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)

Vasoactive intestinal polypeptide (VIP) interaction with the rat brain synaptic membrane was examined using 125I-labeled VIP as a tracer molecule. Ion, pH and incubation time significantly influenced VIP receptor binding. Scatchard analysis suggested that the rat brain membrane had a single binding site with an apparent dissociation constant (Kd) of 9.8 X 10(-9) M. The receptor activity was sensitive to trypsin and phospholipase A digestion, and heating at 100 degrees C for 5 min completely destroyed the binding activity. This indicates that protein and phospholipid moieties are essential for VIP binding. Thiol reagents reduced receptor binding activity, suggesting that an intrachain disulfide bond might be an important constituent of the VIP binding site. High levels of binding were found in the hippocampus, striatum and cerebral cortex, and binding was very low in the medulla/pons and cerebellum. The receptor density did not always parallel the brain distribution of immunoreactive VIP (IR-VIP) concentration. The cerebral cortex had the highest ratio of IR-VIP-to-receptor, and the striatum had the lowest ratio of IR-VIP-to receptor. Although intra-nigral or intra-striatal injection of 6-hydroxydopamine had no effect on striatal VIP-binding, an intra-striatal injection of kainic acid resulted in a substantial lowering of striatal VIP receptors. The neurotoxic effects of kainic acid have been shown to be dependent on the corticostriatal tract, and this suggests that the striatum receives the VIPergic innervation from the cerebral cortex. Our findings indicate that endogenous VIP and VIP receptors might act as a neurotransmission modulator of extrapyramidal function in the striatum.
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PMID:Properties and distribution of vasoactive intestinal polypeptide receptors in the rat brain. 299 38

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

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

The purpose of this study was to determine whether encapsulation of vasoactive intestinal peptide (VIP) in liposomes enhances its vasoactive effects. Liposomes were formed from a solution of VIP in phospholipids and cholesterol, resulting in incorporation of 0.008 mole peptide/mole phospholipid. Leakage of VIP from the liposomes was undetectable over several days of incubation at 4 degrees C in 0.15 M sodium chloride. Under conditions permitting rapid hydrolysis of VIP by trypsin, there was no breakdown of the encapsulated peptide. Increasing concentrations of the liposome-encapsulated VIP administered intravenously to anesthetized hamsters produced a concentration-dependent decrease in the mean arterial blood pressure. The duration and magnitude of the hypotensive effect of the encapsulated VIP was significantly greater (p < 0.05) compared to equivalent concentrations of the unencapsulated peptide. Infusion of empty liposomes was without significant effect on the mean arterial blood pressure. We conclude that encapsulation of VIP in liposomes potentiates the blood pressure-lowering effect of the peptide.
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PMID:Vasoactive intestinal peptide encapsulated in liposomes: effects on systemic arterial blood pressure. 815 24

Association of stress with psoriatic skin symptoms was studied in 13 patients with psoriasis by dividing the patients into low- and high-stress groups based on their clinical examination and answers to three questionnaires (General Health Questionnaire, a somatization scale, and a life change questionnaire). This study focused on skin mast cells and sensory nerves which are the principal components in neurogenic inflammation. Mast cells were stained enzyme-histochemically for tryptase and chymase, and neuropeptides substance P (SP), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP) were demonstrated immunohistochemically. Compared to the low-stress group (n = 7), the patients in the high-stress group (n = 6) had more severe skin and joint symptoms. Furthermore, mast cells positive for chymase activity were prominently reduced, but tryptase-positive mast cells only slightly decreased in the lesional skin of the high-stress group. A similar tendency was also observed in the nonlesional skin. In the papillary dermis of the lesional skin, both VIP- and CGRP-immunoreactive nerves could be observed in the high-stress group whereas in the low-stress group these nerve fibers were hardly visible in the corresponding area. No association of SP with stress was observed. This study suggests that psychic stress is associated with exacerbation of psoriasis, and stress may induce alterations in the psoriatic lesions by increasing the neuropeptide content with a concomitant decrease in the activity of neuropeptide-degrading enzymes, especially mast cell chymase.
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PMID:Association of cutaneous mast cells and sensory nerves with psychic stress in psoriasis. 827 75

