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 Lubrol-dispersed guanylate cyclase from sea urchin sperm was purified and isolated essentially free of detergent by GTP affinity chromatography, DEAE-Sephadex chromatography, and gel filtration. After removal of the detergent, the enzyme remained in solution in the presence of 20% glycerol. The specific activity of the purified enzyme was about 12 mumol of guanosine 3':5'-monophosphate (cyclic GMP) formed - min-1 - mg of protein-1 at 30 degrees, an activity about 4600 times that of a soluble guanylate cyclase purified recently from Escherichia coli (Macchia V., Varrone, S., Weissbach, H., Miller, D.L., and Pastan, I. (1975) J. Biol. Chem. 250, 6214-6217). The cyclic GMP phosphodiesterase activity was negligible and adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase was not detectable in the purified preparation. Cyclic AMP formation from ATP occurred at a rate of 0.002% of that of guanylate cyclase. In the absence of phosphodiesterase or guanosine triphosphatase inhibitors, 100% of the added GTP was converted to cyclic GMP. The purified enzyme required Mn2+ for maximum activity, the relative rates in the presence of Mg2+ or Ca2+ being less than 0.6% of the rates with Mn2+. The purified enzyme displayed classical Michaelis-Menten kinetics with respect to MnGTP (apparent Km is approximately equal to 170 muM) in contrast to the positively cooperative kinetic behavior displayed by the unpurified, detergent-dispersed, or particulate guanylate cyclase. The molecular weight of the purified enzyme was approximately 182,000 as estimated on Bio-Gel A-0.5m columns equilibrated in the presence or absence of 0.1 M NaCl. The unpurified, detergent-dispersed enzyme also migrated with an apparent molecular weight of 182,000 on columns equilibrated with 0.5% Lubrol WX and 0.1 M NaCl, but it migrated as a large aggregate (molecular weight is greater than 5 X 10(5)) on columns equilibrated in the absence of either the detergent of NaCl. After gel filtration, the unpurified, dispersed enzyme still yielded positive cooperative kinetic patterns as a function of MnGTP. Na dodecyl-SO4 gel electrophoresis of the enzyme after the DEAE-Sephadex or the gel filtration steps resulted in two major protein bands with estimated molecular weights of 118,000 and 75,000. Whether or not these protein bands represent the subunit molecular weights of guanylate cyclase is unknown at present.
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PMID:Sea urchin sperm guanylate cyclase. Purification and loss of cooperativity. 0 69

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.
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PMID:Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma. 1 Aug 95

The properties of the guanylate cyclase systems of outer and inner medulla of rat kidney were examined and compared with those of the renal cortex. A gradation in steady-state cyclic guanosine 3',5'-monophosphate (cGMP) levels was observed in incubated slices of these tissues (inner medula greater than outer medulla greater than cortex). This correlated with the proportion of total guanyl cyclase activity in the 100 000 g particulate fraction of each tissue, but was discordant with the relative activities of guanylate cyclase (highest in cortex) and of cGMP-phosphodiesterase (lowest in cortex) in whole tissue homogenates. Soluble guanylate cyclase of cortex and inner medulla exhibited typical Michaelis-Menten kinetics with an apparent Km for MnGTP of 0.11 mM, while the particulate enzyme from inner medulla exhibited apparent positive cooperative behavior and a decreased dependence on Mn2+. Thus, the particulate enzyme could play a key role in regulating cGMP levels inthe intact cell where Mn2+ concentrations are low. The soluble and particulate enzymes from inner medulla were further distinguished by their responses to several test agents. The soluble enzyme was activated by Ca2+, NaN3, NaNo2 and phenylhydrazine, whereas particulate activity was inhibited by Ca2+ and was unresponsive to the latter agents. In the presence of NaNo2, Mn2+ requirement of the soluble enzyme was reduced and equivalent to that of the particulate preparation. Moreover, relative responsiveness of the sollble enzyme to NaNO2 was potentiated when Mg2+ replaced Mn2+ as the sole divalent cation. These changes in metal requirements may be involved in the action of NaNO2 to increase cGMP in intact kidney. Soluble guanylate cyclase of cortex was clearly more responsive to stimulation by NaN3, Nano2, and phenylhydrazine that was soluble activity from either medullary tissue. The effectiveness of the agonists on soluble activity from outer and inner medulla cound also be distinguished. Accordingly, regulation and properties of soluble guanylate cyclase, as well as subcellular enzyme distribution, and distinct in the three regions of the kidney.
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PMID:Properties and subcellular distribution of guanylate cyclase activity in rat renal medulla: correlation with tissue content of guanosine 3',5'-monophosphate. 1 Sep 67

