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Query: CAS:7665-99-8 (cGMP)
 21,074 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thyroid tissue was found to contain at least least two separable cyclic 3',5'-nucleotide phosphodiesterase (cAMP-PDE and cGMP-PDE) activities, as determined by DEAE-cellulose or Sepharose 6B column chromatography. These activities were cAMP- and cGMP-hydrolyzing enzymes. Quantitative differences of cAMP or cGMP hydrolytic activity were observed in tissues from patients with thyroid disorders. Theophylline, modulator protein, and Mg2+ produced similar effects on cAMP or cGMP hydrolytic activity in tissues from patients with a without various thyroid disorders. The mode of the inhibitory effect of cyclic nucleotides on PDE activities in thyroid tissues was competitive, in contrast to the mode seen in other organs. Both cAMP and cGMP hydrolytic activities were elevated in the tissues from patients with hyperthyroidism and thyroid carcinoma compared with the activity in controls. cGMP hydrolysis in hyperthyroidism was 4.4-fold higher than that seen in the controls. The ratio cGMP to cAMP hydrolysis was highest in cases of hyperthyroidism and lowest in cases of thyroid carcinoma, when PDE activities were determined with a substrate concentration of 0.4 microM. Kinetic analysis revealed that higher PDE activity in tissues from patients with hyperthyroidism and thyroid carcinoma was due to an increase in maximal velocity. The apparent Km values for hydrolysis of cyclic nucleotides were similar in normal and pathological thyroid tissues.
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PMID:Cyclic 3',5'-nucleotide phosphodiesterase activities in the thyroid glands of patients with various disorders. 624

The effects of various compounds on homogeneous cyclic CMP phosphodiesterase (cyclic CMP-PDE) from pig liver were compared with the effects on cyclic AMP phosphodiesterase (cyclic AMP-PDE) and cyclic GMP phosphodiesterase (cyclic GMP-PDE). Of the conventional inhibitors for AMP-PDE and cyclic GMP-PDE, only Sch 15280 was found to inhibit cyclic CMP-PDE. Nucleoside monophosphates, orthophosphate, and 2':3'-cyclic nucleotides were rather specific and were more effective in inhibiting cyclic CMP-PDE, compared to their effects on cyclic AMP-PDE and cyclic GMP-PDE. On the other hand, nucleoside di-and triphosphates and pyrophosphate (PPi) were less effective in inhibiting cyclic CMP-PDE and were without marked effect on cyclic AMP-PDE and cyclic GMP-PDE. Orthophosphate (Pi) was more potent than CMP, CDP and CTP in inhibiting cyclic CMP-PDE, with a rank order of inhibitory potency of Pi greater than CMP greater than CDP greater than CTP. Of the 3' :5'-cyclic nucleotides examined, cyclic UMP was more specific in inhibiting cyclic CMP-PDE compared to its effect on cyclic AMP-PDE and cyclic GMP-PDE. In all experiments similar results were obtained when either cyclic CMP or cyclic AMP was used as a substrate for this multifunctional cyclic CMP-PDE, supporting the contention that a single catalytic site on the enzyme is responsible for the hydrolysis of both cyclic CMP and cyclic AMP. The present studies further support our original suggestion that cyclic CMP-PDE is a unique enzyme that is distinguishable from the conventional enzymes for purine cyclic nucleotides.
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PMID:Differential effects of various phosphodiesterase inhibitors, pyrimidine and purine compounds, and inorganic phosphates on cyclic CMP, cyclic AMP and cyclic GMP phosphodiesterases. 627 36

