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)

A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography. Trypsin digestion of the purified pETB-42P gave big ET-1(1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1(1-37) gave ET-1(1-21) in a yield of 74% (overall yield: 52%). Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60%. One milligram of ET-1 or 2.0 mg of big ET-1(1-37) was obtained from 1.8 liters of culture broth. Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1(1-37) had almost the same in vitro and in vivo biological activities as big ET-1(1-38).
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PMID:High-yield production of recombinant endothelin-1. 142 24

The media from cultured microvascular and macrovascular endothelial cells (conditioned media, CM) were collected and tested for constrictor activity in sheep coronary artery rings and tracheal smooth muscle strips in vitro (isometric force), expressed as percentage of contraction produced by 80 mM KCl. Both microvascular (micro) and macrovascular (macro) CM caused a sustained slow-onset contraction (P less than 0.05) of the coronary artery rings by 71 +/- 10% (micro; n = 7) and 67 +/- 8% (macro; n = 6) and tracheal smooth muscle strips by 33 +/- 14% (micro; n = 6) and 34 +/- 6% (macro; n = 11); the calcium antagonist gallopamil (10(-7) M) attenuated these effects by 25-55%. Unconditioned medium and medium conditioned by cultured tracheal smooth muscle cells had no constrictor activity on coronary artery rings or tracheal smooth muscle strips. Synthetic endothelin (ET-1) also produced contraction of coronary artery rings and tracheal smooth muscle strips. The mean levels of ET-1 measured by radioimmunoassay were 1,200 pg/ml in the macro CM and 33 pg/ml in the micro CM. Depleting macro CM of ET-1 by affinity columns constructed with protein A agarose and anti-ET-1 antibody removed the contractile activity for coronary artery rings and tracheal smooth muscle strips. Thus ET-1 did not appear to be the contractile substance in the micro CM. Preliminary characterization of the contractile substance in micro CM revealed that it was heat stable, had a molecular weight of less than 10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze-thawing.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Microvascular and macrovascular endothelial cells produce different constrictor substances. 160 72

The binding and internalization of 125I-endothelin (125I-ET-1) was studied in cultured human vascular smooth muscle cells (hVSMC). Discrimination between surface-bound and internalized radiolabeled ligand was achieved using either acetic acid or trypsin treatment of cell layers, with the two procedures yielding comparable results. Total cellular 125I-ET-1 binding hVSMC at 37 degrees was rapid and reached near equilibrium within 30 min. Such binding could be resolved into surface-bound (acid/trypsin-sensitive) and internalized (acid/trypsin-resistant) components. The accumulation of internalized 125I-ET-1 was temperature dependent and occurred at 37 degrees (t1/2 approximately 15 min) but not at 4 degrees. Internalization of 125I-ET-1 by hVSMC was reversibly inhibited by the transglutaminase inhibitor dansylcadaverine (half-maximal inhibitory concentration, approximately 400 microM). Cytosolic acidification of hVSMC (from pH approximately 6.8 to approximately 6.3) by incubation with potassium acetate in a choline buffer also inhibited 125I-ET-1 internalization. Our observation indicate that smooth muscle cells internalize ET-1 via the clathrin-mediated endocytotic pathway. Dansylcadaverine and other inhibitors of transglutaminase inhibited ET-1-stimulated inositol phospholipid hydrolysis in hVSMC and decreased ET-1-induced vasoconstriction in isolated endothelium-denuded blood vessels. Internalization of ET-1 may, therefore, be relevant to the characteristically protracted physiological effects of this peptide on the vasculature.
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PMID:Internalization of endothelin by cultured human vascular smooth muscle cells: characterization and physiological significance. 220 Sep 54

In human placenta, endothelin (ET) could derive from maternal, fetal, and/or endogenous sources. Therefore, localization of ET-1 was investigated by immunohistochemistry in human term placenta and in cultured trophoblasts. In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblasts of the villi. ET-1 IR was also detected in the decidual cells and in the extravillous cytotrophoblasts of the basal plate. The extravillous cytotrophoblasts of the chorionic plate and of the placental septa also exhibited strong ET-1 IR. For trophoblast culture, cytotrophoblastic cells were obtained from placental villi by trypsin-DNAse dispersion, further purified on a Percoll gradient, and enriched by employing a monoclonal anti-HLA class I antibody. After different periods of culture of purified cytotrophoblastic cells (1-5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblasts. The presence of ET-1,2 immunoreactivity (ET-1,2 IR) in the culture media was demonstrated by radioimmunoassay. A uniform daily production of the peptide was observed over at least 5 days (approximately 50 fmol/10(6) cells/24 h). Furthermore, trophoblastic cells that had been cultured for 5 days contained significant amounts of ET-1,2 IR (24 fmol/10(6) cells). These results suggest a trophoblastic origin for ET-1 and support the hypothesis of a paracrine and autocrine action of the peptide in the physiology of the trophoblast and placenta.
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PMID:Endothelin-1 binding sites and immunoreactivity in the cultured human placental trophoblast: evidence for an autocrine and paracrine role for endothelin-1. 750 48

