Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.
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PMID:Agonist and antagonist properties of calmodulin fragments. 632 72

DEAE-cellulose chromatography of mycelial extracts of Mucor rouxii grown to mid-exponential phase resolves two types of low-Km cyclic AMP phosphodiesterase (EC 3.1.4.17; PDE): PDE I, highly activatable (4-6-fold) by phosphorylation or proteolysis, and PDE II, unresponsive to activation. The enzymic profile of PDE activity obtained from germlings shows only PDE I activity, whereas PDE activity from mycelia grown to stationary phase is eluted from the DEAE-cellulose column at the position of PDE II, and like PDE II is unresponsive to activation. Endogenous proteolysis or controlled trypsin treatment transforms PDE I into PDE II. The insensitive forms of PDE exhibit a slightly smaller sedimentation coefficient than the activatable forms, as judged by sucrose-gradient centrifugation. The basal activity of the highly activatable form of PDE is elevated almost to the value in the presence of trypsin on storage at 4 degrees C in the absence of proteinase inhibitors. Benzamidine, leupeptin, antipain or EGTA prevents the activation produced by storage. PDE I remains strongly activatable by phosphorylation and proteolysis after resolution by polyacrylamide-gel electrophoresis.
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PMID:Regulation of cyclic AMP phosphodiesterase from Mucor rouxii by phosphorylation and proteolysis. Interrelationship of the activatable and insensitive forms of the enzyme. 632 57

A calmodulin-Ca2+-stimulated cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which hydrolyzed both cGMP and cAMP has been purified about 2000-fold from ovaries of the amphibian Xenopus laevis. Gel filtration through Sephadex G-200 indicated a molecular weight of 140,000. A single, major protein band of molecular weight 66,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the stimulation by calmodulin-Ca2+, the enzyme was activated 5- to 10-fold by proteolysis and by certain phospholipids. Trypsin activation of the enzyme caused a reduction in the native molecular weight to 90,000 and a loss of the capacity to be stimulated by calmodulin-Ca2+ or by phospholipids. The phosphodiesterase was stimulated by low concentrations (0.1 microgram/ml) of lysophosphatidylcholine and lysophosphatidylethanolamine. This response did not require calcium ions. Phosphatidylinositol, fatty acids, progesterone, and phospholipase C had little or no effect on activity. Simultaneous addition of 1 mM 2-chloro-10-(3-aminopropyl)phenothiazine and lysophosphatidylcholine to the enzyme did not diminish the stimulatory effect of the phospholipid. The activation of the enzyme by all three agents resulted in an increase in the maximum velocity of the reaction without significant modification of the apparent Km values for cGMP (5 microM) or cAMP (30 microM). It was suggested that trypsin removed an inhibitory domain from the enzyme and that calmodulin and phospholipids interact with this same domain, eliminating its capacity to inhibit the active center of the enzyme.
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PMID:Properties of a cyclic nucleotide phosphodiesterase of amphibian oocytes that is activated by calmodulin and calcium, by tryptic proteolysis, and by phospholipids. 632 99

High affinity (KD approximately 1 X 10(-9) M) monoclonal antibodies (ROS-1 and ROS-2) were prepared to bovine photoreceptor outer segment cGMP phosphodiesterase. ROS-1 immunoadsorbed greater than 95% of the cGMP phosphodiesterase activity from a detergent-solubilized bovine retina extract while ROS-2 immunoadsorbed only a subfraction of the same activity. Sodium dodecyl sulfate gel analysis of these immunoadsorbates demonstrated that ROS-1 and ROS-2 specifically adsorbed only peptides that comigrated with purified rod outer segment phosphodiesterase. Limited trypsin digestion of purified rod outer segment phosphodiesterase greatly reduced its affinity for ROS-1 but not ROS-2. When a crude heat-stable inhibitor fraction was added back to the activated enzyme, the affinity for ROS-1 was restored, suggesting that the inhibitor was necessary for ROS-1 binding. ROS-1 but not ROS-2 was found to inhibit cGMP phosphodiesterase which had been activated either by dilution or guanyl nucleotide. The inhibitory property of ROS-1 may provide a useful probe for directly studying the effects of this phosphodiesterase on the phototransduction response in the retina. Sodium dodecyl sulfate gel analysis demonstrated that the ROS-1 immunoadsorbates from mammals, fish, and amphibia contained peptides of similar mobility. Immunocytochemistry performed with ROS-1 and fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG localized the antigenic determinant to both rod and cone outer segments suggesting the presence of an antigenically similar phosphodiesterase in both types of photoreceptors.
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PMID:Immunologic characterization of the photoreceptor outer segment cyclic GMP phosphodiesterase. 633 Jan 15

