibition of methyltransferases. However, the present studies with mouse lymphocytes show that SIBA also acts as a competitive inhibitor (K_i = 130 μM) of the high-affinity cyclic AMP phosphodiesterase and potentiates the cyclic AMP response of intact cells to several activators of adenylate cyclase. Moreover, SIBA has been found to inhibit lymphocyte-mediated cytolysis, a cellular function known to be sensitive to elevated lymphocyte levels of cyclic AMP, at concentrations (IC₅₀ = 250 μM) similar to those which inhibit cyclic AMP phosphodiesterase. These results indicate the need for caution in attributing biological effects of SIBA singularly to inhibition of methyltransferases and suggest the possible agency of cyclic AMP in the mechanism of SIBA action.     

Some kinetic and chromatographic properties of detergent-dispersed adenylate cyclase

Studies on the reaction kinetics and chromatographic properties of detergent-dispersed adenylate cyclase are described. Detergent-dispersed enzyme was prepared from whole rat cerebellum and from partially purified plasma membranes from rat liver. Data were simulated to fit kinetic models for which an inhibitor is added in constant proportion to the variable substrate. Models were chosen to distinguish whether the adenylate cyclase reaction may be controlled by an inhibitory action of free ATP^?4 (or HATP^?3) or by a stimulatory action of free divalent cations. The various kinetic models were then tested with the dispersed brain adenylate cyclase with both Mg⁺⁺ and Mn⁺⁺ and in two different buffer systems. The experimental data indicate that this enzyme has a distinct cation binding site, but exhibits no significant inhibition by HATP^?3 or ATP^?4. The detergent-dispersed adenylate cyclase both from liver plasma membranes and from brain have been chromatographed on anion exchange material and have been chromatographed on anion exchange material and have been subjected to gel filtration. The presence of detergent was required for elution of cyclase activity from DEAE-Sephadex but was not required when DEAE-agarose was used. Dispersed brain cyclase was also chromatographed on agarose-NH(CH₂)₃ NH(CH₂)₃-NH₂ which exhibits both ionic and hydrophobic properties. Fifty percent of the applied activity was recovered with a fivefold increase in specific activity. The data suggest that the relative effectiveness of a given chromatographic procedure for detergent-dispersed adenylate cyclase may reflect the in fluence of both hydrophobic and ionic factors.     

Two-Step Synthesis and Hydrolysis of Cyclic di-AMP in Mycobacterium tuberculosis

Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5′-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5′-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5′-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.     

Dopamine effect on ionic conduction and activity of adenylate cyclase in the central nervour system of Lymnaea stagnalis

We investigated the action of 3-hydroxytyramine (dopamine) on ionic conduction in membranes of identified neurons of the large pond snailLymnaea stagnalis and adenylate cyclase activity in membranes of the nervous tissue of this mollusc. Stimulatory and inhibitory influences of dopamine upon adenylate cyclase activity were detected. Application of the mediator to cells which produce growth hormone caused inward and outward currents modulated by a phosphodiesterase inhibitor. An influence of cAMP-dependent phosphorylation on dopamine-dependent and dopamine-independent activity of adenylate cyclase is demonstrated. It is suggested that phosphorylation in nervous tissues is one possible mechanism regulating the action of dopamine as a result of inhibition of the sensitivity of adenylate cyclase to the action of G-proteins.Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 437–451, July–August, 1992.     

Effect of PGF2α on progesterone secretion and adenylate cyclase activity in ovine luteal tissue

