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.     

Regulation of Adenosine-Sensitive Adenylate Cyclase from Rat Brain Striatum

An adenosine-sensitive adenylate cyclase has been characterized from rat brain striatum. In whole homogenates as well as in particulate fractions, N⁶-phenylisopropyl adenosine (PIA), 2-chloroadenosine, and adenosine N′-oxide were equipotent in stimulating adenylate cyclase. Although GTP inhibited basal as well as PIA-stimulated activity of whole homogenates, the enzyme showed an absolute dependency on GTP for stimulation by PIA, dopamine, epinephrine, and norepinephrine in a particulate fraction derived from discontinuous sucrose gradient centrifugation. Adenosine exerts two effects on this adenylate cyclase, stimulation at low concentrations and inhibition at high concentrations, suggesting the presence of two adenosine binding sites. The stimulation of adenylate cyclase by PIA was dependent on the concentration of Mg^2-. The degree of stimulation by PIA was greater at a low concentration of Mg²⁺, which suggests that stimulation by PIA was accompanied by increasing the apparent affinity for Mg²⁺. Activation of adenylate cyclase by PIA was blocked by theophylline or 3-isobutyl- 1-methylxanthine (IBMX). The pH optimum for basal or (PIA + GTP)-stimulated activities was broad, with a peak between 8.5 and 9.5. In the presence of GTP, stimulation by an optimal concentration of PIA was additive, with maximal stimulation by the catecholamines. Phospholipase A₂ treatment at a concentration of 1 U/ml for 5 min completely abolished the stimulatory effect of dopamine, whereas PIA-stimulated activity remained unaltered. These data suggest that rat brain striatum either has a single adenylate cyclase, which is stimulated by catecholamines and adenosine by distinct mechanisms, or has different cyclase populations, stimulated by either adenosine or catecholamines.     

Effect of Dopamine on Activation of Rat Striatal Adenylate Cyclase by Free Mg2+ and Guanyl Nucleotides1

Abstract: Stimulation of rat striatal adenylate cyclase by guanyl nucleotides was examined utilizing either MgATP or magnesium 5′-adenylylimidodiphos-phate (MgApp(NH) p) as substrate. GTP and 5′- guanylylimidodiphosphate (Gpp(NH) p) stimulate adenylate cyclase under conditions where the guanyl nucleotide is not degraded. The apparent stimulation of adenylate cyclase by GDP is due to an ATP-dependent transphosphorylase present in the tissue which converts GDP to GTP. We conclude that GTP is the physiological guanyl nucleotide responsible for stimulation of striatal adenylate cyclase. Dopamine lowers the K_a for Gpp(NH) p stimulation twofold, from 2.4 μM to 1.2 μM and increases maximal velocity 60%. The kinetics of Gpp(NH) p stimulation indicate no homotropic interactions between Gpp(NH) p sites and are consistent with one nonessential Gpp(NH) p activator site per catalytic site. Double reciprocal plots of the activation by free Mg²⁺ were concave downward, indicating either two sets of sites with different affinities or negative cooperativity (Hill coefficient = 0.3, K_0.5= 23 mM). The data conform well to a model for two sets of independent sites and dopamine lowers the K_a for free Mg²⁺ at the high-affinity site threefold, from 0.21 mM to 0.07 mM. The antipsy-chotic drug fluphenazine blocks this shift in K_a due to dopamine. Dopamine does not appreciably affect the affinity of adenylate cyclase for the substrate, MgApp(NH) p. Therefore, dopamine stimulates striatal adenylate cyclase by increasing the affinity for free Mg²⁺ and guanyl nucleotide and by increasing maximal velocity.     

Biochemical characterization of histamine-sensitive adenylate cyclase in mammalian brain.

Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (K_a, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the K_m (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg²⁺ stimulated enzymatic activity; the kinetic properties of this activation by Mg²⁺ suggest the existence of a Mg²⁺ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg²⁺. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg²⁺, low concentrations of Ca²⁺ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca²⁺. Other divalent cations, including Zn²⁺, Cu²⁺, and Cd²⁺, were also inhibitory. Of the divalent cations tested, only Co²⁺ and Mn²⁺ could replace Mg²⁺ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F^?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed.     