Evidence is presented for the occurrence of two different non-specific nucleotide-sugar hydrolases in rat liver and other rat tissues. These two enzymes (I and II) were separated by chromatography on a 5'-AMP-aminohexyl-Sepharose column. Enzyme I is most probably identical with phosphodiesterase I (EC 3.1.4.1). Enzyme II appeared to be identical with an enzyme described in literature as 'CMP-sialic acid hydrolase' [Kean & Bighouse (1974) J. Biol. Chem. 249, 7813-7823], since almost all activity with CMP-N-acetylneuraminate as substrate was recovered in this enzyme fraction. CMP-N-acetylneuraminate was a poor substrate for Enzyme I, whereas deoxythymidine-5'-p-nitrophenyl phosphate and all nucleoside-diphosphosugars tested were good substrates for both Enzyme I and II. Therefore it is suggested that CMP-N-acetylneuraminate is used as an additional substrate to discriminate between the activities of Enzyme I and II in homogenates or membrane preparations. The various substrates appeared to be competitive inhibitors of each other, suggesting that, in each enzyme preparation, only one enzyme is responsible for the hydrolysis of the various substrates. The dissimilar properties of the two enzymes are substantiated by studying the subunit molecular masses (Enzyme I, 125 kDa; Enzyme II, 50-55 kDa), the sensitivity towards Triton X-100, Sarkosyl and sodium dodecyl sulphate and towards trypsin treatment. It is discussed whether the alpha-N-acetylglucosamine phosphodiesterase described by Varki & Kornfeld [(1981) J. Biol. Chem. 256, 9937-9943] is identical with one of the nucleotide-sugar hydrolases described here.
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PMID:On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in rat liver. 633 14

Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.
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PMID:Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation. 658 21

Human peripheral blood mononuclear leucocytes (PBML) stimulated with concanavalin A (Con A) or phytohaemagglutinin (PHA) produced a soluble factor which inhibits lung fibroblast DNA synthesis and growth. Lymphocyte enriched preparations produced significant growth inhibitory activity in the presence of PHA whereas media from adherent mononuclear cells incubated in the presence of the mitogen did not contain similar activity. This fibroblast growth inhibitory factor (FGIF) was non-dialysable, heat stable and resistant to pH 5. FGIF was also resistant to treatment with chymotrypsin and phosphodiesterase but partially sensitive to treatment with trypsin. Interestingly, there was significant suppression of FGIF production by PBML cultured with PHA in the presence of low concentrations of chrysotile asbestos (5-25 micrograms/ml). In this regard, asbestos (25 micrograms/ml) was not cytotoxic for lymphocytes but had a damaging effect on monocytes as evidenced by the release of lactate dehydrogenase (LDH) a cytoplasmic enzyme, in their culture media. These findings indicate that stimulated lymphocytes have the ability to inhibit fibroblast proliferation by releasing FGIF and that asbestos interfere with this process. Thus, while FGIF may regulate the extent of connective tissue proliferation during normal repair process, suppression of its production by asbestos may contribute to excessive fibroblast accumulation and fibrosis.
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PMID:In vitro suppression of fibroblast growth inhibitory lymphokine production by asbestos. 687 28

To determine the presence of cone or rod cyclic GMP phosphodiesterase (EC 3.1.4.17) in the mammalian pineal, extracts from adult rat and bovine pineals were injected onto a Mono Q anion-exchange HPLC column and eluted with an NaCl linear gradient. Fractions were immunoadsorbed with monoclonal antibodies specific to rod and cone phosphodiesterases (ROS-1) and to calmodulin-phosphodiesterase complexes (ACC). Profiles were assayed with 10 mumol/L [3H]cyclic GMP in the presence of calcium-calmodulin, histone, or trypsin. Rat and bovine pineals displayed a single peak of activity recognized by ROS-1, which corresponded to the activity of the cone but not to the rod in bovine retina. ROS-1 immunoadsorbed approximately 80% of the activity in the 60-day-old rat pineal but only 26% of the activity in bovine pineal. ACC immunoadsorbed the remaining activity in both species. Western blot analysis of rat pineal extracts revealed three polypeptides of approximately 87, 15, and 10 kDa when probed with a rod/cone phosphodiesterase-specific antiserum. The specific activity of the cone-like phosphodiesterase in 10-day-old rat pineals was twice that of this isozyme in the bovine retina and 150 times that in the bovine pineal. The specific activity of phosphodiesterase in rat pineals decreased with age. We conclude that an enzyme with biochemical and antigenic characteristics similar to cone, but distinct from rod phosphodiesterase, is present in bovine and rat pineals.
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PMID:The mammalian pineal expresses the cone but not the rod cyclic GMP phosphodiesterase. 764 86