A protein modification method has been developed for the production of human big endothelin (ET)-1. Production of a large quantity of big ET-1 by the method described here is expected to facilitate future experiments such as X-ray crystallography and nuclear magnetic resonance studies, aimed at understanding the tertiary structure of big ET-1 and its dynamics. The plasmid pETB-50 used for the synthesis carries the gene for a fusion protein consisting of 34-amino acid (aa) residues of an N-terminal portion of beta-galactosidase and the 38-aa residues of big ET-1. The fusion protein ETB-50P contains an arginine residue in the big ET-1 portion at its second C-terminal site and three lysine residues including the C-terminal site in the beta-galactosidase portion, all of which are susceptible to trypsin. Tryptic digestion of the fusion protein quantitatively produced big ET-1 (1-37), which is depleted in the C-terminal serine. However, a treatment of the fusion protein with 1,2-cyclohexanedione prior to tryptic digestion gave full-length big ET-1 with N7, -N8-(1,2-dihydroxycyclohex-1,2- ylene)-arginine. This modification was reversed to the intact arginine residue when the modified big ET-1 was incubated in 0.5 M TRIS-HCl buffer, pH 8.0. Consequently, a combination of such a reversible protein modification and tryptic digestion gave 1.74 mg of recombinant big ET-1 from 2.01 of culture broth. The procedure described here may be applied to produce other arginine-containing peptides from fusion proteins.
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PMID:High-yield production of human big endothelin-1 by a combination of chemical modification and proteolysis of a fusion protein in Escherichia coli. 776 64

To determine the intrauterine defensive role of urinary trypsin inhibitor (UTI), we studied the effects of UTI in amniotic fluid, fetal membranes and myometrium. The level of UTI was 94 +/- 34 U/ml in neonatal urine (compared to adult urine 8.0 +/- 6.0 U/ml) and 88 +/- 37 U/ml in amniotic fluid. This may indicate that the main source of UTI in the amniotic fluid is the fetal urine. UTI was found to be concentrated in vernix, fetal intestine, amniotic membranes and uterine myometrium. Immunostaining of term amnion revealed a dark staining for UTI, whereas in premature deliveries UTI staining was markedly decreased. In myometrium, the concentration of UTI was found to be increased during pregnancy compared to non pregnant myometrium. Also, placentas were well stained for UTI in term pregnancy. Thus UTI has an important role in amniotic fluid, fetal membranes, placenta and uterine muscles. UTI has an inhibitory effect on several enzymes and cytokines. UTI was found to inhibit neutrophil elastase activity as well as trypsin activity. Its inhibitory activity was increased in the presence of lipid. LPS stimulated amnion cells trapped more UTI than unstimulated amnion cells. UTI in amnion cells was released after addition of 1% meconium solution. UTI was also found to inhibit the effect of IL-1, TNF and interleukin-8 on amnion. These results indicate that UTI localized in amnion is important in the protection of fetal membrane especially against bacterial infections and cytokines. It is known that endothelin (ET), prostaglandin F2 alpha (PGF2 alpha) and oxytocin can induce uterine contraction. UTI could inhibit uterine contractions stimulated by ET, PGF2 alpha and oxytocin in isometric contraction test. UTI could also inhibit cervical maturation induced by interleukin-8. Therefore UTI is essential for maintenance of pregnancy. From the isometric contraction tests, we assumed that UTI might works through regulation of calcium entry or availability in the cells. Initial increase in intracellular calcium was also inhibited by UTI pre incubation dose dependantly. We examined the change in intracellular calcium at single cell level by digital image analysis with Fura 2AM as a calcium probe. At resting level UTI incubation did not produce any significant changes in intracellular free calcium. Thrombin, LPS, interleukin-8 and ET-1, known calcium agonists could increase intracellular calcium in fibroblasts, amnion and uterine myocytes. Whereas as the same doses of those known calcium agonists could not change the intracellular free Ca2+ concentrations in UTI pre incubated fibroblasts, amnion cells and uterine smooth muscle cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Intrauterine defensive mechanism of amniotic fluid and fetal membranes]. 808 4

