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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The peptides substance P (SP) and vasoactive intestinal peptide (VIP) released from peptidergic neurons have potent effects on gland secretion and on smooth muscle tone. Because mast cells release proteases during degranulation, and are located in many of the same tissue microenvironments into which SP and VIP are released, we wished to examine whether mast cell proteases, by cleaving and thus inactivating these peptides, could modulate their effects. We used active site-titrated preparations of the two major neutral proteases of mast cell granules, tryptase and chymase, to determine the sites and rates of cleavage of SP and VIP. The proteases were purified from dog mastocytomas. Tryptase cleaved VIP rapidly at two sites with a kcat/Km of 2.2 X 10(5) sec-1 M-1, but had no effect on SP. Chymase cleaved both SP and VIP at primarily a single site with kcat/Km of 3.9 X 10(4) and 5.4 X 10(4) sec-1 M-1, respectively. Thus, these data show that mast cell proteases degrade SP and VIP. The differences in peptidase activity between tryptase and chymase suggest that the consequences of protease release could vary according to mast cell protease phenotype and location in various tissues and species. Tryptase, by cleaving the bronchodilator VIP but not the bronchoconstrictor SP, might promote bronchial hyper-responsiveness in asthma by decreasing the nonadrenergic neural inhibitory influence mediated by VIP. In skin and other tissues, chymase might interrupt axon reflex-mediated neurogenic inflammation by cleaving SP.
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PMID:Substance P and vasoactive intestinal peptide degradation by mast cell tryptase and chymase. 244 73

Recent evidence suggests that nonadrenergic airway relaxation may be controlled by vasoactive intestinal peptide (VIP). The magnitude and duration of smooth muscle relaxation in response to VIP may be influenced by rates of peptide degradation after release from efferent peptidergic neurons. To explore the potential role of mast cell mediators in modulating neural control of airway tone, we studied the effect of the mast cell proteases tryptase and chymase on airway smooth muscle relaxation induced by VIP in ferret airway. Tracheal rings precontracted by serotonin (10(-6) M) in a muscle bath were relaxed by VIP (10(-7) M). We found that protease-rich supernatant obtained by degranulation of dog mastocytoma cells reversed VIP-induced relaxation, as did highly purified tryptase and chymase incubated with the tracheal rings. Either enzyme completely reversed the effect of VIP, but tryptase was more potent than chymase, paralleling previous test tube observations on the relative rates of VIP cleavage by the two enzymes. Inhibitors of mast cell tryptase and chymase preincubated with the supernatant or with the purified proteases prevented reversal of VIP-induced relaxation. Mast cell proteases did not reverse the tracheal relaxation caused by the nonpeptide adrenergic agonist isoproterenol. These findings show that mast cell proteases tryptase and chymase counteract the smooth muscle relaxant effects of VIP in ferret trachea and suggest a potential role for the mast cell proteases in the modulation of nonadrenergic neural control of airway tone by VIP.
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PMID:Mast cell tryptase and chymase reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret. 249 55

The purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM 125I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1 microM unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound 125I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. The labeling of both components was inhibited by unlabeled VIP or peptide with NH2-terminal histidine and COOH-terminal isoleucine amide (a VIP agonist). The presence of GTP (0.1 mM) in the incubation medium of membranes completely abolished the labeling of the 5.2-nm component but did not affect that of the 3.1-nm one. Moreover, GTP induced dissociation of 125I-VIP from the 5.2-nm component isolated by Sephacryl S-300 chromatography. This effect was time dependent and nucleotide specific. In contrast, GTP did not affect the stability of the 3.1-nm component. After cholera toxin catalyzed [32P]ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of 32P radioactivity was coeluted with the 5.2-nm component.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Solubilization and hydrodynamic characterization of guanine nucleotide sensitive vasoactive intestinal peptide-receptor complexes from rat intestine. 254 61