Potentiation by GTP of hormone-responsive adenylate cyclase in renal cortex plasma membrane preparations

GTP potentiated the stimulation by parathyroid hormone and prostaglandin E₁ of adenylate cyclase in a renal cortex preparation enriched in proximal tubule basal-lateral plasma membranes. Adenylate cyclase in these membranes did not respond to epinephrine nor glucagon, in the absence or presence of GTP. Activation of basal activity by GMP-PNP was strongly inhibited by GTP. GTP also increased the sensitivity of renal adenylate cyclase to parathyroid hormone and prostaglandin E₁. The synergistic effect of GTP was not inhibited by chelating nor thiol-reducing reagents.     

Vasopressin-sensitive kidney adenylate cyclase: Modulation of the hormonal response

Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg²⁺, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg²⁺, and GMPPNP, respectively. Mg^2+·ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg²⁺ interacts with an enzyme regulatory site. Finally, Mg²⁺ can modulate the hormonal response, with Mg²⁺ions affecting the coupling function–that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg²⁺ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg²⁺ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30° C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15° C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10^?8 M to 10^?5 M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg²⁺ concentrations except for the increase in velocity when raising Mg²⁺ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.     

Adenylate cyclase enhancing factor from rat osteosarcoma cytosol.

A cytosol factor from a transplantable rat osteosarcoma stimulates the adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity of partially purified membranes 1.5 fold at pH 7.6 and over 5 fold at pH 6.5. This effect can also be seen at maximum Gpp(NH) stimulation of the enzyme. The cytosol factor is non-dialyzable, ultrafiltrable through a 100,000 dalton exclusion membrane, heat labile and trypsin digestible. The stimulation is immediate, is independent of Ca²⁺, exhibits sygmoidal concentration dependency and is enhanced by GTP. The factor did not bind GTP. The stimulatory activity was fully recovered in two Sephadex G-100 fractions of approximate molecular weights of 55,000 and 29,500. Unlike the starting material the fractions were not stable to freeze-thawing or lyophilization. A similar factor could not be found in embryonic bone, nor did the osteosarcoma factor affect bone 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.     

The hamster adipocyte adenylate cyclase system II. Regulation of enzyme stimulation and inhibition by monovalent cations

In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na⁺>Li⁺>K⁺>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na⁺>Li⁺>K⁺>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E₁ on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E₁, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.     

Calcium potentiates the peroxidation of erythrocyte membrane lipids

A clonal cell line from rat osteosarcoma was found to possess parathyroid hormone and isoproterenol sensitive adenylate cyclase. This study examines the relationship between the two hormones and triphosphoguanine nucleotide with respect to enzyme activation. Concentration-dependence curves, analyzed by computer-aided curve fitting, revealed: (1) in the presence of 5 microM GTP there were two apparent affinities for parathyroid hormone (Km 9 and 89 nM) and isoproterenol (Km 72 and 340 nM; (2) and two affinities for guanosine-5' (beta, gamma-imido)triphosphate (Km 0.25 and 1.3 microM); (3) hormones and guanine nucleotides reciprocally shifted each other's concentration dependence curve to the high affinity sites; (4) parathyroid hormone and isoproterenol interacting with high affinity sites competed for the same adenylate cyclase; (5) parathyroid hormone and isoproterenol, acting on low affinity sites had additive effects and also stimulated adenylate cyclase in the absence of added guanine nucleotides. The findings are consistent with (i) competition of parathyroid hormone and isoproterenol for the activation of the high (hormone) affinity complex containing: receptors, nucleotide subunit, triphosphoguanine nucleotide, catalytic unit (ii) the apparent presence of receptor-nucleotide sub-unit GDP-catalytic unit complexes with low hormone affinity which are stimulated by parathyroid hormone and isoproterenol separately.     

Evidence for an endogenous parathyroid hormone-sensitive adenylate cyclase activator

The middle medullary membrane of canine renal tissue was completely insensitive to parathyroid hormone (PTH) stimulation in the absence of GTP or tissue supernatant. In contrast, removal of endogenous GTP did not eliminate the PTH stimulation of cAMP formation in the renal cortical membrane preparation (P₃). Addition of boiled cortical P₃ to native cortical P₃ enhanced PTH stimulation in a dose-dependent manner. The boiled cortical P₃ was not active in middle medullary tissue. The minimum concentration of GTP which was required to cause stimulation in both cortical and middle medullary preparations was similar. The results suggest that (1) there are two classes of PTH-sensitive adenylate cyclase; one class is GTP dependent, while the other is GTP independent. (2) A new factor, as yet unidentified, in addition to GTP, is important in the regulation of adenylate cyclase by PTH in the renal cortex.     

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.     

