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)

During earlier examination of interleukin-1 (IL-1)-induced matrix metalloproteinase gene expression in human gingival fibroblasts a highly induced immediate early gene, I kappa B-alpha, a NF kappa B DNA-binding inhibitor, was identified. The aim now was to investigate whether recombinant (r)IL-1 beta induces the stimulation of NF kappa B and its inhibitor proteins in human gingival fibroblasts and to understand if inhibition of its activity affects collagenase gene expression. Primary gingival fibroblasts (human) were treated with rIL-1 beta to determine the effect on NF kappa B-like DNA-binding activity. IL-1 induced the production of steady-state mRNA levels of I kappa B-alpha in the cultured fibroblasts. Nuclear run-on transcription studies demonstrated that rIL-1 induction of I kappa B-alpha may be transcriptionally regulated. Using electrophoretic mobility gel-shift assays it was shown that rIL-1 activates NF kappa B-like, DNA-binding activity in these fibroblasts. NF kappa B-like DNA-binding activity was rapidly induced and turned over in gingival fibroblasts with peak activity at 30 min after rIL-1 treatment. Further, treatment with chymotrypsin protease inhibitor and antioxidant inhibitor prevented IL-1-induced, NF kappa B-like, DNA-binding activity and collagenase mRNA production. When coupled with the existence of NF kappa B consensus DNA-binding sites on the collagenase gene promoter, these findings suggest that the stimulation of NF kappa B in gingival fibroblasts by rIL-1 could play an important part in the regulation of their collagenase gene expression. The ability of IL-1 to stimulate this expression may define a pivotal role for this cytokine in the pathogenesis of periodontitis.
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PMID:Association of interleukin-1-induced, NF kappa B DNA-binding activity with collagenase gene expression in human gingival fibroblasts. 880 9

The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. Recombinant C domain (rC domain) (Gly417-Cys631) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted. As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and MatrigelTM were not bound by the rC domain. Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen. Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound. However, binding to heparin and fibronectin (Kd, 1.1 x 10(-6) M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (Kd values, approximately 4-6 x 10(-7) M). The Ca2+ ion, but not the potential structural Zn2+ ion, were found to be essential for maintaining the binding properties of the protein. The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion chelator 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn2+ ion in the rC domain. In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca2+ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important in maintaining the structure and function of the domain.
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PMID:The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding. Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components. 905 49

A bifunctional alpha-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited alpha-amylase from germinating rice seeds weakly. It inhibited rice alpha-amylase more than barley alpha-amylase, and the inhibition of rice alpha-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian alpha-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.
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PMID:Rice bifunctional alpha-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds. 964 30

In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.
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PMID:Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3. 984 7

Clinical situations with release of proteinases from blood cells or tissues into the circulation may result in a marked decrease of blood proteinase inhibitor content which in turn may result in a capillary leak syndrome, shock and even in exitus letalis. Replenishment of blood proteinase inhibitors is of benefit in such situations. In this study the inhibitory potential of fresh plasma, fresh frozen plasma and the liquid plasma protein preparation Biseko has been tested with the following enzymes: human leukocyte elastase, human plasmin, human matrix metalloproteinase-9, bovine trypsin, bovine chymotrypsin and rat liver lysosomal cathepsins. The concentration of the blood protein preparations resulting in 50% inhibition of constant amounts of each of the enzymes has been determined by plotting residual activity vs. log of concentration of blood protein preparation in enzyme assays. From these IC50 values inactivation ratios for 1 mg and 1 ml of the blood protein preparations was calculated. These inactivation ratios show an equal suitability of fresh plasma, fresh frozen plasma or full plasma proteins for replenishment of plasma proteinase inhibitory potential in vitro. As additional finding, commercial preparations of human serum albumin exert a surprisingly high inhibitory potential to lysosomal cathepsins.
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PMID:Comparative determination of the antiproteolytic potential of therapeutically used blood protein preparations. 1056 70