Human cultured T lymphocytes of the Jurkat line and myeloma cells of the U266 line cleaved the 28 amino acid vasoactive intestinal peptide (VIP1-28) preferentially at three sites with time- and temperature-dependence. The fragments VIP4-28 and VIP23-28) from an endopeptidase activity, and VIP15-28 from a trypsin-like peptidase, together represented a range of 26-65% of the VIP1-28 recovered after 2 hr at 37 degrees C or 4 hr at 22 degrees C, based on the absorbance of purified peptides and the radioactivity of [125I]Tyr10 VIP1-28. The endopeptidase activity was associated with membranes recovered after disruption of U266 cells by nitrogen cavitation. Pretreatment of intact U266 and Jurkat cells with diisopropylfluorophosphate (DFP) and the subsequently isolated subcellular particles with phenylmethylsulphonylfluoride (PMSF) and leupeptin inhibited the trypsin-like enzyme by a mean of 80%, without suppressing endopeptidase activity. In contrast, 0.1 mM DL-thiorphan and phosphoramidon blocked selectively a range of 35-70% of the endopeptidase activity in membrane preparations and intact cells. The capacity of lymphocytes to degrade VIP1-28 may substantially alter the effects of this neuromediator on functions of some subsets of T and B cells.
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PMID:Unique pattern of cleavage of vasoactive intestinal peptide by human lymphocytes. 265 11

An improved rat anterior pituitary primary cell culture technique for studying GH-releasing activity of human pancreatic GH-releasing factor (hpGRF) and its analogs is described. Male pituitaries, dispersed by a combination of trypsin digestion and mechanical agitation, were plated at a density of 200,000 cells per well and cultured for 4 days. The attached cells were then stimulated with synthetic hpGRF which was comprised of the first 29 residues of the larger, originally isolated forms and which was amidated at the C-terminal (hpGRF-29). Analogs of hpGRF-29 which were modified in positions 1, 2, 3, or 7, and other secretagogues were similarly tested. Medium was collected after 3 h, and secreted hormone was measured by RIA. The cells were extremely sensitive to hpGRF-29 stimulation, and this effect was specific. The minimal effective dose of hpGRF-29 was an unprecedented 0.4 X 10(-15)M. No stimulation of LH, FSH, or PRL by hpGRF-29 was observed. Bombesin and vasoactive intestinal peptide were ineffective in stimulating GH release. [D-Trp6]LHRH (a potent LHRH agonist), also did not release GH but did stimulate secretion of LH and FSH at doses ranging from 0.4 X 10(-10)M to 1.0 X 10(-9)M. Responses of the cells to hpGRF-29 analogs were characterized by distinct heterologous dose-response curves. [D-Ala2]hpGRF-29 was 50 times more active than its parent 29-amino-acid peptide. [D-Thr7]hpGRF-29, another analog that differed from hpGRF-29 by the insertion of a D-isomer for the naturally occurring L-residue, was about 10,000 times less effective in stimulating GH secretion than was hpGRF-29 itself. Potencies of these and other analogs with respect to GH release in vitro were similar to those estimated in vivo. Thus, this primary cell culture provides an extremely sensitive, selective, and reproducible system for studying hpGRF structure-activity relationships. Further, such tremendous sensitivity to hpGRF can provide a system to study changes in pituitary sensitivity to hpGRF during different physiological states.
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PMID:An extremely sensitive in vitro model for elucidating structure-activity relationships of growth hormone-releasing factor analogs. 285 74

The aim of this study was to determine the effect of wheat bran consumption on exocrine pancreas secretion in pigs. Sixteen Large-White pigs were divided into two groups. The first group (control) was fed a diet without wheat bran and the second one (experimental) a diet containing 40% wheat bran. After one week the animals were fitted with two permanent fistulae (in the pancreatic duct and the duodenum) and/or with a catheter in a carotid artery. After an 8-day recovery period, pancreatic secretion (volume, protein content and output, chymotrypsin, trypsin, lipase and amylase activities) and plasma levels of some gastro-intestinal peptides [secretin, cholecystokinin (CCK), vasoactive intestinal peptide (VIP), somatostatin and pancreatic polypeptide (PP)] were measured over an experimental period of 5 days. The results show that wheat bran intake induced an increase in the volume (+ 115%) and protein output (+ 36%) of the pancreatic juice secreted in a 24-hour period, whereas protein concentration decreased. All enzyme activities were enhanced by wheat bran. The plasma levels of secretin, VIP, somatostatin and PP were higher in the experimental than in the control group. On the contrary, plasma CCK levels were not affected by wheat bran consumption.
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PMID:Effects of wheat bran on exocrine pancreas secretion in the pig. 289 Nov 62