ADP-induced platelet aggregation and shape change were monitored optically in citrated rabbit platelet-rich plasma (PRP) diluted with isotonic salt solutions. Lithium (Li) produced a concentration-dependent reduction in the rate of platelet aggregation but had no discernible effect on the shape change which precedes aggregation. When PRP was pre-incubated with Li, the inhibitory effect of the ion was independent of the duration and temperature of the treatment. The inhibitory effect of Li also was observed in heparinized PRP or when 5-HT was used as the aggregation-inducing agent. When Li was combined with aggregation inhibitors which enhance platelet cyclic AMP content either by activating adenylate cyclase or by inhibiting phosphodiesterase, only additive effects were observed. The inhibitory effect of Li was opposed by added calcium. Kinetic evaluation of the interaction between Li and Ca indicated that their antagonism was competitive. Added calcium also displayed competitive antagonism toward the aggregation inhibiting effect of increased hydrogen ion concentration in the pH range between 6 and 8.
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PMID:Competitive inhibition by lithium and hydrogen ions of the effect of calcium on the aggregation of rabbit platelets. 1 92

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.
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PMID:Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. 1 74

The Ca2+-dependent protein activator of 3':5'-cyclic adenosine monophosphate phosphodiesterase is shown to undergo a conformational transition upon binding of 2 mol of Ca2+/mol of activator. Circular dichroic studies indicate that Ca2+ induces an increase of 5-8% in alpha-helix content with a concomitant decrease in the amount of random coil. In the absence of Ca2+ and in the presence of [ethylenebis(oxoethylenenitrilo)]tetraacetic acid (EGTA), the protein contains 30-35% alpha helix, 50% random coil, and 15-20% beta-pleated sheat. Spectrophotometric titration indicates that the two tyrosyl residues have pK's of 10.4 and 11.9 and are therefore in different environments. The Ca2+-induced conformational change is accompanied by an increased exposure to protons of the partially exposed tyrosine, as shown by a shift in its pK from 10.4 to 10.). Increased solvation is also consistent with a negative difference spectrum at 287 and 279 nm as seen upon Ca2+ binding. Modification in the environment of all or some of the phenylalanine residues also is part of the conformational change accompanying Ca2+ binding. A new and rapid purification procedure which yields large amounts (25-30% yields) of homogenous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.
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PMID:Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase. 1 63

