Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bio-Gel A-5m chromatography has been used to separate apparent multiple forms of cyclic nucleotide phosphodiesterase from rat erythrocytes. Cyclic AMP phosphodiesterase was resolved by gel filtration into three peaks of activity with apparent molecular weights of about 300,000, 225,000 and 100,000, while cyclic GMP phosphodiesterase activity in gel column fractions was too low to permit meaningful estimates of its molecular weight. All three of the separated peaks of cyclic AMP phosphodiesterase activity displayed anomalous kinetic behaviour suggestive of negative cooperativity. The possibility that multiple phosphodiesterase activities could arise from in vitro alterations of a single enzyme was investigated. Similar changes in gel filtration profiles resulted when erythrocyte extracts were treated with trypsin or ammonium sulfate or were incubated at 37 degrees C. After these treatments, a large proportion of the enzyme activity occurred in low (ca. 100,000) molecular weight regions. The low molecular weight phosphodiesterase activities from untreated, incubated, and trypsin-treated extracts possessed similar properties. All were inhibited by methylxanthines, had pH optima of approximately 8.0, and similar kinetic properties and requirements for divalent cations. These observations raise the possibility that preparative procedures or limited proteolysis occurring during preparation and handling of extracts can contribute to the apparent multiplicity of enzyme forms seen after gel filtration of phosphodiesterase from rat erythrocytes and perhaps other cell types.
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PMID:Apparent multiple forms of cyclic AMP phosphodiesterase from rat erythrocytes. 18 74

Resting mammalian fibroblasts, either sparse and maintained in a serum-free medium, or confluent and contact inhibited, are stimulated to divide by treatment with a preparation of 3':5'-cyclic AMP phosphodiesterase. This enzyme preparation contained a low level of trypsin-like and alpha-chymotrypsin-like activity, but its effect on cell growth could not be mimicked by pure, crystallized trypsin and alpha-chymotrypsin at concentration equivalent to their contamination in the above preparation. Preincubation of the 3':5'-cyclic AMP phosphodiesterase preparation with the protease inhibitor, phenyl methane sulfonyl fulride, did not affect, either, its stimulation of DNA synthesis in fibroblasts, or its enzymatic hydrolysis of cyclic AMP.
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PMID:Growth stimulation of sparse, serum deprived and of confluent, contact inhibited mammalian fibroblasts by a preparation of 3':5'-cyclic AMP phosphodiesterase. 19 71

Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.
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PMID:Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells. 20 81

The activity of cyclic AMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in 105 000 X g supernatant fraction from frozen-thawed rat liver was 2.5 times higher than the corresponding preparation from fresh liver. This increased activity of frozen liver enzyme was accompanied by a decreased sensitivity of the enzyme to known activators such as alpha-tocopheryl phosphate and trypsin. Neither membrane-bound cyclic AMP phosphodiesterase, nor supernatant cyclic GMP phosphodiesterase increased in frozen liver preparation. It is unlikely that the activator protein of phosphodiesterase participated in the observed change of enzyme activity. Among rat tissues so far tested, the increased level of cyclic AMP phosphodiesterase was noted only in tissues rich in lysosome content. In the recombination experiment where phosphodiesterase from fresh liver was incubated with lysosomal fraction, stimulation of the enzyme activity was observed with a concomitant loss of sensitivity to above-mentioned activators. Since the stimulation by lysosomal fraction was effectively inhibited by cathepsin B1 inhibitors, leupeptin and antipain, it was deduced cathepsin-B1 (EC 3.4.12.3) type protease(s) was the main causative of activating the cyclic AMP phosphodiesterase. The freezing-thawing process of rat liver made the lysosomal membrane more permeable, and hence lysosomal proteases were released into soluble fraction during phosphodiesterase preparation. These results provide a warning not to use frozen liver for phosphodiesterase preparation, otherwise altered properties of the enzymes will be seen.
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PMID:Increased activity of cyclic AMP phosphodiesterase from frozen-thawed rat liver. A role of lysosomal protease in enzyme activation. 20 22

A protein, present in bovine seminal plasma, initiates forward motility in immature, immotile caput spermatozoa that have been incubated with a cyclic AMP phosphodiesterase inhibitor. An improved motility assay was developed to study this process and the protein involved. This forward motility protein exhibits multiple forms when fractionated on the basis of charge or molecular weight. Molecular sieving in urea or sodium dodecyl sulfate and dithiothreitol results in a single peak of activity which will re-form the larger aggregates in the absence of these agents. The molecular weight of this monomeric motility protein, as estimated from molecular sieving under these dissociating conditions, is 37,500. The forward motility protein can be partially purified by heat treatment, gell chromatography in urea, and affinity chromatography on concanavalin A/agarose. Enzymatic treatments further suggest a glycoprotein nature, i.e. treatment with beta-galactosidase, neuraminidase, alpha-mannosidase, or galactose oxidase reduces its activity by 50%; treatment with trypsin completely abolishes forward motility protein activity. On the basis of concurrent studies on the activity, properties, and distribution of forward motility protein in bovine body fluids, it is suggested that this protein is involved in the development of the capacity for motility as sperm traverse the epididymis.
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PMID:Bovine sperm forward motility protein. Partial purification and characterization. 21 Nov 30

