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)

Human liver type III collagen was prepared by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type III collagen chains yielded nine distinct peptides. Three peptides, alpha1(III)-CB3, alpha1(III)-CB7, and alpha1(III)-CB6, were isolated by carboxymethylcellulose chromatography and Sephadex G-50 SF gel filtration. Automated Edman degradation together with selective hydroxylamine cleavage and chymotrypsin and trypsin digestion enabled determination of their complete amino acid sequence. Compared with type I collagen, the data show tentative homology of alpha1(III)-CB3 with alpha1(I)-CB1, alpha1(I)-CB2, and alpha1(I)-CB4; alpha1(III)-CB7 with alpha1(I)-CB5; and alpha1(III)-CB6 with the amino-terminal portion of alpha1(I)-CB8. Close interspecies homology was found between the sequences presented here with 90 residues of alpha1(III)-CB3 and 26 of alpha1(III)-CB8 of calf aorta. The present study establishes the amino acid sequence of 229 residues near the amino terminus or nearly one-quarter of the type III collagen chains. The disaccharide, Glc-Gal, was convalently bound to hydroxylysine at a position corresponding to the same location in the alpha1(I) chain.
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PMID:Covalent structure of collagen: amino acid sequence of cyanogen bromide peptides from the amino-terminal segment of type III collagen of human liver. 55 35

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
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PMID:Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. 140 Apr 81

We report a case of mild osteogenesis imperfecta in a 56-year-old male undergoing aortic valve replacement surgery. The primary defect in this patient was the substitution of arginine for glycine 85 in one of the two chains of alpha 1(I) procollagen. The thermal stability of the type I collagen synthesized by the patient's cultured skin fibroblasts was examined by enzymatic digestion. Digestion of the mutant type I collagen with trypsin and chymotrypsin at increasing temperatures sequentially generated three discrete collagenous fragments, approximately 90, 170, and 230 amino acids shorter than normal type I collagen. This incremental thermal denaturation is indicative of cooperative melting blocks within the type I collagen. This is the first demonstration of such cooperative blocks of melting in intact, essentially normal post-translationally modified type I collagen. This direct evidence for cooperative melting domains of uncut type I collagen suggests that discrete blocks of amino acids function as core sites stabilizing the collagen helix. The location of mutations of the alpha chains of type I collagen relative to these discrete blocks of amino acids may influence the severity of the disease phenotype.
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PMID:The substitution of arginine for glycine 85 of the alpha 1(I) procollagen chain results in mild osteogenesis imperfecta. The mutation provides direct evidence for three discrete domains of cooperative melting of intact type I collagen. 171 84

Saliva collected from subjects with healthy and with diseased periodontium was assayed for collagenase activity by incubation at 25 degrees C with soluble type I, II or III collagen. The degradation products were analyzed by separation in SDS-polyacrylamide gel electrophoresis followed either by protein staining or by exposure of the dried gel to X-ray film in the case of radioactively labeled type I collagen. Collagenase of vertebrate type was detected in the whole saliva of all subjects but not in parotid, sublingual or submandibular fluids. Most of the collagenase was in the soluble fraction of saliva that also contained factors which both activated and inhibited the enzyme. The salivary collagenase resembled the collagenase of human PMNs and gingival sulcular fluid in its molecular size of 70,000 daltons, in its activation by gold thioglucose and in its tendency to degrade types I and II collagens over type III collagen. Before periodontal treatment, the saliva of periodontitis patients had significantly higher collagenase than after treatment. In periodontitis, collagenase existed mainly in the active form, while in the healthy mouths most of the enzyme was latent but could be activated by sulfhydryl reagents or proteolytically with trypsin, and chymotrypsin but not by human plasma kallikrein or plasmin. In some of the samples from untreated periodontitis patients bacterial collagenase may have been present in small quantities. Most of the collagenase in the saliva from all subjects appeared to originate from PMNs entering the oral cavity through the gingival sulcus.
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PMID:Salivary collagenase. Origin, characteristics and relationship to periodontal health. 216 44