Chemoattractants added to cells of the cellular slime mold dictyostelium discoideum induce a transient elevation of cyclic GMP levels, with a maximum at 10 s and a recovery of basal levels at approximately 25 s after stimulation. We analyzed the kinetics of an intracellular cGMP binding protein in vitro and in vivo. The cyclic GMP binding protein in vitro at 0 degrees C can be described by its kinetic constants K(1)=2.5 x 10(6) M(- 1)s(-1), k(-1)=3.5 x 10(-3)s(-1), K(d)=1.4 x 10(-9) M, and 3,000 binding sites/cell. In computer simulation experiments the occupancy of the cGMP binding protein was calculated under nonequilibrium conditions by making use of the kinetic constants of the binding protein and of the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions by making use of the kinetic constants of the binding protein and the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions the affinity of the binding protein for cGMP is determined by the rate constant of association (k(1)) and not by the dissociation constant (k(d)). Experiments in vivo were performed by stimulation of aggregative cells with the chemoattractant cAMP, which results in a transient cGMP accumulation. At different times after stimulation with various cAMP concentrations, the cells were homogenized and immediately thereafter the number of binding proteins which were not occupied with native cGMP were determined. The results of these experiments in vivo are in good agreement with the results of the computer experiments. This may indicate that: (a) The cGMP binding protein in vivo at 22 degrees C can be described by its kinetic constants: K(1)=4x10(6)M(-1)s(-1) and K(-1)=6x10(-3)s(-1). (b) Binding the cGMP to its binding protein is transient with a maximum at about 20-30 s after chemotactic stimulation, followed by a decay to basal levels, with a half-life of approximately 2 min. (c) The cGMP to its binding proteins get half maximally occupied at a cGMP accumulation of delta[cGMP](10)=2x10(-8) M, which corresponds to an extracellular stimulation of aggregative cells by 10(-10) M cAMP. (d) Since the mean basal cGMP concentration is approximately 2x10(-7) M, the small increase of cGMP cannot be detected accurately. Therefore the absence of a measurable cGMP accumulation does not argue against a cGMP function. (e) There may exist two compartments of cGMP: one contains almost all the cGMP of unstimulated cells, and the other contains cGMP binding proteins and the cGMP which accumulates after chemotactic stimulation. (f) From the kinetics of binding, the cellular responses to the chemoattractant can be divided into two classes: responses which can be mediated by this binding protein (such as light scattering, proton extrusion, PDE induction, and chemotaxis) and responses which cannot be (solely) mediated by this binding protein such as rlay, refractoriness, phospholipids methylation, and protein methylation.
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PMID:Nonequilibrium kinetics of a cyclic GMP-binding protein in Dictyostelium discoideum. 628 89

It has been clearly shown that the action of several hormones is differentially mediated intracellularly by nucleotides containing either adenosine or guanosine base units. To study the protein-nucleotide interactions involved in several complex biological systems our laboratory has synthesized several 8-azido-adenosine (8-N3 A) and 8-azidoguanosine (8-N3 G) derivatives of naturally occurring nucleotides. Modification of the nucleotides in the 8-position of the purine ring was done because: a) 8-substituted derivatives of cAMP and cGMP activated their respective protein kinases at physiological concentrations and were much less susceptible to hydrolysis by specific phosphodiesterases (PDE's) and b) substitution at the 8-position was much less likely to disturb the preferential and selective binding of adenosine versus guanosine nucleotides by enzymes that are specifically regulated by such interactions. This would allow studies of guanosine nucleotide specific binding in the presence of both adenosine nucleotides and adenosine nucleotide binding proteins, and vice-versa. In general, such has been the case and [32P] 8-N3 cAMP and [32P] 8-N3 cGMP have been used effectively to study their respectively activated protein kinases in several systems. Also, [32P] 8-N3 ATP has been used to study several ATPases and kinases while [gamma 32P] 8-N3 GTP has been shown effective for studies on tubulin and the G-regulatory protein (G/N) of adenylyl cyclase (A.C.). Several observations suggest that there must be important physical and energetic tie-ins between external hormone binding and the loading and unloading of specific internal nucleotide binding sites. These binding sites may be activator signals for protein kinases (e.g., cAMP protein kinase regulatory subunit), or cyclases (e.g., G/N proteins of A.C.) or catalytic sites involved in the production or hydrolysis of cyclic nucleotides. The thrust of this article is to detail the use of 8-azidopurine photoaffinity analogs of ATP, GTP, cAMP and cGMP as they may be used to study hormone-mediated events which may or may not involve cyclic nucleotides as a second messenger.
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PMID:Use of nucleotide photoaffinity probes to study hormone action. 629 15

Cyclic AMP (cAMP) and cyclic GMP (cGMP) have been implicated as intracellular signals in the transition from a resting to a growing state. This suggestion comes from observations showing that the addition of growth promoting factors to quiescent cell cultures causes a rapid and transient decrease in cAMP and an increase in cGMP contents [9, 11] and that exogenous cAMP or cGMP congeners reduce or stimulate cell growth respectively [6, 13]. In view of this antagonistic effect elicited by the two nucleotides, it has been suggested that a fall in cAMP/cGMP ratio might be the triggering event for the initiation of cell proliferation [6]. Since polyamines correlate positively with active cell division [7], a possible involvement of these biogenic polycations in the regulation of cellular cyclic nucleotide contents is worthwhile investigating. Our previous reports have shown indeed that in different cultured cell types, spermine, spermidine and putrescine, at relatively low doses, are able to reduce cAMP content [3] by increasing cAMP-dependent phosphodiesterase activity (cAMP-PDE) [4] and to counteract the action of different cAMP-mediated effectors [3]. Besides endogenous polyamines seem to be involved in the cAMP-mediated induction of cAMP-PDE, as observed in heart cell cultures [4]. This report shows that the addition of each individual polyamine to confluent and serum-restricted heart cell cultures, while lowering cAMP content, induces an early and rapid increase of cGMP content by reducing the rate of its degradation.
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PMID:Increased cyclic GMP content in confluent and serum-restricted heart cell cultures exposed to polyamines. 630 28