Ovine luteal slices were used to study the effects of prostaglandins (PG) F₂α on luteinizing hormone (LH)-stimulated secretion of progesterone and adenylate cyclase activity. The accumulation of progesterone in incubation medium and adenylate cyclase activity was similar after incubation of luteal slices with Medium 199 alone or Medium 199 containing PGF₂α (250 ng/ml) for 3 hr. Addition of luteinizing hormone (LH; 100 ng/ml) resulted in a 2–3 fold increase in both the rate of progesterone accumulation and adenylate eyclase activity by 3 hr. When luteal slices were incubated in the presence of both LH and PGF₂α the rates of progesterone accumulation and adenylate cyclase activity were identical to those in flasks containing LH alone after 1 hr; however, after 3 hr both LH stimulated progesterone accumulation and adenylate cyclase activity were inhibited to levels similar to those observed in control slices.In a second experiment, after 60–120 min of exposure to PGF₂α the rate of progesterone accumulation in the medium was not different from that in untreated control slices. In addition, after this experiment the luteal slices were homogenized and the basal, sodium fluoride, LH, isoproterenol (ISO) and PGE₂ sensitive adenylate cyclase activities were determined to evaluate the hormonal specificity of the negative effect of the pretreatment with PGF₂α. Both LH and ISO stimulated adenylate cyclase activities were reduced after PGF₂α pretreatment. However, fluoride ion stimulated adenylate cyclase activity was not significantly effected by PGF₂α pretreatment and PGE₂ sensitive adenylate cyclase was effected only slightly.     

Variations du taux d'amp cyclique et des activités spécifiques de l'adénylate cyclase et de l'amp cyclique-phosphodiestérase pendant le cycle cellulaire d'un actinomycète

The variations in the concentrations of intra- and extracellular cyclic AMP and in the specific activities of adenylate cyclase (EC 4.6.1.1) and cyclic AMP phosphodiesterase (EC 3.1.4.17) have been monitored in synchronized culture of Nocardia restricta, a prokaryote belonging to the group of Actinomycetes. At the beginning of the cell cycel, during a first period of RNA and protein synthesis, there is an increasing synthesis of adenylate cyclase which can be suppressed in the presence of chloramphenicol or rifampicin. Simultaneously, the specific activity of cyclic AMP phosphodiesterase decreases and the concentrations of intra- and extracellular cyclic AMP rise. After the end of DNA replication, during a second period of RNA and protein synthesis, the specific activity of cyclic AMP phosphodiesterase increases; during the same time, the specific activity of adenylate cyclase and the level of intracellular cyclic AMP drop. It appears that the overall metabolism of cyclic AMP is coordinated so that the cyclic AMP level will be high at the beginning of DNA replication and will fall thereafter. The results are discussed in comparison with known data about the variations of cyclic AMP during the cell cycle of mammalian cells in cultures.     

Studies on the interconversion of 2,3'-anhydro-1-β-D-xylofuranosyluracil and 2,2'-anhydro-1-β-D-arabinofuranosyluracil

2,3'-Anhydro-1-β-D-xylofuranosyluracil (10) is converted, reversibly, into 2,2'-anhydro-1-β-D-arabinofuranosyluracil (1) in the presence of sodium tert-butoxide. The reaction probably involves 2',3'-anhydrouridine as an intermediate and equilibrium is strongly in favour of 1. The behaviour of 1 and 10 towards sodium hydroxide and sodium methoxide is described. Reaction of 3-azido-3-deoxy-2,5-di-O-p-nitrobenzoyl-β-D-xylofuranosyl chloride with monochloromercuri-4-ethoxy-2(lH)-pyrimidinone afforded crystalline 1-(3-azido-3-deoxy-2,5-di-O-p-nitrobenzoyl-β-D-xylofura-nosyl)uracil (24) in 57% yield. Alkaline methanolysis of 24 gave crystalline 1-(3-azido-3-deoxy-β-D-xylofuranosyl)uracil, which yielded 1-(3-amino-3-deoxy-β-D-xylofuranosyl)uracil (27) on hydrogenation. Deamination of 27 with nitrous acid gave mainly uracil (55%) and not the epoxide 5 or compounds derived from it.     

Hepatic microsomal enzyme induction by 2, 2', 3, 3', 4, 4'- and 2, 2', 3', 4, 4', 5-hexachlorobiphenyl

In an attempt to resolve conflicting reports in the literature, the effects of 2, 2', 3, 3', 4, 4'-hexa- and 2, 2', 3', 4, 4', 5-hexachlorobiphenyl on the hepatic microsomal drug-metabolizing enzymes were evaluated in the immature male rat. By comparison with the effects of the classical enzyme inducers, phenobarbitone (PB) and 3-methylcholanthrene (MC), 2, 2', 3, 3', 4, 4'-hexachlorobiphenyl, irrespective of its synthetic route, exhibited PB- and MC-type characteristics; the most prominent feature of the latter being a seven-fold increase in 4-chlorobiphenyl hydroxylase activity. 2, 2', 3', 4, 4', 5-Hexachlorobiphenyl also resembled a mixed (PB + MC)-type inducer although its MC-type characteristics were more pronounced than those of 2, 2', 3, 3', 4, 4'-hexachlorobiphenyl.     

