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)

Trypsin increases intracellular levels of cylic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induced the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.
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PMID:Trypsin-induced increase in intracellular cyclic AMP of lymphocytes. 18 38

Gangliosides were recently shown to bind to calmodulin (Higashi, H., Omori, A., and Yamagata, T. (1992) J. Biol. Chem. 267, 9831-9838). This prompted us to investigate the effects of gangliosides on the calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. Several species of gangliosides competitively inhibited calmodulin-stimulated phosphodiesterase activity, with GD1b, GT1b, and GD1a being noted to do so particularly (group 1). GM1, GQ1b, and GM2 (group 2) were less inhibitory, and GM3, GM3(NeuGc), GalCer, sulfatide, GgOse4Cer, and oligosaccharide portions of inhibitory gangliosides showed no inhibition in accordance with the binding specificity of calmodulin to gangliosides. Trypsin-activated phosphodiesterase was inhibited by gangliosides with similar specificity, indicating interactions of gangliosides with the enzyme. Inhibition, however, was less than that of calmodulin-dependent activity by these compounds and, in both cases, was eliminated by excess calmodulin. In the absence of calmodulin, group 1 gangliosides at lower concentrations activated the intact enzyme but inhibited it over a certain range of increase in concentration. Ganglioside-dependent modulation of calmodulin-dependent phosphodiesterase activity is thus shown to be due to interactions of gangliosides with both calmodulin and the enzyme, and consequently, ganglioside-calmodulin binding is likely the mechanism for regulation of the enzyme.
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PMID:Mechanism for ganglioside-mediated modulation of a calmodulin-dependent enzyme. Modulation of calmodulin-dependent cyclic nucleotide phosphodiesterase activity through binding of gangliosides to calmodulin and the enzyme. 131 72

Two factors were separated from rat liver particulate fraction treated with insulin, one of them having a stimulating effect on low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase activity of crude microsomal fraction (P-2 fraction) and the other having an inhibiting effect on the activity of low-Km cAMP phosphodiesterase solubilized with 0.3% Brij 58 from P-2 fraction. Trypsin and heat treatments had essentially no effect on these two factors. The stimulating factor did not significantly change the apparent Km value of enzyme in P-2 fraction but increased the maximal velocity of the reaction. The inhibiting factor raised the Km value of solubilized enzyme without affecting the maximal velocity of the reaction. The stimulating factor level in diabetic rat was larger than that in normal rat while the inhibiting factor level in diabetic rat was smaller than that in normal rat. Possible participation of both factors in insulin action is discussed.
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PMID:Separation and some characteristics of two factors from rat liver particulate fraction which stimulate and inhibit the low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase of rat fat cells. 132 56

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.
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PMID:The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system. 282 42

A partially purified preparation (200-fold) of cAMP phosphodiesterase (PDE) was obtained from Mucor rouxii grown and extracted under conditions minimizing endogenous proteolysis. Four purification steps were applied: batch DEAE-Sepharose, DEAE-Sepharose chromatography, Sephadex G-150 super-fine gel filtration and sucrose gradient centrifugation. The final PDE preparation was activatable by cAMP-dependent phosphorylation and controlled trypsin treatment. A careful correlation of protein patterns with PDE activity was done throughout the whole procedure by analyzing the active fractions of each step by mini-polyacrylamide non-denaturing gel electrophoresis. The final preparation displayed four major protein bands, none of which corresponded to PDE, although PDE activity comigrated with two of them. Some properties of this preparation were studied. Vmax increased around 10-15 fold by activation of PDE by phosphorylation or proteolysis; Km values were unaffected. PDE had Stokes radius of 3.5 nm, sedimentation coefficient of 4.3 S and molecular weight of 70,000 daltons. The treatment of sucrose gradient fractions with [gamma-32P] ATP and cAMP-dependent protein kinase catalytic subunit and further analysis through minigels showed that none of the visible bands was phosphorylated, and that among the four phosphorylated bands there was one that cosedimented and comigrated with PDE activity. Trypsin treatment of the phosphorylated samples removed the label but did not modify the staining pattern.
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PMID:Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii. 285 14

Cyclic GMP phosphodiesterase (PDE) in rod disk membranes has three subunits of molecular weight 88 000 (alpha), 84 000 (beta), and 13 000 (gamma). Physiological activation of the enzyme by light is mediated by a GTP binding protein (G protein). The enzyme can also be activated by controlled digestion with trypsin, which destroys the gamma subunit, leaving the activated enzyme as PDE alpha beta [Hurley, J. B., & Stryer, L. (1982) J. Biol. Chem. 257, 11094-11099]. Addition of purified gamma subunit to PDE alpha beta inhibited the enzyme fully. This suggested the possibility that G protein could also activate PDE by removing the gamma subunit and leaving the active enzyme in the form of PDE alpha beta. Should this be true, the properties of light- and trypsin-activated enzymes should be comparable. We found this not to be the case. The Km of light-activated enzyme for cyclic GMP was about 0.9-1.4 mM while that of trypsin-activated enzyme was about 140 microM. The cyclic AMP Km was also different for the two enzymes: 6.7 mM for light-activated enzyme and 2.0 mM for trypsin-activated enzyme. The inhibition of both enzymes by the addition of purified gamma subunit also differed significantly. Trypsin-activated enzyme was fully inhibited by the addition of about 200 nM gamma, but light-activated enzyme could not be fully inhibited even with 2600 nM inhibitor subunit. The Ki of the trypsin-activated enzyme for gamma was 15 nM and of the light-activated enzyme 440 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits. 300 65