About 2000 PDE molecules are gradually activated by one bleached rhodopsin molecule, R* on a toad disk membrane to yield a final enzyme velocity of about 2.5 x 10(6) cGMP hydrolyzed sec-1 bleached rhodopsin-1. This amplified effect of a single photon requires GTP, whose function we originally proposed (Yee and Liebman, 1978; Liebman and Pugh, 1979) to serve as a "memory" label attached to each PDE as it is contacted via lateral diffusion by R*. Thus, the binding of GTP was explicitly seen as an identically-amplified casual link in the amplified PDE activation. We have subjected our GTP-PDE coupling hypothesis to both stoichiometric and kinetic tests using radioactive GTP labelling techniques. We find agreement in principle with our original hypothesis with modifications to allow for (1) GTP binding to a separate G-protein (gamma) which activates PDE; (2) evidence that there are fewer PDE's activated than GTP's bound in response to a light flash; (3) evidence of reversible binding of gamma to PDE with incomplete activation of the latter; (4) multisecond delay of GTP binding compatible with lateral diffusionally-mediated activation of thousands of gamma's by single R*'s; (5) gain regulation by ATP that reduces both PDE activation and GTP binding.
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PMID:Gain, speed and sensitivity of GTP binding vs PDE activation in visual excitation. 630 23

Extracts of a mutant S49 lymphoma cell line, termed K30a, hydrolyze cAMP and cGMP at rates much faster than do wild type S49 extracts. This elevated phosphodiesterase activity, called K-PDE, elutes as a single peak of activity on DEAE-cellulose columns (Brothers, V. M., Walker, N., and Bourne, H. R. (1982) J. Biol. Chem. 257, 9349-9355). Direct photoaffinity labeling of K30a extracts with [32P]cGMP results in radiolabeling of a unique polypeptide, not observed in wild type extracts, which migrates in sodium dodecyl sulfate polyacrylamide gels with an Mr = 106,000. The 106-kDa band was identified as the catalytic K-PDE polypeptide based on the following observations: competitive inhibitors and substrates of K-PDE inhibit photolabeling of the 106-kDa band, indicating that [32P] cGMP photolabels the enzyme at its catalytic site; on DEAE-cellulose chromatography the polypeptide that is susceptible to photolabeling co-elutes with K-PDE activity; the 106-kDa band is detectable in extracts of WT X K30a hybrids (where WT denotes wild type) in amounts proportional to the K-PDE activity in the hybrids, but is undetectable in wild type. The hybrid phenotype strongly suggests that the K30a phenotype is not due to mutations that affect either a diffusible regulator of transcription or an enzyme that modifies K-PDE. Although wild type cells contain a minor cGMP phosphodiesterase activity distinct from the major cAMP phosphodiesterase, the wild type cGMP phosphodiesterase is not susceptible to radiolabeling with [32P]cGMP; this rules out the possibility that the K30a phenotype is caused by overexpression of a wild type phosphodiesterase. We conclude that the K30a mutation produced expression of a new species of phosphodiesterase molecule that is not detectably expressed in the parental S49 wild type cell line.
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PMID:Identification by direct photoaffinity labeling of an altered phosphodiesterase in a mutant S49 lymphoma cell. 630 83

Kidney function is regulated by several hormones which act through adenylate cyclase-cyclic AMP system. The present study was undertaken to investigate cyclic AMP- and cyclic GMP-phosphodiesterase (cAMP-PDE and cGMP-PDE respectively) activities in the rat kidney, and also the effect of several hormones affecting the kidney function on these enzyme activities in vitro. Rat kidneys were separated into cortex and medulla. These were homogenized in 50 mM Tris-HCl buffer, pH 7.5, containing 0.32 M sucrose and fractionated by centrifugation. PDE activity was measured in all fractions, using the two-step assay system. A low substrate concentration (0.5 microM) was used, unless otherwise stated. Substantial activity was present in all of the fractions and most of the activity existed in the soluble fraction (105000 X g supernatant). Cyclic GMP-PDE activity was dominant in both cortex and medulla. The rat kidney contained two forms of cAMP-PDE, one of which had a Km of 2.0 X 10(-4) M and another which had a low Km of 2.5 X 10(-5) M, and one form of cGMP-PDE with a Km of 2.5 X 10(-5) M. These cAMP-PDE and cGMP-PDE were purified by Sepharose-6B column chromatography. Cyclic AMP-PDE activity was found in a broad area associated with two peaks and cGMP-PDE activity had one peak corresponding to the same peak as the high molecular weight cAMP-PDE. Calmodulin was eluted after the peak of cGMP-PDE activity. Both cAMP-PDE and cGMP-PDE activities were inhibited by calcium ion at a concentration of more than 5.0 X 10(-4) M. Cyclic GMP-PDE activity was not activated by calmodulin in the presence of enough calcium ion. The effect of 1 alpha, 25(OH)2 Vit D3, parathyroid hormone (PTH), antidiuretic hormone (ADH), calcitonin (CT), angiotensin II, and trichlormethiazide on the partially purified cAMP-PDE and cGMP-PDE activities were examined. 1 alpha, 25(OH)2 Vit D3 activated cAMP-PDE activity and did not affect cGMP-PDE activity. The concentrations of 1 alpha, 25(OH)2 Vit D3 producing 50% activation of cAMP-PDE activity were 5.0 X 10(-11) M (cortex) and 6.7 X 10(-10) M (medulla). CT and ADH inhibited both cAMP-PDE activities. The concentrations of CT producing 50% inhibition of cAMP-PDE activity were 4.0 X 10(-5) M (cortex) and 3.3 X 10(-7) M (medulla), and those of cGMP-PDE activity were 1.0 X 10(-5) M (cortex) and 1.0 X 10(-4) M (medulla). Concerning ADH, the concentrations required for 50% inhibition of cAMP-PDE activity were 5.3 X 10(-6) M (cortex) and about 1.0 X 10(-3) M (medulla), and those of cGMP-PDE activity were 5.3 X 10(-3) M (cortex) and 5.3 X 10(-8) M (medulla).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Effect of several hormones on cyclic 3',5'-nucleotide phosphodiesterase in rat kidneys]. 631 6