The chymotrypsinlike protease gene (prtA) from Treponema denticola ATCC 35405 was isolated from a lambda gt11 clone bank as one of several clones expressing protease activity. The DNA from one positive clone capable of hydrolyzing type IV collagen was subcloned into plasmid vector pUC119 for further analysis. Deletion analysis of subclone pXQ27.2 revealed the approximate location of the prtA gene on the DNA insert. DEAE-Sephadex chromatography of crude cell extracts of the subclone revealed two distinct T. denticola enzymes, one hydrolyzing SAAPNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide [chymotrypsin substrate]) and the other hydrolyzing PZ-PLGPA (phenylazobenzyl-oxycarbonyl-L-leucylglycyl-L-prolyl-D -arginine [collagenase substrate]). Each activity was purified to near homogeneity and exhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis estimated molecular sizes of 67 and 36 kDa, respectively. Western blot (immunoblot) analysis demonstrated that only the 67-kDa SAAPNA-hydrolyzing enzyme reacted with antibody against the T. denticola chymotrypsinlike protease. The purified SAAPNA-hydrolyzing enzyme degraded type IV collagen, laminin, and fibronectin, but not type I collagen. These results indicate that the prtA gene coding for the chymotrypsinlike protease from T. denticola has been isolated. Another distinct gene encoding an enzyme hydrolyzing PZ-PLGPA appears to be adjacent to the prtA gene.
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PMID:Isolation and characterization of the Treponema denticola prtA gene coding for chymotrypsinlike protease activity and detection of a closely linked gene encoding PZ-PLGPA-hydrolyzing activity. 217 32

The existing forms of neutral proteases present in inflamed human gingiva were examined. Neutral 2 M K Cl extracts of inflamed human gingival tissue were fractionated by gel filtration on Sephacryl S-200 and the fractions were assayed for collagenase, trypsin-, chymotrypsin-, and elastase-like proteases. Apparent molecular weights of 80-85 kDa were obtained for trypsin-, chymotrypsin-, and elastase-like proteases, and 70-75 kDa for latent collagenase. Further fractionation of high molecular weight proteases on Con A-Sepharose revealed that, unlike collagenase, chymotrypsin- and elastase-like proteases, the trypsin-like protease was bound by the affinity column. Native human placental type IV (basement membrane) collagen was degraded by chymotrypsin-like and elastase-like proteases but not by the trypsin-like protease. This degradation was inhibited by phenylmethyl sulfonyl fluoride and EDTA. The serine proteases also degraded efficiently denatured type I collagen. No correlation of the activities of trypsin-like protease and the other proteolytic enzymes was found in extracts of 18 individual gingival specimens. Significant correlation, however, was noted between collagenase and gelatinase. The gingival culture studies showed that, while the highest activity of the trypsin-, chymotrypsin-, and elastase-like enzymes were measured in medium during first days of the culture, collagenase and gelatinase activities increased up to the fourth day of culture and stayed high until the end of the culture. These results suggest that the neutral proteases that may participate in the periodontal tissue destruction are produced by different cell types of gingiva.
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PMID:Characteristics of neutral proteases present in inflamed human gingiva. 255 68

The binding of highly purified bovine aortic lysyl oxidase to native fibrils of type I collagen has been measured by assay of unbound lysyl oxidase activity in the supernatants of enzyme-collagen mixtures after centrifugation. The apparent binding affinity of lysyl oxidase for native fibrils is quite similar to that for fibrils prepared from pepsin- or chymotrypsin-digested type I collagen, demonstrating that the enzyme binds to the triple-helical portion of collagen molecules. The data also indicate that the enzyme binds predominantly to the fibrillar surface. The results suggest that lysyl oxidase initiates crosslink formation at an early stage in collagen fibrillogenesis.
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PMID:Binding of lysyl oxidase to fibrils of type I collagen. 286 30

Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain. 295 85

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of Mr 80,000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.
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PMID:Identification of a surface protein of the rabbit blood platelet with high affinity for collagen. 302 23

Sporothrix schenckii, mainly in the yeast form of the organism, produced extracellular proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing medium was performed by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-200. Proteinase I had a molecular weight of 36,500, an optimal pH at 6.0, and a pI at 4.8. Despite its activities in weakly acidic conditions, proteinase I demonstrated chymotrypsinlike characteristics, these being indicated by strong inhibitory activity by phenylmethylsulfonyl fluoride and chymostatin and good kinetic constants for a synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA. Proteinase II had a molecular weight of 39,000, an optimal pH at 3.5, and a pI at 3.8. Proteinase II showed cathepsin D-like characteristics, these being indicated by strong inhibitory activity by pepstatin, an acidic optimal pH, and good kinetic constants for hemoglobin. These two enzymes hydrolyzed natural substrates such as stratum corneum, type I collagen, and elastin although not type IV collagen. Proteinase production and cell growth in collagen-containing medium and the enzymatic digestion of skin constituents by isolated proteinases suggested that these two proteinases cooperatively enable the organism to invade skin and to obtain peptides from insoluble proteins.
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PMID:Isolation and properties of extracellular proteinases from Sporothrix schenckii. 330 79


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