The influence of the divalent cations Mn2+ and Mg2+ on the activation of particulate and digitonin-solubilized adenylate cyclase from rat fat cell membranes

A comparison was made between the activation of membrane-bound adenylate cyclase from rat fat cell membranes and the enzyme solubilized with digitonin. The isoprenaline stimulation of the particulate enzyme was enhanced by GTP, both in the presence of Mg²⁺ and Mn²⁺, but no effect of the metal ion nor of GTP was found on the K_a of isoprenaline. The K_a of sodium fluoride for enzyme stimulation was shifted to 3-fold higher concentrations when Mg²⁺ was replaced by Mn²⁺, whereas V decreased. GTP did not influence the K_a of sodium fluoride but reduced V, irrespective of the metal ion. After digitonin solubilization the enzyme was no longer responsive to isoprenaline or GTP; however, V of the sodium fluoride activation was higher in the presence of Mn²⁺ than in the presence of Mg²⁺, and the K_a was found at 15-fold higher concentrations. Both the solubilized and the particulate adenylate cyclase were inhibited by adenosine; this inhibition was also seen with the fluoride stimulated enzyme. We conclude that solubilization with digitonin did not result in an enzyme preparation which preferentially turns over MnATP²⁺, although the fat cell adenylate cyclase possesses a metal ion regulatory site with a higher affinity for Mn²⁺ than for Mg²⁺. The data suggest that the guanyl nucleotide regulatory site and the sodium fluoride-sensitive site are located on different subunits while there is an interaction between the metal ion regulatory site and the fluoride-sensitive site.     

Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of 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.     

Mechanism of action ofVibrio cholerae enterotoxin

Summary The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30°C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximateK _m for ATP, Mg⁺⁺ increases from about 1.8 to 3.4mm in the toxinstimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg⁺⁺ concentrations, from 20% at low levels (0.2mm) to 500% at high concentrations (greater than 3mm). Addition of GTP, Mg⁺⁺ (0.2mm) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation ofV _max. GTP enhances the toxin-treated enzyme activity two-to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6mm ATP and 5mm MgCl₂ the apparentK _a for GTP, Mg⁺⁺ is 5 to 10m. The control (unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide. 5-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg⁺⁺, decreasing the apparentK _a from about 25 to 5mm. (–)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparentK _a for free metal. Sodium fluoride, which cause a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparentK _a for Mg⁺⁺; moreover, it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three- to fivefold stimulation by toxin becomes a 30 to 60% inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (–)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.Part of this work was reported at the 1974 meeting of the Federation of American Societies for Experimental Biology (Bennett & Cuatrecasas, 1974).     

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.     

Changes in pH sensitivity of adenylate cyclase specifically induced by fluoride and vanadate

The interdependent effects of divalent cations, pH, and various activators of adenylate cyclase were examined in partially purified plasma membranes from rat liver. This adenylate cyclase was found to exhibit largely alkaline pH optima, in the range of 8.3 to 9.3, for the expression of basal activity, and activities with GTP, GPP(NH)P, prostaglandin E₁ and GTP, and N⁶-(phenylisopropyl)adenosine and GTP. Glucagon and GTP, while increasing activity 8- to 10-fold, shifted the optimum activity to about pH 7.5. However, stimulation of the enzyme by 10 mm NaF or 3 mm Na₃VO₄ was strikingly dependent on pH. In both cases activation was optimal at pH values between 6.3 and 7.3, though above about pH 8.5 fluoride was barely stimulatory and vanadate was slightly inhibitory. This effect of elevated pH to reduce fluoride- or vanadate-stimulated activity could be prevented by glucagon plus guanine nucleotide, but could not be reversed once activity was lowered during preincubation. The data suggest that this effect was not due to the formation of an inhibitor of adenylate cyclase per se, nor to an artifact of assay methods. The effect of elevated pH was more pronounced with Mn²⁺ as activating cation than with Mg²⁺. With fluoride and lower pH adenylate cyclase was essentially Mn²⁺ requiring, whereas with fluoride and higher pH activity was comparable with either cation. The data suggested that combinations of pH, divalent cation, and activating ligand dictate the interactions of the constitutive subunits of the adenylate cyclase and provide additional criteria with which current models for the regulation of adenylate cyclase may be tested.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the ?EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F^?, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in ?EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in ?EFA membranes than in +EFA. Total Na⁺, K⁺ (Mg²⁺)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent K_m and V_max. 5′-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient,heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg²⁺ or Mn²⁺. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds.