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1.
Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides 总被引:2,自引:0,他引:2
A potent (Ki = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N6,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg2+ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg2+ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E1, E2, and F2α, histamine, and glucagon. 相似文献
2.
《Experimental mycology》1984,8(4):334-341
The ascomyceteSaccobolus platensis Gamundi´& Ranalli requires light to produce apothecia. It has now been found that this light requirement can be satisfied by a 24-h pulse of white light at certain stages of the sexual cycle. The addition of exogenousN6,O2′-dibutyryl adenosine 3′,5′-cyclic monophosphate (db-cyclic AMP) to the dark growing mycelia could replace rather efficiently the inductory effect of light; cyclic AMP,N6-monobutyryl cyclic AMP, andO2′-monobutyryl cyclic AMP were less effective, while guanosine 3′,5′-cyclic monophosphate (cyclic GMP) was a very weak inducer. An inducing effect similar to that of db-cyclic AMP was obtained by the addition of 3-isobutyl-1-methylxanthine (MIX) or theophylline to cultures developing in darkness. In the presence of theophylline, endogenous cyclic AMP levels of dark-grown mycelia were several fold higher than those of control cultures. The cyclic AMP content of mycelia growing under different light regimes was measured and no significant differences were observed. However, cultures submitted to white light showed an increase in adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) and a decrease in cyclic AMP phosphodiesterase (3′,5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) specific activities compared with the activities of dark-grown mycelia. The cyclic AMP phosphodiesterase activity was strongly inhibited by theophylline and by MIX. The possible role of cyclic AMP in the induction of apothecia in this species is discussed. 相似文献
3.
Adenosine 3',5' cyclic monophosphate in Euglena gracilis 总被引:2,自引:0,他引:2
contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg. 相似文献
4.
Arrest of mammary tumor growth in vivo by L-arginine: stimulation of NAD-dependent activation of adenylate cyclase 总被引:1,自引:0,他引:1
Y S Cho-Chung T Clair J S Bodwin D M Hill 《Biochemical and biophysical research communications》1980,95(3):1306-1313
growth of hormone-dependent rat mammary tumors was arrested by daily injections of L-arginine (L-arginine·HCl 50 mg/200g rat s.c.). Arginine + N6,O2′-dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) acted synergistically to enhance the growth inhibitory effect. Growth arrest by arginine was accompanied by a sharp increase in cellular cAMP content, which was preceded by parallel increases in NAD-dependent ADP-ribosylation of the membrane proteins and NAD-dependent activation of adenylate cyclase. The ADP-ribosylation of the membrane proteins required GTP and was catalyzed similarly by the 105,000 × g supernatant fraction of the tumor and by cholera toxin. These results suggest a specific role for arginine in the cAMP-mediated inhibition of mammary tumors. 相似文献
5.
Y S Cho-Chung T Clair J P Zubialde 《Biochemical and biophysical research communications》1978,85(3):1150-1155
Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N6,O2′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors. 相似文献
6.
An exogenous supply of N6,O2′-dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) produces regression of one type of Walker 256 mammary carcinoma cell population (DBcAMP-responsive); a second type of cell population continues to grow despite DBcAMP treatment (DBcAMP-unresponsive). A correlation was found between altered cAMP-binding of the tumor cytosol and DBcAMP-unresponsiveness. It was found that there was: a) a higher apparent dissociation constant (Kd) for cAMP-binding in unresponsive tumor cytosol , and b) unsaturability of cAMP-binding by unresponsive tumor cytosol in response to elevated cAMP levels . Cycloheximide abolished the saturation of cAMP binding as well as tumor regression produced by DBcAMP. 相似文献
7.
Effects of cyclic AMP on the kinetics of serum protein synthesis in cultured mouse hepatoma cells 总被引:1,自引:0,他引:1
The mouse hepatoma cell line, Hepa, was cultured in the presence of either 1 mM N6, O2′-dibutyryl cyclic AMP (Bt2cAMP), 0.5 mM 3-isobutyl-1-methylxanthine, or 1 μg/ml cholera toxin. The synthesis and secretion of albumin, α-fetoprotein, and transferrin were elevated above controls by 24 h reaching two- to fourfold stimulations within 72 h. These effects were reversible and were specific for the serum proteins. The stimulation of serum protein synthesis was accompanied by a decrease in the rate of cell proliferation. Protein synthetic parameters were analyzed in Hepa cells 72 h after exposure to N6,O2′-dibutyryl cyclic AMP. The cellular rate of albumin synthesis was increased fourfold and the relative rate of albumin synthesis was increased approximately threefold. N6,O2′-Dibutyryl cyclic AMP did not affect the size distribution of either total Hepa polyribosomes or of albumin-synthesizing polyribosomes. The elongation rate on total mRNA and on albumin mRNA was decreased by approximately 40%. These results indicate that the rate of initiation of total Hepa mRNA and of albumin mRNA also decreased by 40%. The nonspecific nature of the N6,O2′-dibutyryl cyclic AMP effect on Hepa protein synthetic parameters must be due to an alteration in the level of a common substrate, perhaps ATP. The specific threefold increase in the relative rate of albumin synthesis with no alteration in polyribosome sizes requires a threefold increase in the relative amount of functional albumin mRNA in Hepa cells. This prediction was confirmed by cell-free translation of Hepa polyribosomes. 相似文献
8.
A specific antibody to phenylalanine:pyruvate transaminase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by glucagon and N6,O2′-dibutyryl cyclic AMP. Cycloheximide given simultaneously with glucagon or dibutyryl cyclic AMP blocked the increase in [3H]leucine incorporation when it was injected along with glucagon, but had no effect when given 4 h after the glucagon. This finding suggests that the mRNA synthesis for phenylalanine:pyruvate transaminase may be completed in 4 h. 相似文献
9.
M K Haddox J H Stephenson M E Moser D B Glass J G White B Holmes-Gray N D Goldberg 《Life sciences》1979,24(17):1555-1566
Chronic ascorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80%); ascorbate (1 mM) addition to these cells restored the cellular concentration to control levels. Splenic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to exogenous ascorbate whereas thiol reducing agents diminished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of ascorbate while agents known to interfere with or promote H2O2 production had no effect. Guanylate cyclase activity in cell lysates increased after treatment of intact cells with ascorbate; dithiothreitol reversed this effect. Ascorbate also enhanced guanylate cyclase activity in cell lysates. The results suggest that oxidizing equivalents in the form of the monoanionic free radical of ascorbate alter cyclic GMP metabolism in these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component. 相似文献
10.
Masafumi Fujimoto Atsushi Ichikawa Kenkichi Tomita 《Archives of biochemistry and biophysics》1974,161(1):54-63
Cyclic AMP phosphodiesterase from Saccharomyces cerevisiae was purified about 20,000-fold to homogeneity. The purified enzyme had a molecular weight of about 60,000 as estimated by gel filtration.The enzyme activity was optimal at pH 8.5–9.0 and was not stimulated by imidazole. Among cyclic 3′,5′-nucleotides, cyclic AMP was the most active substrate for the purified enzyme (Km = 0.25 mM), but it was inhibitory at concentrations above 4 mm. N6,O2′-dibutyryl cyclic AMP was not hydrolyzed at all.Unlike other cyclic AMP phosphodiesterases from various sources, the purified yeast enzyme did not require divalent metal ions for maximal activity and was rather inhibited in various degrees by added metal ions. The enzyme was not very sensitive to thiol inhibitors.The purified yeast enzyme was strongly inhibited by theophylline and slightly by caffeine. In contrast to the enzyme from S. carlsbergensis, the enzyme from S. cerevisiae was not inhibited at all by ATP or PPi.The enzyme activity was not released into the growth medium, and the intracellular distribution studies indicated that the enzyme was located mainly in the cytosol fraction. 相似文献
11.
Cyclic GMP-dependent protein kinase from bovine lung and cyclic AMP-dependent protein kinase from bovine heart are inactivated by Nα-tosyl-L-lysine chloromethylketone (TLCK) in the presence of cyclic GMP and cyclic AMP, respectively. The inactivation of both protein kinases is pseudo-first order, suggesting the rate limiting step is beyond the binding of TLCK. Cyclic GMP-dependent protein kinase is inactivated less than as rapidly as cyclic AMP-dependent protein kinase, although it shows a higher apparent affinity for TLCK. Cyclic AMP stimulated the rate of inactivation of cyclic AMP-dependent protein kinase 10-fold but cyclic GMP stimulated the rate of inactivation of cyclic GMP-dependent protein kinase only 1.5-fold. The rate of inactivation of cyclic GMP-dependent protein kinase by TLCK is sufficiently rapid (half-time of about 30 min at 37°C with 2 mM TLCK) to account for the effects of TLCK on cell growth observed by others. 相似文献
12.
Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca2+ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10?8 M and 5.0 × 10?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. , 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca2+-dependently and independently. 相似文献
13.
Gene I. Higashi Peter W. Kreiner James J. Keirns Mark W. Bitensky 《Life sciences》1973,13(9):1211-1220
Homogenates of adult (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E1, E2 or B1. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP () over cyclic GMP (). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen. 相似文献
14.
M. Shinmei P. Ghosh T.K.F. Taylor 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,437(1):94-105
The stimulatory effects of N6,O2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases. 相似文献
15.
Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the Mr of the enzyme was 145,000. This protein was composed to two identical subunits each with Mr of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N2-monobutyryl cyclic GMP > N2, O2-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis. 相似文献
16.
P M Silverman 《Biochemical and biophysical research communications》1976,70(2):381-388
Levels of guanylate cyclase activity in extracts of the unicellular eukaryote differed by at least 100-fold at different stages of the cell cycle, paralleling changes in the cyclic GMP content of this organism (Proc. Natl. Acad. Sci. U.S.A. , 442 (1975)). Extracts of vegetative cells lacked appreciable guanylate cyclase activity, whereas the specific activity of the enzyme in zoospore extracts was 2 nmol cyclic GMP synthesized/min/mg protein at 35°. Guanylate cyclase activity increased at least 50-fold during the period of zoospore formation when cyclic GMP begins to accumulate . Since actinomycin D or cycloheximide added at the beginning of this period blocked any increase in enzyme activity, it appears that synthesis of guanylate cyclase during sporulation is responsible for the accumulation of cyclic GMP that occurs at that time. 相似文献
17.
Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae () have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells. 相似文献
18.
Adherence and phagocytosis of 51chromium labeled sheep red blood cells ([51Cr]-sRBC) by P388 D1 cells in tissue culture were studied under various conditions and were found to possess certain requirements including opsonization, temperature, microfilaments and cyclic nucleotide levels. Exogenous administration of 10?2 M N6, O2-dibutyryl adenosine 3′–5′ cyclic monophosphoric acid (db-cAMP) or adenosine 3′–5′ cyclic monophosphoric acid (cAMP) inhibited phagocytosis of opsonized [51Cr]-sRBC by 36 and 42%, respectively. Aminophylline potentiated the inhibitory response to both cAMP and db-cAMP. The measurement of endogenous cyclic nucleotide levels during phagocytosis of opsonized sRBC showed a rise in guanosine 3′–5′ cyclic monophosphate (cGMP) during the first 5 min with a gradual decline to control levels at 45 min and a rise in cAMP levels reaching a peak at 30 min which remained above control values for the duration of the experiment. As the rate of phagocytosis decreased the ratio of increased. These observations emphasize the importance of metabolic functions and cyclic nucleotides during phagocytosis by the P388 D1 cells and strengthen the usefulness of the P388 D1 cells as a model in evaluating various macrophage activities. 相似文献
19.
N6,O2-′Dibutyryl cyclic AMP (dibutyryl cyclic AMP), a derivative of 3′,5′-adenosine monophosphate (cyclic AMP) resistant to phosphodiesterase inactivation, has been reported to stimulate serotonin and melatonin synthesis in the pineal gland in vitro1–3. In brain adenyl cyclase and phosphodiesterase, which catalyse the formation and the inactivation of cyclic AMP, are found chiefly in the synaptosomal fraction of the tissue homogenates4, where vesicles containing monoamine are also present5. These factors prompted us to study the effects of cyclic AMP and its dibutyryl derivative on the synthesis of brain monoamines. 相似文献
20.
T E Donnelly 《Archives of biochemistry and biophysics》1976,173(1):375-385
The effects of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by crude and partially purified phosphodiesterases obtained from bovine heart and rat liver were studied in order to determine if imidazole has an activity on cyclic nucleotide hydrolysis under conditions which might explain its ability to antagonize the effects of several hormones. Imidazole-Cl (40 mm, pH 7.4) had no effect on the hydrolysis of cyclic AMP or cyclic GMP at substrate levels below 10 μm by the crude enzymes but increasing stimulation was observed with increasing substrate concentrations reaching a twofold stimulation at 1 mm cyclic nucleotide. Three phosphodiesterases with varying substrate specificities were partially purified from bovine heart by ammonium sulfate precipitation and diethyl aminoethyl cellulose chromatography. With these enzymes imidazole had less stimulatory activity and some inhibitory effect on the hydrolysis of 10?4m cyclic AMP and cyclic GMP but was without significant effect on the hydrolysis of 10?6m cyclic AMP or cyclic GMP. The stimulatory activity of imidazole on the hydrolysis of high levels of cyclic nucleotide was dependent on the presence of phosphodiesterase activator. The stimulatory effect of the activator and imidazole plus activator on the hydrolysis of 10?4m cyclic GMP by the rather cyclic GMP-specific enzyme could be eliminated by the addition of ethylene glycol-bis-(β-aminoethyl ether)N,N′-tetraacetate (EGTA) and restored by Ca2+. Imidazole was without effect on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase from bovine heart. The lack of effect of imidazole on the hydrolysis of physiological levels of cyclic AMP or cyclic GMP suggests that the activity of imidazole to antagonize the effects of various hormones is probably not due to a direct action of imidazole on the hydrolysis of cyclic AMP or cyclic GMP. 相似文献