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)

Proteoglycan from pig costal cartilage and fragments obtained by proteolytic digestion were characterized by equilibrium ultracentrifugation and amino acid analysis. The proteoglycan extractable in 4 M guanidinium chloride yielded, after proteolytic digestion with trypsin and chymotrypsin, a chondroitin sulfate peptide containing four chains of polysaccharide. The unextractable residue yielded chondroitin sulfate peptide containing only two chains. The amino acid composition indicated a fairly uniform spacing between all four chains with an average of eight amino acid residues between the serine residues involved in linkage. Following the alkaline sulfite elimination-addition reaction, free peptide was isolated and found to contain one unsubstituted serine residue for every two linked glycosidically. Glycine and glutamic acid were the only two amino acids sufficiently abundant to be part of an invariant sequence near to serine residues destined to be glycosylated. The linkage region of the polypeptide also contains some substituted serine residues which do not carry a full chondroitin sulfate chain.
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PMID:The linkage region in the polypeptide of pig costal cartilage proteoglycan. 12 39

After chondroitinase digestion of bovine nasal and tracheal cartilage proteoglycans, subsequent treatment with trypsin or trypsin followed by chymotrypsin yielded two major types of polypeptide-glycosaminoglycan fragments which could be separated by Sepharose 6B chromatography. One fragment, located close to the hyaluronic acid-binding region of the protein core, had a high relative keratan sulfate content. This fragment contained about 60% of the total keratan sulfate, but less than 10% of the total chondroitin sulfate present in the original proteoglycan preparation. The weight average molecular weight of the keratan sulfate-enriched fragment was 122,000, as determined by sedimentation equilibrium centrifugation. The chemical and physical data indicate that this fragment contains an average of 10 to 15 keratan sulfate chains, if the average molecular weight of individual chains is assumed to be about 8,000, and about 5 chondroitin sulfate chains attached to a peptide of about 20,000 daltons. The other population of fragments was derived from the other end of the proteoglycan molecule, the chondroitin sulfate-enriched region, and contained mainly chondroitin sulfate chains. About 90% of the total chondroitin sulfate, but only 20 to 30% of the total keratan sulfate was recovered in these fragments. On the average, approximately 5 chondroitin sulfate chains and 1 keratan sulfate chain could be linked to the same peptide. Another 10 to 20% of the total keratan sulfate, originally found in or near the hyaluronic acid-binding region, was not separated from the chondroitin sulfate-enriched fragments. Hydroxylamine could be used to liberate a large molecular size, chondroitin sulfate-enriched fragment (Kav 0.54 on Sepharose 2B) from the proteoglycan aggregates. The remainder of the protein core, containing the keratan sulfate-enriched region, was bound to hyaluronic acid with the link proteins and recovered in the void volume on the Sepharose 2B column.
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PMID:Distribution of keratan sulfate in cartilage proteoglycans. 13 6

Assays of several proteases, incorporating guanidinium chloride extracts of human femoral head cartilage and intervertebral disc, demonstrated that both tissues contain inhibitors of certain serine proteases. Trypsin, chymotrypsin and a granule extract of human polymorphonuclear leukocytes containing elastase and cathepsin G activities, were inhibited by low molecular weight fractions prepared by Sephadex G-75 chromatography. Using a radioassay, it was further shown that these fractions inhibit proteolysis of cartilage proteoglycan. The inhibitor in intervertebral disc is concentrated in the nucleus pulposus, with a decreasing gradient to the periphery of the annulus fibrosus. It is proposed that these inhibitors confer at least partial protection against pathological proteolysis of the proteoglycans in human articular cartilage and nucleus pulposus.
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PMID:Neutral protease inhibitors from human intervertebral disc and femoral head articular cartilage. 15 73

