Characterization of adenylate and guanylate cyclases in rat peritoneal mast cells

Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn⁺⁺ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg⁺⁺ or Mn⁺⁺. The apparent Km values of adenylate cyclase for Mn⁺⁺-ATP and Mg⁺⁺-ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn⁺⁺-GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.     

Adenylate cyclase activity in porcine sperm in response to female reproductive tract secretions

Adenylate cyclase activities were studied in porcine sperm in the presence and absence of Mn⁺⁺ before and after incubation in vivo and in vitro. Incubation of sperm in vivo for 30 min increased the Mg⁺⁺-stimulated adenylate cyclase activity from 35.1 pmoles cyclic AMP formed per mg protein per 10 min to 50.4 pmoles. The activity stimulated by Mg⁺⁺ and Mn⁺⁺ increased from 392 to 729 pmoles after 30 min of in vivo incubation. Activity after incubation in vivo for 120 min was not different from activity after 30 min. In vitro incubation of porcine sperm in Ca⁺⁺-free Ringer-fructose resulted in no change, but incubation in oviductal and uterine flushings obtained from gilts soon after ovulation increased Mg⁺⁺-stimulated activity by 24% and Mg⁺⁺?+ Mn⁺⁺-stimulated activity by 49%. In vitro incubations in preovulatory flushings plus follicular fluid or in bovine serum albumin also increased adenylate cyclase activity.     

Properties of adenylate cyclase activity during early sea urchin development

The total adenylate cyclase activity in homogenates of eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus was assayed in vitro and found to remain constant in eggs before and at intervals after fertilization. In S. purpuratus egg homogenates virtually all of the enzyme activity was sedimented by centrifugation at 20 000 g. The enzyme specific activity in the 20 000 g pellet remained unchanged at each point through first cleavage, though it was several-fold higher than in the whole homogenate. The adenylate cyclase from both fertilized and unfertilized eggs was maximally active in vitro when assayed with 10 mM MgSO₄ and 10 mM NaF at pH 8 using 0.2 mM AMP-PNP (an ATP analog) as the substrate. Sucrose density gradient centrifugation of egg homogenates showed that adenylate cyclase activity was present in fractions which sedimented at a variety of densities. The adenylate cyclase specific activity in cortices isolated by the method of Sakai [10] from eggs at first cleavage was 4- to 6-fold higher than in unfertilized egg cortices. The increased enzyme activity in egg cortices at first cleavage suggests that adenylate cyclase-containing membranes may become localized within the egg cortex after fertilization.     

Particulate and soluble adenylate cyclase activities of mouse male germ cells

Germ cells from the mouse testis possess both a particulate and a soluble form of adenylate cyclase (EC 4.6.1.1). Germ cell adenylate cyclase activity is Mn⁺⁺ dependent and is not stimulable with either NaF or 5′guanylylimidodiphosphate. Both particulate and soluble adenylate cyclase specific activities increase as germ cells progress through their differentiative stages, but epididymal spermatozoa seem to lack a significant amount of soluble activity. Somatic cells of the seminiferous tubule possess only a membrane bound activity, which is Mg⁺⁺ and Mn⁺⁺ dependent, NaF and 5′guanylylimidodiphosphate stimulable. It is suggested that germ cell adenylate cyclases represent incomplete forms of the enzyme, devoid of regulative subunits.     

Solubilization of adenylate cyclase of brain membranes by lipid peroxidation

Adenylate cyclase in the membrane fractions of bovine and rat brains, but not in rat liver plasma membranes, was solubilized by treatment with Fe²⁺ (10 μM) plus dithiothreitol (5 mM). Solubilization of the enzyme by these agents was completely prevented by simultaneous addition of N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation. Ascorbic acid also solubilized the enzyme from the brain membranes. Lipid peroxidation of the brain membranes was characterized by a selective loss of phosphatidylethanolamine. Solubilization of membrane-bound enzymes by Fe²⁺ plus dithiothreitol was not specific for adenylate cyclase, because phosphodiesterase, thiaminediphosphatase and many other proteins were also solubilized. Solubilized adenylate cyclase had a high specific activity and was not activated by either NaF, 5′-guanylyl imidodiphosphate (Gpp[NH]p) or calmodulin. These results suggested that lipid peroxidation of the brain membranes significantly solubilized adenylate cyclase of high specific activity.     

Modifications of adenylate and guanylate cyclase activities during multiplication of KB cells.

Adenylate and guanylate cyclase activities do not vary in concert during the multiplication of KB cells. Adenylate cyclase activity is low and slightly increases at cell confluency, guanylate cyclase activity, great in sparce cells, decreases during cell multiplication period. These variations are not caused by a modification of catalytic sites because the apparent Km for ATP or GTP is not changed, but by a modification of the dependance on Mg⁺⁺ or Mn⁺⁺ ions. Fresh serum increases guanylate cyclase activity but does not affect adenylate cyclase.     

Calcium and adenosine affect human sperm adenylate cyclase activity

The adenylate cyclase activity of human ejaculated spermatozoa in broken-cell preparations was investigated. In the presence of 5 mM metal cations and 0.1 mM ATP, the relative enzyme activity with Mn²⁺, Ca²⁺, Mg²⁺, Ba²⁺ was 1.00, 0.28, 0.22, and 0.03, respectively. Added Ca²⁺ appeared to activate the enzyme in the presence of Mn²⁺ or Mg²⁺. The human sperm adenylate cyclase was stimulated by ～ 2-fold by free Ca²⁺ (lmM) in the presence of Mg²⁺ (5 mM). If the GTP analogue, 5′-guanylyl imidophosphate (Gpp(NH)p) was added to the sperm homogenate in the presence of 200 μM ethylene-glycol-bis (β-aminoethylether) N,N′-tetraacetic acid (EGTA), the adenylate cyclase activity was increased by approximately 25%, but with the addition of 280 μM Ca²⁺ there was a decrease in enzyme activity. A similar response to low concentrations of Ca²⁺ was obtained after complementation of the sperm enzyme with the guanine nucleotide regulatory component from human erythrocytes, where the addition of 40 μM Gpp(NH)p, 200 μM EGTA, and Ca²⁺ (≤ 160 μM) stimulated the sperm enzyme ～ 3–4-fold, but the further addition of Ca²⁺ (280 μM, final) neutralized the stimulatory effect. The addition of adenosine, and the nucleotides 5′-AMP and 5′-ADP inhibited the enzyme, whereas guanine and 5′-GMP had no appreciable effect. Human follicular fluid and serum also had little direct effect on the sperm adenylate cyclase. These resuls suggest that Ca²⁺ might be an important physiological modulator of the human sperm adenylate cyclase.     

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.     

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.     

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.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Isolation and characterization of the plasma membrane from Yoshida hepatoma cells

Summary Plasma membranes isolated from Yoshida ascites hepatoma AH-130 by a modification of the method of T. K. Ray (Biochim. Biophys. Acta 196: 1, 1970), were subfractionated into three fractions having densities (d) 1.12, 1.14 and 1.16 by discontinuous sucrose density-gradient. Membrane subfractions were characterized by electron-microscopy, by assay of marker enzymes and by lipid composition. All subfractions appeared to be essentially free from whole mitochondria, lysosomes and nuclei. Subfraction d 1.16 had, the highest 5-nucleotidase, Mg⁺⁺-ATPase and (Na⁺+K⁺)-ATPase activities; cytochromec oxidase was undetectable in any fraction and glucose-6-phosphatase was measurable only in fraction d 1.14. Adenylate cyclase had the highest activity in fractions d 1.14 and 1.16. Cyclic AMP phosphodiesterase was nearly equally distributed in the fractions. Adenylate, cyclase, 5-nucleotidase and Mg⁺⁺-ATPase activities of tumor membrane were lower with respect to liver plasma membrane, while cyclic AMP phosphodiesterase and (Na⁺+K⁺)-ATPase were found to have similar activities in the two membrane preparations. With respect to liver membrane, hepatoma membrane contained a higher amount of glycolipids and a higher amount of phospholipids accounted for mainly, by sphingomyelin, phosphatidylserine and phosphatidic acid. The possible significance of the decrease of adenylate activity in the hepatoma membrane is briefly discussed.     

Retention of the speract receptor by isolated plasma membranes of sea urchin spermatozoa

Membrane vesicle preparations enriched in plasma membrane marker proteins, such as adenylate cyclase, were prepared from spermatozoa of the sea urchin, Lytechinus pictus. These membranes, prepared by nitrogen cavitation and subsequent sucrose gradient centrifugation, retained the capacity to bind [125I]-Bolton-Hunter speract (nonspecific binding was less than 5% of specific binding). Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly, Tyr-Asp-Leu-Thr-Thr-Gly-Gly-Gly-Val-Gly and Gly-Phe-Ala-Leu-Gly-Gly-Gly-Val-Gly caused a 50% decrease in [125I]-Bolton-Hunter speract binding at 10, 600, 1260 and 3160 nM concentrations, respectively. One analogue (Phe-Asp-Leu-Asn-Gly-Gly-Gly), which had no biological activity, failed to compete at concentrations as high as 10 microM. To demonstrate that the binding was due to the isolation of membranes with an intact receptor, the speract analogue (Gly-Gly-Gly-Gly-Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) was synthesized, radiolabeled with 125I at the position of tyrosine, and covalently cross-linked to the receptor with disuccinimidyl suberate. A single radiolabeled band at an apparent molecular weight of 77,000 was detected on Na X dodecyl X SO4 gels. These studies are the first to identify a receptor for egg-associated peptides in isolated spermatozoan membranes.     

A thermodynamic analysis of the correlation between active Na+ transport and the rate of oxygen consumption in epithelia

Summary Insulin decreased markedly the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation in sucrose gradients. The hormone effect was not readily evident in crude membrane preparations.The kinetics of this effect indicate that some time was required for the onset of the insulin-induced inactivation. This lag period decreased when the insulin concentration was increased. The hormone dose dependence for adenylyl cyclase inactivation measured at a fixed time (3 min) showed a 10 to 15% decrease in activity at 1 to 30 U per ml insulin; 30 to 40% at 100 to 1000 U per ml; and 75% at 0.1 U per ml.The insulin effect was completely abolished by 0.1mm GMP-P(NH)P, 10mm fluoride, or 50 ng per ml glucagon, or by increasing the Mn⁺⁺ concentration to 4mm. In addition, it was partially reversed by the addition of a fraction from the sucrose gradient, which contained soluble factors.The kinetics of the adenylyl cyclase-catalyzed reaction were studied using ATP or AMP-P(NH)P as adenylyl donor, and Mn⁺⁺ or Mg⁺⁺ as divalent cation, in the absence or presence of insulin. With ATP and Mg⁺⁺ there was a striking reduction of the transient reaction rates after 1.5 min of incubation. Under these conditions the insulin effect was not evident. On the contrary, with ATP and Mn⁺⁺ this spontaneous reduction of activity was less evident; however, in the presence of insulin there was a clear and marked reduction of the transient reaction rate measured after 1.5 min of incubation. With AMP-P(NH)P the kinetic data were qualitatively similar to those observed with ATP.It is concluded that under certain assay conditions adenylyl cyclase may be converted to an inactive enzyme form, and that such a conversion is more evident in the presence of Mg⁺⁺ than with Mn⁺⁺. In the latter case, insulin appears to enhance the rate of this conversion.     

New ATP incorporating activities from rat liver mitochondria: the attachment of a portion of ATP to a pronase-sensitive receptor

Studies using a Brij 58 detergent extract of rat liver mitochondria reveal that these organelles can catalyze the time-dependent incorporation of a portion of [³H]ATP into an acid-insoluble product. The activities studied using 8 mM Mn⁺⁺ or 15 mM Mg⁺⁺ are stimulated by dithiothreitol and by CTP, GTP or UTP, while that studied using 2 mM Mg⁺⁺ is not. The incorporated tritium remains bound after incubation in the presence of excess unlabeled ATP and chromatography on Sephadex G-25. The labeled product is insensitive to ribonuclease A and snake venom phosphodiesterase, but is sensitive to pronase. The attached portion of the ATP molecule released upon treatment of the product has been tentatively identified as adenosine for the activities studied using 2 mM Mg⁺⁺ or 8 mM Mn⁺⁺ and as AMP (80%) and adenosine (20%) for the reaction studied using 15 mM Mg⁺⁺.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes

A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 · 10⁻¹⁰−1 · 10⁻⁷ M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 ± 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive inteetinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 μg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5–10 mM Mg²⁺ and was inhibited by 1 · 10⁻⁵ M Ca²⁺. At sufficiently high concentrations, both ATP (3 mM) and Mg²⁺ (40 mM) inhibited the enzyme.Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E₁ and E₂ at 1 · 10⁻⁵ M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Isolation and characterization of a liver plasma membrane fraction enriched in glucagon-sensitive adenylate cyclase

Partially purified liver plasma membranes were fractionated further on sucrose layers. Three membrane populations, numbered Peaks 1, 2 and 3, were isolated at densities of 1.23, 1.16, and 1.03, respectively. Peaks 1 and 2 were enriched to a similar degree in 5′-nucleotidase activity, a plasma membrane marker, relative to membranes in Peak 3. Electron micrographs indicated that Peak 1 possessed desmosomes and bile canaliculi, while Peak 2 contained large vesicles as well as smaller vesicular structures attached to membranes. The latter have been attributed to hepatocyte sinusoidal surfaces. All three membrane fractions contained adenylate cyclase activity with the highest specific activity found in Peak 2. The enzyme in all three peaks was F sensitive with higher sensitivity in Peaks 1 and 2. Glucagon sensitivity of adenylate cyclase in Peak 2 membranes was four times that of Peak 1. Only Peak 2 membranes were sensitive to epinephrine. The Peak 2 membranes were three times more sensitive to glucagon than the partially purified membranes from which they were derived. These findings indicate that, while both bile canalicular and sinusoidal faces of hepatocytes possess adenylate cyclase, the sinusoidal fraction is more sensitive to glucagon. Solubilized adenylate cyclase of the Peak 2 membranes, obtained as the 165,000g supernate of membranes treated with Lubrol-PX, was sensitive to stimulation by guanyl nucleotide analogs. Guanyl nucleotide sensitivity thus resides in the catalytic site and is not dependent on membrane integrity. All three membrane fractions possessed similar activities of nucleotide phosphohydrolase activity.