This study has examined the localisation and receptor-binding of the endothelins in retina and choroid of human and rat origin. Immunoreactivity to anti-ET1 and anti-ET3 was investigated in trypsin digests, frozen sections and ultrathin sections using immunocytochemistry and immunogold labelling techniques. In addition, receptor binding of 125I-ET1 and 125I-ET3 was visualised and quantified using autoradiography and image analysis. Intense immunoreactivity to anti-ET1 and anti-ET3 was observed in the photoreceptor inner segments and in the outer plexiform layer (OPL) of human and rat retina. Ultrastructural localisation using immunogold labelling confirmed the presence of ET1 and ET3 in the photoreceptor cells. In retinal vascular digests, ET1 was visualised in the arteries, arterioles and at the pre-arteriolar sphincters, however, immunoreactivity to anti-ET3 was absent in the retinal vasculature. Both ETA and ETB-type receptor binding sites to 125I-ET1 and 125I-ET3 were detected in the vascular smooth muscle of choroidal and retinal vessels with the former being predominant. Extravascular binding sites of the ETB-type were found in the ganglion cell layer.
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PMID:Endothelin-like immunoreactivity and receptor binding in the choroid and retina. 863 Nov 98

Endothelin (ET) receptors activate heterotrimeric G proteins that are members of the Gi, Gq, and Gs families but may also activate members of other families such as Galpha12/13. Galpha13 has multiple complex cellular effects that are similar to those of ET. We studied the ability of ET receptors to activate Galpha13 using an assay for G protein alpha-chain activation that is based on the fact that an activated (GTP-bound) alpha-chain is resistant to trypsinization compared with an inactive (GDP-bound) alpha-chain. Nonhydrolyzable guanine nucleotides and AlMgF protected Galpha13 from degradation by trypsin. In membranes from human embryonic kidney 293 cells that coexpress ETB receptors and alpha13, ET-3 and 5'-guanylylimidodiphosphate [Gpp(NH)p] increased the protection of alpha13 compared with Gpp(NH)p alone. The specificity of ETB receptor-alpha13 coupling was documented by showing that beta2 receptors and isoproterenol or ETA receptors and ET-1 did not activate alpha13 and that a specific antagonist for ETB receptors blocked ET-3-dependent activation of alpha13.
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PMID:Endothelin-B receptors activate Galpha13. 1019 25

Our pilot study disclosed that tryptase-positive mast cells (MC) were densely distributed around the intrahepatic bile ducts (peribiliary MC). In this study, the pathophysiologic roles of these MC were examined with respect to the microcirculation around the bile duct in 71 cases of histologically normal liver, 24 cases of chronic hepatitis, and 45 cases of liver cirrhosis. The tryptase-positive MC were very close to the microvessels of the peribiliary vascular plexus (PVP), which supply the intrahepatic biliary tree. The tryptase-positive MC were frequently found adjacent to vascular smooth muscle cells, including pericytes. The location of the tryptase-positive MC was confirmed by ultrastructural analysis. In cirrhosis, the numbers of both microvessels of PVP and peribiliary MC increased in parallel. Peribiliary MC were immunoreactive for endothelin 1 (ET-1), and were variably immunoreactive for histamine, chymase, inducible nitric oxide synthase (iNOS), and endothelin A and B (ET(A) and ET(B)) receptors, particularly in cirrhotic livers. On vascular endothelial cells of PVP, endothelial nitric oxide synthase (eNOS) and ET-1 were consistently detectable, and ET(A) receptors, ET(B) receptors, and iNOS were variably detectable. Pericytes of PVP expressed ET(A) and ET(B) receptors in addition to ET-1 and iNOS. Biliary epithelial cells also focally expressed iNOS, ET-1, and ET(A) and ET(B) receptors. These vasoactive substances were strongly expressed on the cellular components in cirrhotic liver. By in situ hybridization, iNOS mRNA signals were observed on iNOS-immunoreactive cell components, including peribiliary MC. These morphologic and immunohistochemical findings suggest that the cellular components displaying vasoactive substances in the milieu of the intrahepatic biliary tree are very dynamic in the vasoregulation of PVP in normal livers, even more so in cirrhosis, and that peribiliary MC exert local effects on the microcirculation of PVP, directly and indirectly.
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PMID:Evidence of the participation of peribiliary mast cells in regulation of the peribiliary vascular plexus along the intrahepatic biliary tree. 1090 46

When mast cells are activated they can respond by releasing their secretory granule compounds, including mast cell-specific proteases of chymase, tryptase and carboxypeptidase A (MC-CPA) type. MC-CPA is a dominant protein component of the mast cell granule and the MC-CPA gene is extremely highly expressed. Despite this, relatively little has been known of its biological function. However, the recent generation of mouse strains lacking MC-CPA has opened up new possibilities for investigations related to this protease. This recent development has revealed a role for MC-CPA in regulating innate immunity responses, including the degradation of harmful substances such as the vasoconstrictive factor endothelin 1 and snake venom toxins. Here, we summarize the current knowledge of MC-CPA.
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PMID:Novel insights into the biological function of mast cell carboxypeptidase A. 1964 69


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