Monomer proteoglycan was isolated from porcine ovarian follicular fluid by isopycnic CsCl centrifugation in the presence of 4 M guanidine HCl and protease inhibitors. The elution profile of the D1 preparation on Sepharose 2B was similar to that of monomer proteoglycan from bovine nasal cartilage, indicating a similar molecular size. Follicular fluid proteoglycans consist of about 20% protein, 50% dermatan sulfate, and 20% oligosaccharides rich in sialic acid, galactose, mannose, glucosamine, and galactosamine. The amino acid composition of this proteoglycan is significantly different from that of cartilage proteoglycans, with a higher proportion of aspartic acid, threonine, and lysine, and lower amounts of proline and glycine. Alkali-released dermatan sulfate chains are larger on Sepharose 6B (average Mr = 56,000) than chondroitin sulfate chains from cartilage proteoglycans (average Mr = 25,000), and iduronic acid accounts for 9% of total hexuronic acid. Disaccharide units released by chondroitinase ABC consists of 67% 4-sulfated, 22% 6-sulfated, 5% non-sulfated, and 5% disulfated disaccharides. After treatment with 0.05 M NaOH, 1 M NaBH4 at 45 degrees C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to penta- and hexasaccharides. The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfate-peptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.
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PMID:Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. 76

We investigated the effect of ulinastatin, a candidate anti-osteoarthritic drug, in comparison with indomethacin and triamcinolone, two well-known drugs for osteoarthritis, on IL-1 production by monocytes, proteoglycan synthesis by chondrocytes and superoxide generation by leukocytes. Ulinastatin, a glycoprotein purified from human urine, suppressed both the IL-1 production and the IL-1 induced reduction of proteoglycan synthesis. In addition, ulinastatin inhibited superoxide generation. These actions of ulinastatin seemed to be related to its inhibitory actions against serine proteases such as trypsin, alpha-chymotrypsin, plasmin, leukocyte elastase and leukocyte cathepsin G. Triamcinolone suppressed the IL-1 production more potently than ulinastatin and it also suppressed the IL-1 induced reduction of proteoglycan synthesis. Triamcinolone alone, however, reduced the proteoglycan synthesis, and it did not affect the superoxide generation. In contrast, indomethacin had no effect on proteoglycan synthesis and superoxide generation, although it accelerated the IL-1 production. These results indicate that these three drugs have different mechanisms of action on the factors involved in the pathogenesis of osteoarthritis. Since ulinastatin has broad actions, which are considered to be beneficial for preventing some process of osteoarthritic pathogenesis, ulinastatin is expected to be an useful drug for the treatment of osteoarthritis.
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PMID:[Mechanism of the anti-osteoarthritic action of ulinastatin in comparison with those of indomethacin and triamcinolone]. 131 79

Monoclonal antibodies were raised that specifically recognize the NH2-terminal neoepitope sequence present in link protein cleavage products derived from stromelysin-degraded proteoglycan aggregate. Competitive enzyme-linked immunosorbent assay, using synthetic peptides as inhibitors, showed that one of these antibodies (CH-3) required, for antibody recognition, the free NH2-terminal amino acid isoleucine (residue 17 of the intact protein) in the sequence NH2-IQAENG at the stromelysin cleavage site of link protein 3. Human proteoglycan aggregate was digested with recombinant human stromelysin, bovine chymotrypsin, bovine trypsin, and porcine elastase, and their respective link protein degradation products were tested for immunoreactivity with antibody CH-3. Only stromelysin- and chymotrypsin-generated link protein 3 were recognized by antibody CH-3. Both of these enzymes generate link protein NH2 termini with the sequence 17IQAENG. . .; hence these studies indicated that monoclonal antibody CH-3 recognized this neoepitope sequence in only specific proteolytically modified link protein molecules. Since the occurrence of link protein 3 increases with aging, the incidence of CH-3 epitope in proteoglycans isolated from human knee articular cartilage of individuals of different ages was investigated. The prevalence of CH-3 epitope was found to be highest in newborn and adolescent articular cartilage samples. However, little CH-3 epitope was detected in older adult cartilage, although considerably more link protein 3 was present in these samples. These results suggest that additional proteolytic agents are responsible for the increased occurrence of link protein degradation products with aging.
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PMID:Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin. 137 86