Vasoactive intestinal polypeptide (VIP) is a neurotransmitter of nonadrenergic noncholinergic (NANC) nerves in the cat gastric fundus. A possible additional role for nitric oxide (NO) was investigated in circular and longitudinal muscle strips from this tissue. Incubation with the inhibitor of NO-synthesis, NG-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) M), inhibited the relaxant response to both short- and long-lasting electrical field stimulation. The substrate for NO synthesis, L-arginine (2 x 10(-3) M), prevented this inhibition. In experiments with long-lasting electrical field stimulation, trypsin (3 x 10(-6) M) and L-NAME inhibited the beginning of the NANC relaxation to the same extent, but the inhibition by trypsin was more pronounced at the end of the stimulation period. The inhibitory effect of L-NAME and trypsin was additive. L-NAME did not influence the relaxations induced by VIP (10(-7) M), NO (10(-5) M) and isoprenaline (3 x 10(-6) M). NG-nitro-L-arginine (3 x 10(-4) M) also did not change the response to VIP. The relaxation induced by 10(-5) M NO was not transient but was sustained. The plateau phase of this relaxation was reduced by trypsin but not by 3 x 10(-6) M tetrodotoxin. It is concluded that NO is involved in NANC relaxation of the cat gastric fundus. During sustained NANC relaxation in the cat gastric fundus, cotransmission of NO and VIP is possible.
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PMID:Involvement of the L-arginine: nitric oxide pathway in nonadrenergic noncholinergic relaxation of the cat gastric fundus. 833 56

Tryptase and chymase released from activated mast cells degrade the neuropeptides calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) to peptide fragments. We have examined whether nedocromil sodium can modulate the ability of rat activated peritoneal mast cells to degrade 125I-CGRP and 125I-VIP. Mast cell-dependent degradation of both 125I-CGRP and 125I-VIP was observed with compound 48/80 (0.03-1 microgram/ml) and in the case of 125I-VIP with anti-IgE (1-20 micrograms/ml). Nedocromil sodium (10(-6)-10(-4) M) caused significant inhibition of neuropeptide degradation, with the most effective inhibition observed against anti-IgE-induced degradation of 125I-VIP. Nedocromil sodium had no inhibitory effect on the ability of lysed mast cells, bovine trypsin or chymotrypsin to breakdown 125I-VIP. These results suggest that nedocromil sodium inhibits mast cell-dependent degradation of neuropeptides, such as VIP, as a secondary consequence of inhibiting the release of mast cell proteases.
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PMID:The modulation by nedocromil sodium of proteases released from rat peritoneal mast cells capable of degrading vasoactive intestinal peptide and calcitonin gene-related peptide. 839 43

A rapid, sensitive, semiautomated method to measure the activity of mast cell-derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N-terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While the Km value was unaffected, the kcat value decreased, yielding a kcat/Km ratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in the kcat/Km ratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.
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PMID:High-performance liquid chromatographic assay for tryptase based on the hydrolysis of dansyl-vasoactive intestinal peptide. 861 98

Extracts from ragweed pollen grains contain novel trypsin and chymotrypsin-like serine peptidases which are described in this report. The molecular mass of the chymotrypsin-like enzyme was 82 kDa, had a pH optimum near 9.0, and its activity was unaffected by chelating or reducing agents. It was inhibited by diisopropyl fluorophosphate (DFP), a general serine class inhibitor, and more specifically N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like proteinase inhibitor. In addition to various synthetic substrates, the neuropeptides, vasoactive intestinal peptide (VIP) and substance P, which are required for normalized lung functions, were also rapidly hydrolysed. Activity toward protein substrates was not detected with the exception of the inactivation of alpha-1-proteinase inhibitor (alpha-1-PI) which occurred through cleavage within the reactive site loop. The 'trypsin-like' enzyme has a molecular mass near 80 kDa, a blocked N-terminus, a pH optimum near 9.0, and requires Ca++ for stability and activity, but not reducing agents. It is inhibited by DFP, and more specifically the trypsin-like proteinase inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK). Again, activity toward protein substrates was not detected, but various synthetic substrates and biologically active peptides were efficiently cleaved. Significantly, atrial natriuretic peptide (ANP) and angiotensin 2 (ATII), whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed.
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PMID:Ragweed pollen proteolytic enzymes: possible roles in allergies and asthma. 946 75


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