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)

The binding of [3H]cGMP (guanosine 3',5'-monophosphate) to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity has been measured under the cGMP-binding assay conditions, in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 microM concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP (adenosine 3',5'-monophosphate) and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 microM cGMP range and only at a fiftyfold higher cAMP concentration. Our data with comparative series of analogs (e.g. 5'-amino-5'-deoxyguanosine 3',5'-monophosphate and 3'-amino-3'-deoxyguanosine 3',5'-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. Those experiments suggest that the cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binding site.
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PMID:Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue. 258 80

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).
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PMID:Acid sphingomyelinase from human urine: purification and characterization. 282 97

Mannosylphosphodolichol phosphodiesterase, which catalyzes the release of mannose from mannosylphosphodolichol, was solubilized from chicken liver microsomes by treatment with the non-ionic detergent, Emulgen 909. The enzyme was partially purified using ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration on Sepharose 6B. The enzyme showed absolute requirement for sulfhydryl reducing agents. The enzyme activity was stimulated by the addition of CaCl2 and Emulgen 909 and exhibited a pH optimum around 5.3. The Km value for mannosylphosphodolichol was found to be 0.43 microM. The activity was competitively inhibited by dolichyl phosphate and dolichol and the Ki value for dolichyl phosphate was estimated to be 12.5 microM. The purified preparation had no activity toward N-acetylglucosaminyldiphosphodolichol, glucosylphosphodolichol, mannose 1-phosphate, or artificial substrates for mannosidases, glucosidases, acid phosphatase, and acid phosphodiesterase. A heat-stable factor which stabilizes the mannosylphosphodolichol phosphodiesterase was separated from the enzyme by DEAE-cellulose chromatography. It was precipitated by trichloroacetic acid and not extracted into lipid solvents. The separation resulted in the complete loss of the enzyme activity and the restoration of the activity was not observed when the factor was added back to the enzyme solution.
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PMID:Characterization and partial purification of a novel mannosylphosphodolichol phosphodiesterase from chicken liver microsomes. 282 59

Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.
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PMID:Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets. 283 85

An apparent increase of calmodulin (CaM) activity was previously observed in the heart and kidney but not in the liver of spontaneously-hypertensive rats (SHR) and mice compared with their corresponding normotensive controls. As this change was due to an elevated recovery of CaM in the organs of the hypertensive animals, the present study was designed to evaluate its activity in hypertension. A CaM activator, detected in heart and kidney supernatants from hypertensive animals, was found to be responsible for this enhanced recovery. Similar results were obtained with passaged, cultured aortic smooth muscle cells from SHR, indicating that the anomaly was not a mere consequence of elevated blood pressure but rather a genetic expression of cells of hypertensive origin. The activator was heat stable, nondialyzable, and recovered in the fraction precipitated with 30-50% ammonium sulfate. Preliminary extraction studies suggest that the activator is contained in a glycolipid fraction. The stimulation of phosphodiesterase by this activator was calcium and CaM dependent. The activator appears to affect the affinity of the phosphodiesterase for CaM rather than the maximal stimulation. The activator was also present at a low concentration in the heart and kidney of normotensive animals. These findings indicate that at least some of the calcium abnormalities implicated in the pathogenesis of hypertension could be the result of interactions between CaM, calcium, and this activator.
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PMID:Abnormality of calmodulin activity in hypertension. Evidence of the presence of an activator. 283 48

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.
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PMID:[Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver]. 284 24

Male ICR mice, young (25-days old), mature (3-months old), and old (22 months), were injected with morphine sulfate (10 mg/kg, s.c.) or were implanted with morphine pellets (75 mg). Controls received saline injections or placebo pellets. One hour after injections and 72 h after pellet implantations, the mice were decapitated and striatal regions were removed for the following analyses: calmodulin (CaM) levels via radioimmunoassay and activities of cyclic nucleotide phosphodiesterases, adenylate and guanylate cyclases, and Ca2+, Mg2+-ATPase. Acute morphine treatment produced the following: (1) increases in calmodulin levels in the young and old mice while having no effect on mature levels; (2) increases in activities of guanylate cyclase of mature mice while decreasing those of the old mice; (3) no effects on activity of adenylate cyclase; (4) decreased activity of cyclic AMP-phosphodiesterase in young mice only; (5) decreased activity of Ca2+, Mg2+-ATPase in the old mice only. The only changes found in striata from morphine-tolerant mice when compared with age-matched controls were elevations in cyclic GMP-phosphodiesterase activities in all three age groups. Differences in control values of the three age groups were as follows: CaM levels, mature greater than old greater than young; Ca2+, Mg2+-ATPase activity, old greater than mature-young. The results indicate age-induced changes in cellular regulation and biochemical responses to morphine.
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PMID:Effects of aging and morphine administration on calmodulin and calmodulin-regulated enzymes in striata of mice. 285 71

During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.
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PMID:Beta gamma-subunit of bovine transducin composed of two components with distinctive gamma-subunits. 292 42

Immunochemical, chromatographic, and sodium dodecyl sulfate gel electrophoresis studies suggest that immunologically related but distinct cyclic GMP phosphodiesterases are present in rod and cone outer segments of the retina. Immunocytochemical studies demonstrated that one monoclonal antibody (ROS-1) recognized a determinant present in both rod and cone outer segments, while another monoclonal antibody (ROS-2) only recognized rod outer segments. At least two peaks of phosphodiesterase activity could be separated by high-performance anion-exchange chromatography of retinal extracts. Both peaks were recognized by ROS-1. None of the first peak and only 80% of the second broad peak of activity were recognized by ROS-2. High-performance liquid chromatography profiles from human fovea and several other types of cone-enriched retina showed that most of the activity was contained in the first peak, suggesting that this activity was derived from cone outer segments. Conversely, the phosphodiesterase in rod-enriched preparations migrated predominately in the second peak. Sodium dodecyl sulfate-gel electrophoresis indicated that this first peak contained a single large immunoreactive polypeptide (alpha') that migrated with the same mobility as a phosphorylase b standard and was distinct from the more rapidly migrating large immunoreactive polypeptides (alpha and beta) present in a broad second peak. The second peak could be further separated into a first part that contained a doublet of two immunoreactive polypeptides (alpha and beta) that migrated faster than phosphorylase b and a later part that contained only the most rapidly migrating polypeptide (beta). All of the peaks could be activated by histone or transducin:GTP, implying that all contained a small 11-kDa inhibitory subunit (gamma) of the enzyme. Since the larger (alpha') and smaller (beta) immunoreactive polypeptides could be completely separated from the alpha polypeptide and from each other, yet still retain the ability to be activated by histone or transducin, the data suggest that only a single species of polypeptide-inhibitor complex (e.g. alpha' gamma, alpha gamma, or beta gamma) was required for histone or transducin:GTP activation.
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PMID:cGMP phosphodiesterase in rod and cone outer segments of the retina. 298 Dec 19

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.
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PMID:Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas. 298 11


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