The present data are consistent with the following mechanism of activation of the cGMP-stimulated PDE: cGMP is first bound to an allosteric site of the enzyme; this is followed by a conformational change in protein structure, a shift of kinetic behavior, and sequential activation of cAMP PDE hydrolysis (18). Evidence for the existence of distinct activating and catalytic sites is obtained from the use of several cyclic nucleotide derivatives to elucidate the essential molecular interactions at both of these sites (4). Since the liver PDE under study exhibits positive homotropic cooperativity by cAMP, the stimulatory effect of low concentrations of MIX is consistent with its binding at the substrate-active sites. As shown previously, this enzymatic mechanism is reproduced by an analog of the substrate, c6clPMP, but not by cGMP. Since the order of potency of a series of competitive inhibitors of two PDE (i.e., the cyclic GMP-sensitive enzyme and a calmodulin-sensitive enzyme), is not parallel, it is suggested that the active sites of these enzymes are distinct. The interaction of MIX at the active site of two PDE studied here could reflect a general binding mechanism of the xanthine due to similar chemical forces. Since we propose that the allosteric-activation site is specific for cGMP, the xanthine does not bind to that site. This is also suggested from substrate-velocity relationships measured in the presence of MIX and cGMP (Table 3). Complete characterizations of the cGMP-activating site and the xanthine-sensitive catalytic site are required in order to elucidate the exact chemical interactions at both sites on the cGMP-stimulated PDE.
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PMID:Cyclic nucleotide derivatives as probes of phosphodiesterase catalytic and regulatory sites. 632 17

The cyclic nucleotide PDE activity in crude tissue extracts is due to the composite activity of several distinct isozymes, each having different kinetic and functional characteristics. Most of these isozymes will hydrolyze the same substrates, that is, cyclic AMP and cyclic GMP. Therefore, it is difficult to conclusively identify and quantitate any given isozyme in crude systems. Since the cyclic nucleotide PDE are present in low concentrations in cells, it is also difficult to isolate these proteins without having specific solid phase affinity probes. This chapter described the use of monoclonal antibodies as probes to specifically identify, measure, characterize, and isolate the PDE isozymes in impure preparations. Monoclonal antibodies have been produced to the cyclic-GMP-stimulated PDE, ROS PDE, and a calcium-calmodulin-dependent PDE of bovine tissues. A polyclonal antiserum has also been produced to the cyclic-GMP-stimulated PDE. Sensitive immunoprecipitation assays for the measurement of PDE activity and cyclic GMP binding have been developed that have allowed the detection of femtomole amounts of each specific PDE isozyme. None of the antibodies appear to recognize antigenic determinants on other isozymes, suggesting that the PDE are antigenically distinct and therefore different proteins. Most of the monoclonal antibodies do not appear to affect kinetic parameters of the enzymes and have been used to specifically identify, measure, and characterize each isozyme in crude systems. A monoclonal antibody to the calcium-calmodulin-dependent PDE requires calcium and calmodulin for high-affinity binding to the enzyme. This apparent conformational requirement has been used to purify the isozyme from both bovine brain and heart. These two tissues contain enzymes with differing subunit MW on SDS-polyacrylamide gel electrophoresis, although no other biochemical differences were observed.
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PMID:Immunologic approaches to the study of cyclic nucleotide phosphodiesterases. 632 39


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