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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been shown that specific trypsin inhibitors exhibit also antichymotrypsin activity in the presence of high NaCl concentrations. Taking advantage of this phenomenon a simple procedure of separation of the virgin forms of trypsin inhibitors from squash seeds and porcine pancreas (Kazal) was elaborated. In a typical experiment the inhibitor sample was loaded onto immobilized chymotrypsin equilibrated with 5 M NaCl at pH 8. After washing out unadsorbed material the virgin forms of inhibitors could be eluted either with water, buffer pH 8.0 or 0.02 M citrate buffer pH 2.6 containing no NaCl.
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PMID:High concentrations of sodium chloride facilitate the use of immobilized chymotrypsin for separating virgin forms of specific trypsin inhibitors. 1048 Feb 47

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.
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PMID:Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1. 1056 88

The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.
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PMID:Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome. 1078 26

alpha(1)-Antichymotrypsin is a member of the serine proteinase inhibitor, or serpin, family that typically forms very long-lived, enzymatically inactive 1:1 complexes (denoted E*I*) with its target proteinases. Serpins share a conserved tertiary structure, in which an exposed region of amino acid residues (called the reactive center loop or RCL) acts as bait for a target proteinase. Within E*I*, the two proteins are linked covalently as a result of nucleophilic attack by Ser(195) of the serine proteinase on the P1 residue within the RCL of the serpin. This species is formally similar to the acyl enzyme species normally seen as an intermediate in serpin proteinase catalysis. However, its subsequent hydrolysis is extremely slow as a result of structural changes within the enzyme leading to distortion of the active site. There is at present an ongoing debate concerning the structure of the E*I* complex; in particular, as to whether the enzyme, bound to P1, maintains its original position at the top of the serpin molecule or instead translocates across the entire length of the serpin, with concomitant insertion of RCL residues P1-P14 within beta-sheet A and a large separation of the enzyme and RCL residue P1'. We report time-resolved fluorescence energy transfer and rapid mixing/quench studies that support the former model. Our results indicate that the distance between residue P1' in alpha(1)-antichymotrypsin and the amino terminus of chymotrypsin actually decreases on conversion of the encounter complex E.I to E*I*. These results led us to formulate a comprehensive mechanism that accounted both for our results and for those of others supporting the two different E*I* structures. In this mechanism, partial insertion of the RCL, with no large perturbation of the P1' enzyme distance, is followed by covalent acyl enzyme formation. Full insertion can subsequently take place, in a reversible fashion, with the position of equilibrium between the partially and fully inserted complexes depending on the particular serpin-proteinase pair under consideration.
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PMID:Formation of the covalent chymotrypsin.antichymotrypsin complex involves no large-scale movement of the enzyme. 1102 95

Myoepithelial cells surround incipient ductal carcinomas of the breast and exert anti-invasive and antiangiogenic effects in vitro through the elaboration of suppressor molecules. This study examines one putative molecule, solubilized CD44 produced by myoepithelial shedding of membrane CD44. Studies with different human myoepithelial cell lines demonstrate that myoepithelial cells express and shed both the 85-kDa standard (CD44s) and the 130-kDa epithelial (CD44v8-10) isoforms, findings further confirmed by the use of isoform-specific antibodies. PMA pretreatment enhances CD44 shedding detected by two different methods at different time points: a reduction in surface CD44 at 2 h by flow cytometry and a marked decrease in both total cellular CD44 and plasma membrane CD44 at 12 h by Western blot. This shedding is both specific for CD44 and specific for myoepithelial cells. This shedding is inhibited by the chymotrypsin inhibitors chymostatin and alpha(1)-antichymotrypsin but not by general metallo-, cysteine, or other serine proteinase inhibitors. Myoepithelial-cell-conditioned medium and affinity-purified solubilized CD44 from this conditioned medium block hyaluronan adhesion and migration of both human carcinoma cell lines and human umbilical vein endothelial cells.
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PMID:Myoepithelial-specific CD44 shedding contributes to the anti-invasive and antiangiogenic phenotype of myoepithelial cells. 1108 85

Our previous studies have demonstrated that myoepithelial cells, which surround incipient carcinomas in situ of the breast and other organs, exert antiinvasive and antiangiogenic effects in vitro through the elaboration of a number of different suppressor molecules among which include the shed membrane CD44. The present study addresses the mechanism of this myoepithelial CD44 shedding. This CD44 shedding is enhanced by PMA pretreatment, is specific for myoepithelial CD44, and inhibited by chymotrypsin-like inhibitors (chymostatin, alpha(1)-antichymotrypsin, TPCK, and SCCA-2) but not by trypsin-like inhibitors (TLCK), nor papain-like inhibitors (SCCA-1) nor hydroxamate-based or general metalloproteinase inhibitors (BB2516 (marimastat), 1,10-phenanthroline, and TIMP-1). The effect of PMA can be mimicked by exogenous chymotrypsin but not by other proteases. The CD44 shedding activity cannot be transferred by conditioned media, cell-cell contact, peripheral membrane, or integral membrane fractions. However, cell-free purified integral plasma membrane fractions obtained from myoepithelial cells pretreated with PMA also exhibit CD44 shedding which is inhibited by chymotrypsin-like inhibitors. These findings support the presence and activation of a putative chymotrypsin-like sheddase as the mechanism of CD44 shedding in myoepithelial cells.
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PMID:Myoepithelial-specific CD44 shedding is mediated by a putative chymotrypsin-like sheddase. 1111 26

Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid beta (A beta), are considered to be central to the pathology of Alzheimer's disease (AD). Soluble A beta is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble A beta present in circulation can cross the blood-brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that A beta originating from circulation may contribute to vascular and parenchymal A beta deposition in AD. Enhancing of A beta catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble A beta. To launch A beta clearance we have exploited the A beta-degrading activity of diverse alpha 2-macroglobulin (alpha 2-M)-proteinase complexes. Complexes with trypsin, alpha-chymotrypsin, and bromelain strongly degrade (125)I-A beta 1--42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. A beta degradation by the complexes was not inhibited by alpha 1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for A beta degradation is its binding to specific binding sites in alpha 2-M that may direct A beta to the active site of the caged proteinase. Ex vivo, enhanced degradation of (125)I-A beta 1--42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of A beta catabolism could probably reduce the risk of developing AD by preventing A beta accumulation in brain and vasculature.
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PMID:Alpha 2-macroglobulin-mediated degradation of amyloid beta 1--42: a mechanism to enhance amyloid beta catabolism. 1116 27

Prostate specific antigen, the clinical marker for prostate cancer, is a neutral serine protease whose function is to lyse seminal proteins. Recent work by our laboratory has suggested that prostate specific antigen stimulates the generation of reactive oxygen species in prostate cancer cells. Using 2',7'-dichlorofluorescin diacetate, a dye that fluoresces in the presence of hydrogen peroxide or hydroxyl radicals, we found that prostate specific antigen markedly stimulated reactive oxygen species generation in LNCaP cells. The effect was concentration dependent and its specificity was supported by the fact that anti-prostate specific antigen antibodies abolished the response. Since testosterone stimulates the production of prostate specific antigen, we considered that the reactive oxygen species response to testosterone may be linked to prostate specific antigen. We found that the testosterone effect on reactive oxygen species was blocked by flutamide and by anti-prostate specific antigen antibody. Additionally, though PC3 and DU145 could not respond to testosterone, they readily increased reactive oxygen species in response to prostate specific antigen. Focusing on the mechanism of the prostate specific antigen effect, we tested two other serine proteases, trypsin and chymotrypsin, but found no effect on reactive oxygen species in LNCaP cells. Nevertheless, serine protease inhibitors, alpha(1)-antichymotrypsin, alpha(2)-macroglobulin and Bowman-Birk inhibitor, blocked reactive oxygen species generation stimulated by prostate specific antigen. This apparent paradox was investigated with the use of a specific anti-'prostate specific antigen' antibody which recognizes an epitope away from the catalytic site and which does not inhibit protease activity. Despite the lack of inhibition of proteolytic activity, this antibody blocked the effect of prostate specific antigen on reactive oxygen species generation. These findings suggest that although the integrity of the prostate specific antigen molecule is necessary for stimulating reactive oxygen species generation, its proteolytic activity is not. The underlying mechanism is currently under investigation.
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PMID:Testosterone and prostate specific antigen stimulate generation of reactive oxygen species in prostate cancer cells. 1169 38

The large size of the serpin reactive site loop (RSL) suggests that the role of the RSL in protease inhibition is more complex than that of presenting the reactive site (P1 residue) to the protease. This study examines the effect on inhibition of relocating the reactive site (Leu-358) of the serpin alpha(1)-antichymotrypsin either one residue closer (P2) or further (P1') from the base of the RSL (Glu-342). alpha(1)-Antichymotrypsin variants were produced by mutation within the P4-P2' region; the sequence ITLLSA was changed to ITLSSA to relocate the reactive site to P2 (Leu-357) and to ITITLS to relocate it to P1' (Leu-359). Inhibition of the chymotrypsin-like proteases human chymase and chymotrypsin and the non-target protease human neutrophil elastase (HNE) were analyzed. The P2 variant inhibited chymase and chymotrypsin but not HNE. Relative to P1, interaction at P2 was characterized by greater complex stability, lower inhibition rate constants, and increased stoichiometry of inhibition values. In contrast, the P1' variant inhibited HNE (stoichiometry of inhibition = 4) but not chymase or chymotrypsin. However, inhibition of HNE was by interaction with Ile-357, the P2 residue. The P1' site was recognized by all proteases as a cleavage site. Covalent-complexes resistant to SDS-PAGE were observed in all inhibitory reactions, consistent with the trapping of the protease as a serpin-acyl protease complex. The complete loss in inhibitory activity associated with lengthening the Glu-342-reactive site distance by a single residue and the enhanced stability of complexes associated with shortening this distance by a single residue are compatible with the distorted-protease model of inhibition requiring full insertion of the RSL into the body of the serpin and translocation of the linked protease to the pole opposite from that of encounter.
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PMID:The effects of reactive site location on the inhibitory properties of the serpin alpha(1)-antichymotrypsin. 1205 88

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.
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PMID:Novel secretory vesicle serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities. 1243 89


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