Effects of adenosine analogs on rat cerebral cortical neurons

Adenosine and the adenosine 5'-phosphates (5'-AMP, 5'-ADP and 5'-ATP) depress the spontaneous firing of cerebral cortical neurons. In this study adenosine analogs, adenosine transport blockers and adenosine deaminase inhibitors have been used to gain further insight into the nature of the adenosine receptor and the likely routes of metabolism of extracellularly released adenosine. The firing rate of cortical neurons, including identified corticospinal neurons, was depressed by 2-substituted derivatives of adenosine. 2-Halogenated derivatives of adenosine were potent depressors while 2-aminoadenosine and 2-hydroxyadenosine (crotonoside) were slightly less potent than adenosine. The α,β-methylene isosteres of 5'-ADP and 5'-ATP were almost devoid of agonist activity while the β,γ-methylene analog was an active agonist. This suggests that ADP and ATP must be converted to AMP or possibly adenosine before they can activate the adenosine receptor. 2'-, 3'- and 5'- deoxyadenosine depressed spontaneous firing without antagonizing the effect of adenosine. Adenosine deaminase inhibitors, deoxycoformycin and $\text{erythro}$-9-(2-hydroxy-3-nonyl) adenine had potent, long lasting depressant actions on the spontaneous firing of cortical neurons and concurrently potentiated the actions of adenosine or 5'-AMP. Inhibitors of adenosine transport, papaverine and 2-hydroxy-5-nitrobenzylthioguanosine, prolonged the duration of action of adenosine and 5'-AMP. Intracellular recordings show that 5'-AMP hyperpolarizes cerebral cortical neurons and suppresses spontaneous and evoked excitatory postsynaptic potentials, in the absense of any pronounced alterations in membrane resistance.     

The IgG3 subclass of β1-adrenergic receptor autoantibodies is an endogenous biaser of β1AR signaling

Dysregulation of immune responses has been linked to the generation of immunoglobulin G (IgG) autoantibodies that target human β1ARs and contribute to deleterious cardiac outcomes. Given the benefits of β-blockers observed in patients harboring the IgG3 subclass of autoantibodies, we investigated the role of these autoantibodies in human β1AR function. Serum and purified IgG3(+) autoantibodies from patients with onset of cardiomyopathy were tested using human embryonic kidney (HEK) 293 cells expressing human β1ARs. Unexpectedly, pretreatment of cells with IgG3(+) serum or purified IgG3(+) autoantibodies impaired dobutamine-mediated adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) generation while enhancing biased β-arrestin recruitment and Extracellular Regulated Kinase (ERK) activation. In contrast, the β-blocker metoprolol increased AC activity and cAMP in the presence of IgG3(+) serum or IgG3(+) autoantibodies. Because IgG3(+) autoantibodies are specific to human β1ARs, non–failing human hearts were used as an endogenous system to determine their ability to bias β1AR signaling. Consistently, metoprolol increased AC activity, reflecting the ability of the IgG3(+) autoantibodies to bias β-blocker toward G-protein coupling. Importantly, IgG3(+) autoantibodies are specific toward β1AR as they did not alter β2AR signaling. Thus, IgG3(+) autoantibody biases β-blocker toward G-protein coupling while impairing agonist-mediated G-protein activation but promoting G-protein–independent ERK activation. This phenomenon may underlie the beneficial outcomes observed in patients harboring IgG3(+) β1AR autoantibodies.     

Insulin exerts actions through a distinct species of guanine nucleotide regulatory protein: inhibition of adenylate cyclase.