Ligneous conjunctivitis (LC) is a rare disease of unknown etiology characterized by the growth of "woody" plaques on ocular and extraocular mucosa. These lesions are comprised of fibrin and both direct and indirect evidence implicates hypofibrinolysis as the primary defect in LC. To further elucidate the pathophysiology of LC we investigated the biochemical aspects of ligneous lesions with respect to the fibrinolytic system. Ligneous lesions were obtained from the right eye of a 15 year-old female patient with longstanding LC since age 2.5 year. Ligneous conjunctivitis in this patient has exhibited a chronic recurrent coarse and has involved multiple muscosal sites. Samples analyzed included an abundant mucoid thread from the conjunctival fornix and the ligneous plaque attached to the inferior tarsus. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to characterize protein profiles and by a variety of zymographic methods to visualize fibrinolytic enzymes. We found that mucoid and ligneous samples were distinct entities. Specifically, ligneous samples contained polypeptides with electrophoretic profiles characteristic of intact fibrin, and were replete in fibrin-bound tissue plasminogen activator (t-PA). Despite the presence of ample t-PA, ligneous samples were essentially devoid of fibrinolytic activity. In contrast, neither proteins nor t-PA could be detected in mucoid samples when fractionated by 7.5-15% SDS-PAGE or analyzed by fibrin zymography, respectively. Despite the absence of t-PA, mucoid samples were replete in fibrinolytic activity. This activity was plasminogen independent, heterogenous and inhibited by PMSF. Degradation profiles suggested that this activity represented in part alpha-chymotrypsin, consistent with this patient's treatment regime, as well as plasmin, elastase and an unidentified neutrophil-derived activity. Interestingly, ligneous samples contained both latent and activated forms of matrix metalloproteinase-9 (MMP-9), whereas mucoid samples contained predominantly activated forms of MMP-9. LC is characterized by defective fibrinolysis, despite the presence of ample t-PA and intact fibrin, and by an abundant mucoid thread which binds both endogenous and exogenous enzymes including serine protease(s) and collagenase(s). The implications of these results with respect to a role for exuberant mucus production or abnormal mucins in the development of a relative mucosal-site specific plasmin(ogen) deficiency is discussed.
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PMID:Ligneous conjunctivitis: biochemical evidence for hypofibrinolysis. 1070 63

The assay for the cross-linked carboxyterminal telopeptide of type I collagen (ICTP) has been shown to reflect increased type I collagen degradation in such pathological conditions as bone metastases and rheumatoid arthritis, but to be rather insensitive to the changes in physiological bone collagen turnover (e.g., induced by estrogen or bisphosphonate treatment). To determine the reasons for this discrepancy we localized the antigenic determinant recognized by the ICTP assay and studied the effects of two major osteoclastic proteinases, cathepsin K (EC 3.4.22.38) and matrix metalloproteinase-9 (MMP-9; gelatinase B; EC 3.4.24.35), on immunoreactivity. The antigenic determinant was shown to reside within the hydrophobic phenylalanine-rich regions of the carboxyterminal telopeptides of the two alpha1 chains of human type I collagen, situated between the triple helical domain and the lysine-derived trivalent cross-link. This conclusion was based on differences between the amino acid sequences and cross reactivities of the corresponding human and bovine antigens before and after proteolytic treatments with chymotrypsin. A trivalent cross-link is necessary for providing such a structure, because the divalently cross-linked and monomeric natural and synthetic peptides from the same region, but containing only one phenylalanine-rich sequence, showed poor immunoreaction. Recombinant human cathepsin K cleaved the trivalently cross-linked ICTP structure at two sites between the phenylalanine-rich region and the cross-link, destroying the reactivity with ICTP antibodies. On the contrary, the treatment of isolated ICTP by the matrix metalloproteinases MMP-9 (gelatinase B), MMP-1 (collagenase 1), or MMP-13 (collagenase 3) had no effect on the immunoreaction. Our results indicate that the increased circulating concentrations of ICTP found in several clinical situations are most likely produced by matrix metalloproteinases, whereas cathepsin K-mediated, osteoclastic bone resorption destroys ICTP antigenicity.
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PMID:Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: loss of antigenicity by treatment with cathepsin K. 1071 80

This article reviews the current status of research on proteases of the enamel layer that are capable of processing and degrading proteins of the enamel matrix. Following a brief survey of the historical development of this discipline, a summary is presented of the current status. Two proteases have recently been cloned: EMSP-1 (enamel matrix serine protease-1), a serine protease, and enamelysin, a metalloprotease. These two are placed into their appropriate families: the chymotrypsin family S1 of clan SA of the serine protease class and the matrixin family or matrix metalloproteinase family, M10 of clan MB (the metzincins) of the metalloprotease class. The major features of these two families are outlined. The article concludes with some suggested areas for future research--identifying further proteases and characterizing those now known.
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PMID:Role of matrix proteases in processing enamel proteins. 1106 89