Heparan sulphate proteoglycans are rapidly released from VACO 10MS colon cancer cells that are triggered with phorbol esters to undergo terminal differentiation. This lag-free temperature-sensitive process is correlated with a conversion of the lipophilic proteoglycans of the cell surface into non-lipophilic proteoglycans that accumulate in the culture medium. The released proteoglycans are very similar to their lipophilic precursors in size, buoyant density and glycosaminoglycan characteristics; however, they exhibit slightly smaller core proteins after chemical and enzymic deglycosylation. The lipophilicity of the larger-sized core proteins of the cell-associated proteoglycans is also correlated with the presence of an easily iodinatable domain; this domain is missing in the released proteoglycans. Exogenous proteases (i.e. chymotrypsin, V8, trypsin and proteinase K) readily cleave this segment from the larger protease-resistant region of the proteoglycan structure. It is also released intact by treatment of the isolated proteoglycans with methanolic HCl. This component appears to be peptide in character, in that proteases readily degrade it and release iodotyrosines when the precursor has been iodinated. No evidence for the presence of covalently attached fatty acids in the cell-associated proteoglycans was found. These results are consistent with the hypothesis that the altered proteoglycan metabolism that is associated with the phorbol-ester-induced terminal differentiation of certain human colon cancer cells ensues upon the activation of a membrane-localized protease that cleaves a lipophilic anchor segment from the cell surface proteoglycans.
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PMID:Release of cell surface proteoglycans from differentiating colon cells proceeds by cleavage of lipophilic anchor peptides. 141 65

The specific binding and nature of the epitope recognized by monoclonal antibody (Mab) 1H10, which binds an antigen expressed on human cervical tumors, was characterized by enzyme digestion, lectin competition assay and immuno-electron microscopy. Membrane homogenates of CaSki cervical carcinoma cells were digested with various enzymes, then analysed by SDS-PAGE and immunoblotting. Cells grown on coverslips were treated with various enzymes and in situ binding of Mab 1H10 to cells was analysed by electron microscopy. The ability of lectin-conjugates to block Mab 1H10 binding to CaSki cells was also examined. Treatment of samples with sodium periodate abrogated antigen recognition by Mab 1H10. Neuraminidase and hyaluronidase digestion decreased but did not eliminate Mab 1H10 binding to cells in situ. Chondroitinase ABC digestion, in contrast, removed Mab 1H10 binding sites both in vitro and in situ. Trypsin and chymotrypsin digestion of cell membrane homogenates decreased the molecular weight of the Mab 1H10 antigen but did not decrease the binding intensity. Wheat germ agglutinin (WGA) strongly bound to CaSki cells and partially blocked Mab 1H10 binding, indicating that the antigen contains N-acetyl-galactosamine residues at or near the epitope recognized by Mab 1H10. Ricinus communis agglutinin (RCA) exhibited a similar binding pattern to WGA. However, concanavalin A bound only weakly to CaSki cells and was ineffective at blocking Mab 1H10 binding. The tumor-associated antigen recognized by Mab 1H10 is concluded to be a chondroitin sulphate glycoprotein or proteoglycan rather than a mucopolysaccharide or lipoprotein.
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PMID:Characterization of a human cervical carcinoma-associated antigen by lectin binding and immuno-electron microscopy. 142 5

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.
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PMID:Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern. 154 Dec 70

Newly synthesized 35S-labeled chondrocytic keratan sulfate chains were generated by chondrocytes of human chondrosarcoma cell line 105KC and were analyzed for heterogeneity of regional substitution, hydrodynamic size, and charge density. After isolation of the high density large chondrocytic proteoglycans and sequential digestions with chondroitinase ABC, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, and alpha-chymotrypsin, followed by Superose 6 chromatography, two populations of keratan sulfate-containing proteoglycan fragments were identified and pooled separately. Keratan sulfate chains from each of the regions were compared after release by Pronase digestion, and differences in substitution patterns were observed; keratan sulfate chains of greater polydispersity, as well as a population of larger hydrodynamic size, were present in only one of the two regions. Alkaline/borohydride treatment confirmed both the existence of a population of uniquely large keratan sulfate chains and its restriction to a single region of proteoglycan fragments. In addition to heterogeneity of hydrodynamic size, the keratan sulfate chains exhibited regional heterogeneity of charge density and hence, of sulfation patterns. Analysis by Mono Q chromatography identified distinct groups of keratan sulfate that segregated by charge density and whose proportionate composition differed between the proteoglycan regions. Furthermore, the most highly charged species were unique to a single region and encompassed the chains of larger hydrodynamic size. This suggests that there may be regional heterogeneity of keratan sulfate chains substituted along a single class of proteoglycans and identifies a novel population of large, highly sulfated chondrocytic keratan sulfate chains.
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PMID:Heterogeneity of keratan sulfate substituted on human chondrocytic large proteoglycans. 155 68


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