Insulin failed to exert an effect on the basal and glucagon- and guanosine 5'-[beta, gamma-imido]-triphosphate-stimulated adenylate cyclase activities of hepatocyte membranes. In the presence of high GTP (0.1 mM) concentrations, however, insulin was shown to inhibit adenylate cyclase activity. This effect was dose-dependent, exhibiting an EC50 (median effective concentration) of 3 microM for GTP. Elevated glucagon concentrations blocked the inhibitory effect of insulin in a dose-dependent fashion, with an EC50 of 1 nM. The insulin inhibition was dose-dependent (EC50 = 90 pM). The inhibitory effects of insulin were abolished using membranes from either glucagon-desensitized hepatocytes or cholera-toxin-treated hepatocytes. If either Mn2+ replaced Mg2+ in adenylate cyclase assays or Na+ was removed from the assay mixtures then insulin failed to exert any inhibitory effect. It is suggested that insulin exerts its action on adenylate cyclase through an inhibitory guanine nucleotide protein. This is integrated with the proposal [Heyworth, Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & Houslay (1983) Biochem. J. in the press] that insulin mediates a variety of cellular effects through a specific guanine nucleotide regulatory protein and associated protein kinase(s).     

Muscarinic cholinergic receptor mediated inhibitory transduction of adenylate cyclase activity in subcellular fractions from rat heart: improved detection in sodium phosphate buffer

Summary Cholinergic inhibition of myocardial adenylate cyclase activity in cell-free fractions has been known for many years, although the reported degrees of inhibition have been rather modest (20–30%), notably in rat heart fractions. The present study conducted with rat heart subcellular fractions document following major findings: (1) Myocardial adenylate cyclase activity and notably its cholinergic inhibition in cell-free fractions are notoriously labile to storage at 4°C whereas its stimulation by beta adrenergic receptor agonists or forskolin are reasonably well preserved during storage. (2) Among four buffers (Tris, glycylglycine, imidazole and sodium phosphate) examined, sodium phosphate buffer afforded the best preservation of cholinergic inhibitory response of adenylate cyclase. (3) The commonly used biochemical buffers, notably imidazole, exerted deleterious effect on the cholinergic inhibition of myocardial adenylate cyclase such that it was considerably attenuated or barely detectable; this explains, in part, the reported poor inhibition of myocardial enzyme by others. (4) Imidazole buffer, on the other hand, augmented beta adrenergic and forskolin stimulated adenylate cyclase activity. The likely significance of these findings is discussed from consideration that the observed differential influence of buffers results from differential actions on the interactions between the components (receptor/coupling G proteins/catalyst) comprising autonomic receptor coupled adenylate cyclase system in rat heart.     

Evaluation of the effects of adenosine on hepatic and adipocyte adenylate cyclase under conditions where adenosine is not generated endogenously.

Direct effects of adenosine on adipocyte and hepatic adenylate cyclase have been demonstrated in an assay system where adenosine is not generated. The substrate used, 2'-deoxy ATP may, on metabolism, only give rise to 2'-deoxyadenosine, which does not act at adenosine receptors. With a slight modification of existing assay techniques this assay system has been used to detect a hitherto undiscovered adenosine receptor on liver plasma membranes, which is antagonised by methylxanthines and which stimulates adenylate cyclase activity in a GTP-dependent manner. The potent inhibitory effects of purine-modified adenosine analogs on fat cell adenylate cyclase are reproduced by adenosine in this assay system. An application of this approach to the study of adenylate cyclase not only simplifies detection of the role of adenosine, but also yields insights into the interaction between guanine nucleotides and hormones.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Effect of thyroid status on α- and β-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased β-catecholamine responsiveness in hyperthyroid animals is due to a decrease in α-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the α₂-adrenergic inhibition of adenylate cyclase or the α₁-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamster. No effect of hyperthyroidism was found on the K_d of [³H]dihydroegocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to β-catecholamines was enhanced in adipocytes from hyperthyroid hamster, as was lipolysis. However, in adipocytes from hyperthyroid hamster the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on α-catecholamine action but enhances the activation of lipolysis by β-catecholamine agonists.     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.     

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N₃-ATP) and 8-azidoadenosine 5′-monophosphate (8-N₃-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N₃-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N₃-ATP at ATP-binding site 2. The adenylate kinase active center probe P¹,P⁵-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.     

Adenylate cyclase system of human skeletal muscle: Characteristics of catecholamine stimulation and nucleotide regulation

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.