The receptor for vasoactive intestinal peptide (VIP) was identified in rat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis(propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80,000 and 56,000 daltons (Da). Both the large and small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 degrees C for 30 min with 1 microgram/mL chymotrypsin, trypsin, or elastase to fragments that that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80- and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (approximately 50%). The WGA-bound fraction (eluted with N-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80- and 56-kDa species to endo-beta-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydrate chains. The receptor species do not appear to have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-beta-acetylglucosaminidase H.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. 300 58

Venom from Gila monster (family Helodermatidae) contains a pancreatic secretagogue. In dispersed acini from guinea pig pancreas, the venom increased enzyme secretion to the same extent as did vasoactive intestinal peptide, secretin, or PHI. The abilities of vasoactive intestinal peptide and Gila monster venom to stimulate enzyme secretion were not altered by boiling but were abolished by incubation with trypsin or chymotrypsin. Like vasoactive intestinal peptide, secretin, and PHI, the venom caused a 50- to 60-fold increase in cellular cAMP and inhibited binding of 125I-vasoactive intestinal peptide to its membrane receptors on pancreatic acini. The action of venom on enzyme secretion was inhibited by [Gln9]secretin-(5-27), a vasoactive intestinal peptide receptor antagonist, but was not altered by atropine, a cholinergic receptor antagonist, or by dibutyryl cGMP, a cholecystokinin receptor antagonist. Gila monster venom contained no immunoreactive vasoactive intestinal peptide by radioimmunoassay. These results indicate that venom from Gila monster contains a peptide that can stimulate pancreatic enzyme secretion by interacting with vasoactive intestinal peptide receptors on pancreatic acinar cells and thereby activating adenylate cyclase and increasing cellular cAMP.
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PMID:Actions of Gila monster venom on dispersed acini from guinea pig pancreas. 617 52

Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.
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PMID:Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences. 773 67

This study set out to identify the neurotransmitters involved in autonomic vasodilatation of the guinea pig uterine artery. Non-noradrenergic, paracervical neurons supplying this artery contain at least four neuropeptides: vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), dynorphin A (1-17) and somatostatin, probably in addition to acetylcholine. Transmural nerve stimulation of arterial segments precontracted with phenylephrine (3 x 10(-7) mol l-1 and treated with guanethidine (10(-6) mol l-1), produced relaxations which varied in form with the frequency of stimulation and the length of the pulse train. The relaxations were monophasic at low frequencies (< 2 Hz), and were biphasic at higher frequencies (> 5 Hz) and with longer pulse trains (> 50 pulses). Neither phase of the relaxations was reduced by hyoscine (10(-6) mol l-1), or by removal of the endothelium. The faster phase of the relaxations was selectively reduced (by 61%) during treatment with L-nitro-arginine methyl ester (L-NAME; up to 3 x 10(-5) mol l-1). This reduction was reversed by an excess of L-arginine, indicating that the fast relaxation was mediated by nitric oxide, possibly acting as a neurotransmitter. The slower phase of the neurogenic relaxation was preferentially reduced (by 43%) by the endopeptidase, trypsin (1-3 micrograms.ml-1). As VIP is the only currently identified peptide present in the paracervical neurons which causes vasodilatation, it is likely that VIP, or a closely-related peptide, is the transmitter responsible for the slow relaxation. Acetylcholine and an opioid peptide also seem to be released from the vasodilator neurons, but their effects were small, and may have been restricted to pre-synaptic sites. The slower neurogenic relaxations were inhibited by exogenous neuropeptide Y (68% reduction in amplitude), and were slightly potentiated by somatostatin (21% increase in amplitude). Therefore, endogenous stores of these peptides may also contribute to the sum effect of stimulating the paracervical vasodilator neurons. In conclusion, many different substances may act as autonomic co-transmitters from these pelvic vasodilator neurons.
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PMID:Co-transmission from autonomic vasodilator neurons supplying the guinea pig uterine artery. 809 24


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