The interaction between the GTP-bound form of the transducin alpha-subunit (G alpha t) and the gamma-subunit (P gamma) of cGMP phosphodiesterase (PDE) is a key event in effector activation during photon signal transduction. The carboxyl-terminal half of P gamma is involved in interaction with G alpha t as well as in inhibition of PDE activity. Here we have utilized a combination of synthetic peptide and mutagenesis approaches to localize specific regions of the carboxyl-terminal region of P gamma interacting with G alpha t and P alpha beta and have determined residues involved in inhibition of PDE activity. We found that synthetic peptide corresponding to residues 68-87 of P gamma completely inhibit trypsin-activated PDE. The peptide P gamma-63-87 bound to G alpha t GTP gamma S with a Kd of 2.5 microM, whereas the binding of P gamma-68-87 to G alpha tGTP gamma S was approximately 15-fold less (Kd = 40 microM) suggesting that carboxyl-terminal P gamma region 68-87 contains a site for interaction with P alpha beta and also a part of the alpha t binding site. To map G alpha t and P alpha beta sites more precisely within the carboxyl-terminal region, a set of carboxyl-terminal mutants was generated by site-directed mutagenesis. Deletion of residues 63-69 and 70-76 diminished the binding of mutants to alpha t while binding to carboxyl-terminally truncated mutants lacking up to 11 amino acid residues was unchanged. In contrast, carboxyl-terminal truncations of P gamma from delta 1 to delta 11 resulted in a gradual decrease of its inhibitory activity. Thus, the extreme carboxyl-terminal hydrophobic sequence -Ile86-Ile87 together with 9 adjacent residues provides inhibitory interaction of P gamma with P alpha beta. The carboxyl-terminal G alpha tGTP gamma S binding site of P gamma is different from but adjacent to its PDE inhibitory site. During the visual transduction process, G alpha tGTP likely binds to this region of P gamma inducing a displacement of the extreme carboxyl terminus from the inhibitory site on P alpha beta, leading to PDE activation.
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PMID:The carboxyl terminus of the gamma-subunit of rod cGMP phosphodiesterase contains distinct sites of interaction with the enzyme catalytic subunits and the alpha-subunit of transducin. 776 19

Knowledge of the kinetics of the rod cyclic GMP phosphodiesterase is essential for understanding the kinetics and gain of the light response. Therefore, the interactions between Mg2+, cyclic GMP, and purified, trypsin-activated bovine rod cyclic GMP phosphodiesterase (EC 3.1.4.17) were examined. The effects of Mg2+ and of cyclic GMP on the rod phosphodiesterase activity were mutually concentration-dependent. Formation of a free Mg-cyclic GMP complex is unlikely due to its high dissociation constant (Kd = 19 mM). Plots of 1/velocity versus 1/[cyclic GMP] as a function of [Mg2+] and 1/velocity versus 1/[Mg2+] as a function of [cyclic GMP] intersected to the left of the 1/velocity axis. This is consistent with the formation of a ternary complex between the phosphodiesterase, Mg2+, and cyclic GMP. A competitive inhibitor of the phosphodiesterase relative to cyclic GMP, 3-isobutyl-1-methylxanthine, non-competitively inhibited the enzyme relative to Mg2+, Pb2+, a competitive inhibitor of the phosphodiesterase relative to Mg2+ [D. Srivastava, R.L. Hurwitz and D. A. Fox (1995) Toxicol. Appl. Pharmacol, in the press] non-competitively inhibited the enzyme relative to cyclic GMP. Collectively these results are suggestive of a rapid equilibrium random binding order of Mg2+ and cyclic GMP to the rod phosphodiesterase.
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PMID:Effects of magnesium on cyclic GMP hydrolysis by the bovine retinal rod cyclic GMP phosphodiesterase. 777 55


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