The specific activity of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase of leukemic lymphocytes was 5-10-fold greater than that of purified normal lymphocytes or of homogenates of spleen, thymus or lymph nodes of normal mice. This rise was demonstrable over a wide range of substrate concentrations. Both normal and leukemic lymphocytes contained a heat-stable, calcium-dependent activator of phosphodiesterase. However, the increased activity of phosphodiesterase in leukemic lymphocytes was not due to this protein activator since (a) phosphodiesterase activity from these cells was not stimulated by this activator and (b) phosphodiesterase activity of leukemic lymphocytes was not inhibited by the calcium chelater, ethylene-glycol-bis,(beta-aminoethylether)-N,N'-tetraacetic acid, suggesting that the enzyme was not already maximally activated. A comparison of several other properties of phosphodiesterase from normal and leukemic lymphocytes showed that the enzymes have similar pH optima, similar stabilities to freezing and thawing and similar sensitivities to inhibition by the phosphodiesterase inhibitors, chlorpromazine, papaverine and isobutylmethylxanthine. However, the subcellular distribution of the phosphodiesterases was different, and the phosphodiesterase of leukemic lymphocytes was significantly more resistant to heat than that of normal lymphocytes. Although no differences were found between the phosphodiesterases of normal and leukemic lymphocytes in their sensitivities to drugs, there were marked differences in drug sensitivity between the phosphodiesterase of lymphocytes and that of other tissue. For example, concentrations of chlorpromazine which inhibited phosphodiesterase of cerebrum by 70% had no effect on phosphodiesterase activity of lymphocytes. On the othere hand, the papaverine-induced inhibition of phosphodiesterase was similar in lymphocytes and cerebrum. Since an optimal concentration of cyclic nucleotides is essential to maintain normal cell growth, these results suggest that the abnormal growth characteristics of leukemic lymphocytes may be explained by their high activity of phosphodiesterase. Furthermore, the qualitative and quantitiative differences between the phosphodiesterases of leukemic lymphocytes and other tissues raise the possibility of selectively inhibiting the phosphodiesterase of the leukemic lymphocytes, thereby reducing their rate of growth, without affecting other tissues.
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PMID:Characteristics of the cyclic nucleotide phosphodiesterases of normal and leukemic lymphocytes. 1 11

Modern data on the nature, properties and pathways of regulation enzymes, participated metabolism of cyclic, adenosine-3',5'-monophosphate, are described. Much consideration is being given to the problem of regulation of the phosphodiesterase activity due to the effect of Ca2+-binding proteins.
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PMID:[Adenyl cyclase and adenosine-3',5'-monophosphate phosphodiesteraze: their nature, properties and regulation]. 1 91

In chick embryo cells (CEC) and plasma membranes (PM) isolated therefrom, three forms of 3',5'-cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE) were demonstrated: (1) PDE activated only by Ca2+ (Ca-PDE); (2) PDE activated only by Mg2+ (Mg-PDE); and (3) PDE stimulated in the presence of 2 mM Mg2+ by low concentrations of Ca2+ (Ca-Mg-PDE). Purified influenza A virus, under suitable conditions, lowered the activities of these PDEs. The decrease was greater in samples incubated in the presence of adenosine triphosphate (ATP) than in those incubated in its absence.
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PMID:Interaction of plasma membranes with influenza virus. VII. Effect on 3',5'-cyclic adenosine monophosphate phosphodiesterase activity. 1 92

On the basis of the information presented in this review, it is difficult to reach any firm decision regarding the role of cyclic AMP (or cyclic GMP) in synaptic transmission in the brain. While it is clear that cyclic nucleotide levels can be altered by the exposure of neural tissues to various neurotransmitters, it would be premature to claim that these nucleotides are, or are not, essential to the transmission process in the pre-or post-synaptic components of the synapse. In future experiments with cyclic AMP it will be necessary to consider more critically whether the extracellularly applied nucleotide merely provides a source of adenosine and is thus activating an extracellularly located adenosine receptor, or whether it is actually reaching the hypothetical sites at which it might act as a second messenger. The application of cyclic AMP by intrcellular injection techniques should minimize this particular problem, although possibly at the expense of new diffulties. Prio blockade of the adenosine receptor with agents such as theophylline or adenine xylofuranoside may also assist in the categorization of responses to extracellularly applied cyclic AMP as being a result either of activation of the adenosine receptor or of some other mechanism. Utimately, the developement of highly specific inhibitor for adenylate cyclase should provide a firm basis from which to draw conclusions about the role of cyclic AMP in synaptic transmission. Similar considerations apply to the action of cyclic GMP and the role of its synthesizing enzyme, guanylate cyclase. The use of phosphodiesterase inhibitors in studies on cyclic nucleotides must also be approached with caution. The diverse actions of many of these compounds, which include calcium mobilization and block of adenosine uptake, could account for many of the results that have been reported in the literature.
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PMID:The role of cyclic nucleotides in the CNS. 1 46


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