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide phosphodiesterase on linear sucrose gradients, a 7-S form capable of hydrolyzing both cyclic AMP and cyclic GMP. Treatment of rat uterine supernatant with trypsin leads to the appearance of a 4-S, cyclic AMP-specific form with properties similar to that of BHK peak I. These data suggest that the kinetically complex, higher molecular weight cyclic nucleotide phosphodiesterases may consist of more than one catalytically active site and that multiple forms of the enzyme arise through dissociative mechanisms, possibly as a means of in vivo regulation.
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PMID:Activation of mammalian cyclic AMP phosphodiesterases by trypsin. 21 13

Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demonstrate the appearance of some factor(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase. The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75 degrees C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.
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PMID:Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development. 44 79

1. Four cyclic AMP phosphodiesterase-activating activities, designated as A, B, C and D, were isolated from lugworm, Arenicola cristata, by preparative flat-bed isoelectric focusing. Activators C and D were further purified by TSK 3000SW HPLC to homogeneity. 2. Activators A, B, C, and D corresponded to pIs of 4.4, 5.0, 5.2 and 5.4; their mol wts were estimated to be 36,200, 30,500, 30,200 and 28,300 respectively. 3. The protease nature of these activities were confirmed by the inhibition by several trypsin inhibitors of their activation of phosphodiesterase and by their hydrolysis of TAME, a synthetic trypsin substrate. Only protease A also hydrolyzed BTEE, a chymotrypsin substrate.
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PMID:Isolation from lugworm (Arenicola cristata) of four proteases that activate cyclic AMP phosphodiesterase. 282 20

Extraction of frozen canine cardiac muscle rendered soluble over 90% of the cyclic AMP phosphodiesterase activity. The residual activity was membrane-bound. Ion exchange chromatography of the soluble activity on DE-52 allowed for the resolution of three distinct cyclic AMP phosphodiesterase fractions termed PDE-I, PDE-II and PDE-III in order of elution from the column by a linear NaCl gradient. The relative ratio of cyclic AMP phosphodiesterase activity exhibited by these three peaks was 1:0.65:0.82 and of cyclic GMP phosphodiesterase activity was 1:0.52:0.05 for PDE-I, PDE-II and PDE-III respectively. PDE-II and PDE-III were further purified by re-chromatography on DE-52. Fractions PDE-II and PDE-III were thermolabile at 50 degrees, decaying as single exponentials with half lives of 180 sec and 77 sec respectively. All three species exhibited non-linear Lineweaver-Burke plots for the hydrolysis of cyclic AMP, exhibiting both high and low affinity components. Hydrolysis of cyclic GMP by all three components obeyed normal kinetics, yielding linear plots. PDE-I was a Ca2+/calmodulin-activated species which exhibited a low Km for both cyclic AMP and cyclic GMP but hydrolysed cyclic GMP with a higher Vmax than for cyclic AMP. PDE-II exhibited a much lower Km for cyclic AMP than for cyclic GMP and a much higher Vmax for the hydrolysis of cyclic AMP. PDE-III exhibited a low Km for both cyclic AMP and cyclic GMP, however, its Vmax for cyclic AMP was about 40-fold higher than for cyclic GMP. Cyclic GMP acted as a potent inhibitor (IC50 = 6.3 microM) of cyclic AMP hydrolysis catalysed by PDE-III but not of the hydrolysis of cyclic AMP by PDE-II (IC50 = 33.2 microM). The phosphodiesterase inhibitors milrinone, CI-930, UK-35,493, carbazeran and buquineran acted as potent inhibitors of cyclic AMP hydrolysis catalysed by both PDE-II and PDE-III enzymes. They did not inhibit PDE-I activity. PDE-II, when prepared in the absence of protease inhibitors exhibited a reduced potency to inhibition by these compounds. Treatment of purified PDE-II with trypsin caused a reduction in enzyme activity and reduced dramatically the sensitivity of PDE-II activity to inhibition by these various compounds. The action of proteolysis in attenuating the inhibitory effect of these compounds on PDE-II was most dramatic with CI-930, milrinone, amrinone, buquineran and UK35,493 and least dramatic with carbazeran and IBMX.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Proteolysis of cyclic AMP phosphodiesterase-II attenuates its ability to be inhibited by compounds which exert positive inotropic actions in cardiac tissue. 282 12

Ecto-cyclic AMP phosphodiesterase activity was determined from freshly isolated and cultured liver cells. The cells were capable of hydrolyzing cyclic AMP in the medium. The ecto-phosphodiesterase represents a low Km phosphodiesterase which was activated by physiological concentrations of insulin. The product, 5'-AMP, was recovered in the medium and not with the cells. The enzyme was inhibited with aminophylline and trypsin. The ecto-phosphodiesterase activity was proportional to cell number, and total phosphodiesterase activity increased 5- to 10-fold when the cells were ruptured. About one-third of the ecto-phosphodiesterase activity from freshly isolated liver was due to phosphodiesterase in the medium. No phosphodiesterase was in the medium of cultured liver cells.
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PMID:Hormonally sensitive cyclic AMP phosphodiesterase in liver cells. An ecto-enzyme. 626 46


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