Soluble enzymes, extracted from bovine retinal rod outer segments (ROS), were recombined with native ROS discs and discs which had been modified either by protease treatment or phosphorylation with rhodopsin kinase. The effect of these modifications on rhodopsin's ability to light-activate the ROS phosphodiesterase was determined. Trypsin, short-term thermolysin, and papain-digested discs were more effective in activating the phosphodiesterase than were undigested discs, whereas phosphorylated discs showed reduced ability to activate the phosphodiesterase. When a non-hydrolyzable analogue was employed in place of GTP in the assay, the same differences in the activation of phosphodiesterase as described above were observed between control discs and discs which were digested with thermolysin or phosphorylated. The proteolysis treatments remove various segments of amino acids from the carboxyl terminus of rhodopsin. In addition, at least seven phosphorylation sites are located in the terminal 15 amino acid residues of the carboxyl terminus of rhodopsin. Hence, it would appear from these studies that modifications of rhodopsin which affect the carboxyl terminus result in marked changes in the level of light-activatable phosphodiesterase activity, strongly suggesting a regulatory involvement in the light-activation process for this portion of rhodopsin.
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PMID:Activation of rod outer segment phosphodiesterase by enzymatically altered rhodopsin: a regulatory role for the carboxyl terminus of rhodopsin. 608 65

Bovine serum albumin inhibits the light activation of bovine rod disc membrane (RDM) cyclic GMP phosphodiesterase. The KI for inhibition is 32 microM at pH 8 and 37 degrees C. Trypsin-activated phosphodiesterase was not inhibited under these conditions, suggesting that albumin does not alter substrate access to the enzyme. Light titration curves of phosphodiesterase activity were vertically displaced downwards by albumin. The lack of displacement along the bleach axis indicated no loss in relative light sensitivity, but rather loss of a constant fraction of the normal activity for each bleach level. Thus, activated rhodopsin appeared to be functional in the presence of albumin. However, the metarhodopsin II yield with less than 10% bleached was reduced in the presence of albumin. This effect was quantitatively explained by albumin elution of GTP-binding protein from the RDM. Similarly, the reduction in light-induced phosphodiesterase activity quantitatively matched GTP-binding protein elution by albumin. beta-Lactoglobulin, which, like albumin, is known to bind hydrophobic molecules, also inhibits phosphodiesterase activation. In contrast, ovalbumin, which has little hydrophobic binding affinity, had little or no inhibitory effect on phosphodiesterase light activation. We conclude that albumin and other molecules capable of hydrophobic interactions inhibit light activation of RDM-phosphodiesterase by selectively eluting GTP-binding protein from the membrane into the surrounding medium where it is unable to efficiently gain access to activated rhodopsin.
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PMID:Albumin inhibits light activation of cGMP phosphodiesterase on rod disc membranes. 609 63

The first stage of amplification in the cyclic GMP cascade in bovine retinal rod is carried out by transducin, a guanine nucleotide regulatory protein consisting of two functional subunits, T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). Limited trypsin digestion of the T beta gamma subunit converted the beta polypeptide to two stable fragments (Mr approximately 26,000 and approximately 14,000). The GTPase and Gpp(NH)p binding activities were not significantly affected by the cleavage. Trypsin digestion of the T alpha subunit initially removed a small segment from the polypeptide terminus and resulted in the formation of a single 38,000-Da fragment. When this fragment was recombined with the intact T beta gamma subunit in the presence of membranes containing photolyzed rhodopsin, the reconstituted transducin exhibited greatly reduced GTPase and Gpp(NH)p binding activities. The loss in activities was due to the inability of the cleaved T alpha to bind to the photolyzed rhodopsin. Prolonged digestion converted the 38,000-Da fragment to a transient 32,000-Da fragment and then to two stable 23,000-Da and 12,000-Da fragments. The cleavage of the 32,000-Da fragment, however, can be blocked by bound Gpp(NH)p. The 32,000-Da fragment contains the Gpp(NH)p binding site and retains the ability to activate phosphodiesterase. These results indicate that the guanine nucleotide binding and rhodopsin binding sites are located in topologically distinct regions of the T alpha subunit and proved evidence that a large conformational transition of the molecule occurs upon the conversion of the bound GDP to GTP.
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PMID:Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin. 613 10

Trypsin increased cyclic AMP levels of the pig skin. This effect was markedly potentiated by the cyclic nucleotide phosphodiesterase inhibitor theophylline. Without theophylline this trypsin-induced cyclic AMP accumulation was transient and the maximal accumulation was noted by 5 min. Soybean trypsin inhibitor inhibited this trypsin-induced cyclic AMP accumulation. After the trypsin treatment, marked acantholysis was noted histologically and the decreased responsiveness to other adenylate cyclase stimulators was seen. The decrease of the epinephrine response was most marked and that of histamine response was much less. Both low and high Km cyclic nucleotide phosphodiesterase activities were decreased by the trypsin treatment. However, at 5 min incubation time, when the increase in cyclic AMp level was most marked, the decrease in the phosphodiesterase activities was minimal. Trypsin seems to reveal its action through the proteolytic activation of adenylate cyclase system of the skin.
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PMID:Effects of trypsin on the cyclic AMP system of the pig skin. 626 81


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