Our aim was to correlate the activity of matrix metalloproteinases (MMPs) with denaturation and the turnover of collagen in normal and pathological human tendons. MMPs were extracted from ruptured supraspinatus tendons (n=10), macroscopically normal ("control") supraspinatus tendons (n=29) and normal short head of biceps brachii tendons (n=24). Enzyme activity was measured using fluorogenic substrates selective for MMP-1, MMP-3 and enzymes with gelatinolytic activity (MMP-2, MMP-9 and MMP-13). Collagen denaturation was determined by alpha-chymotrypsin digestion. Protein turnover was determined by measuring the percentage of D-aspartic acid (% D-Asp). Zymography was conducted to identity specific gelatinases. MMP-1 activity was higher in ruptured supraspinatus compared to control supraspinatus and normal biceps brachii tendons (70.9, 26.4 and 11.5 fmol/mg tendon, respectively; P<0.001). Gelatinolytic and MMP-3 activities were lower in normal biceps brachii and ruptured supraspinatus compared to control supraspinatus (gelatinase: 0.18, 0.23 and 0.82 RFU/s/mg tendon respectively; P<0.001; MMP-3: 9.0, 8.6 and 55 fmol/mg tendon, respectively; P<0.001). Most gelatinase activity was shown to be MMP-2 by zymography. Denatured collagen was increased in ruptured supraspinatus compared to control supraspinatus (20.4% and 9.9%, respectively; P<0.001). The % D-Asp content increased linearly with age in normal biceps brachii but not in control supraspinatus and was significantly lower in ruptured supraspinatus compared to age-matched control tendons (0.33 and 1.09% D-Asp, respectively; P<0.01). We conclude that the short head of biceps brachii tendons show little protein turnover, whereas control supraspinatus tendons show relatively high turnover mediated by the activity of MMP-2, MMP-3 and MMP-1. This activity is thought to represent a repair or maintenance function that may be associated with an underlying degenerative process caused by a history of repeated injury and/or mechanical strain. After tendon rupture, there was increased activity of MMP-1, reduced activity of MMP-2 and MMP-3, increased turnover and further deterioration in the quality of the collagen network. Tendon degeneration is shown to be an active, cell-mediated process that may result from a failure to regulate specific MMP activities in response to repeated injury or mechanical strain.
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PMID:Matrix metalloproteinase activities and their relationship with collagen remodelling in tendon pathology. 1185 34

Degradation of the extracellular matrix by proteolytic enzymes is a central aspect of physiological and pathologic tissue-remodeling processes such as trophoblastic implantation, wound healing, and tumor invasion. We have hypothesized that prostate adenocarcinoma cell invasion through the normal basal lamina is attributable in part to metalloproteinase-induced cleavage of laminin-5 (Ln-5) and enhanced motility of the cancer cells. We studied the role of membrane type-1-matrix metalloproteinase (MT1-MMP) expressed on the surface of prostate tumor cells in cleaving Ln-5 and enhancing the migration of prostate tumor cells. We also determined the nature of the MT1-MMP cleavage of human Ln-5 and how this altered Ln-5 changes the migration of prostate carcinoma cells. We found that human MT1-MMP cleaves purified human Ln-5 to an 80-kDa fragment. Mass spectrometry analyses of the 80-kDa cleaved product by trypsin and chymotrypsin gave 14 and 9 different peptide sequences, respectively, that were identical to the expected amino acid sequence of the Ln-5-beta3 chain. The recovered peptides represent 14.4% (trypsin) and 10.3% (chymotrypsin) of Ln-5-beta3 chain by amino acid count. Both trypsin and chymotrypsin digestion of MT1-MMP-cleaved product of Ln-5 did not show any other peptides that were identical to the other chains of Ln-5. Using a linear migration assay we found that the Ln-5 cleaved by MT1-MMP enhanced the migration of DU-145 prostate carcinoma cells by 2-fold compared with uncleaved Ln-5. The use of blocked antisense MT1-MMP oligonucleotides inhibited the migration of DU-145 cells on Ln-5. We also found that the prostate carcinoma cells expressing high levels of MT1-MMP, such as PC3N and PPC, demonstrated enhanced migration on human Ln-5-coated substrate, and this migration was inhibited using blocked antisense MT1-MMP oligonucleotides. In conclusion, this is a novel and important finding where we have shown that beta3-chain is cleaved by MT1-MMP, and this cleavage enhances migration of prostate cancer cells.
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PMID:Membrane type-1-matrix metalloproteinase expressed by prostate carcinoma cells cleaves human laminin-5 beta3 chain and induces cell migration. 1272 52


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