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)

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.
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PMID:The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis. 60 25

A peptide with hyaluronic acid-binding properties was isolated from trypsin digests of bovine articular cartilage proteoglycan aggregate. This peptide originated from the N-terminus of the proteoglycan core protein, retained its function of forming complexes with hyaluronate and link protein and contained at least one keratan sulfate chain. Amino acid sequence data demonstrated that the first six amino acid residues of the N-terminus of bovine articular cartilage proteoglycan core protein differed from the same region from the rat chondrosarcoma proteoglycan. Further sequence data indicate areas of considerable sequence homology in the hyaluronic acid-binding regions of proteoglycans from the two species.
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PMID:The N-terminal sequence of the large proteoglycan of articular cartilage. 225 58

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.
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PMID:Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins. 260 92

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.
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PMID:The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans. 316 89

The ternary complex consisting of a 65-kDa peptide originating from the proteoglycan core protein and a 43-kDa link protein bound to hyaluronic acid was purified from a clostripain digest of the rat chondrosarcoma aggregating proteoglycan and 14C-carbamylated with potassium [14C]cyanate. At a pH of 8.0, 14C-carbamylation of the alpha-NH2 groups in the N-terminal amino acids was favored over carbamylation of epsilon-NH2 groups in the lysinyl residues for both the 65- and 43-kDa species. Two-dimensional tryptic peptide maps revealed a single major, distinctly different, fluorographic spot for each. These tryptic peptides had approximate masses of 4.5 kDa (from the 65-kDa species) and 3.0 kDa (from the 43-kDa species) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and each contained greater than 60% of the total radioactivity associated with its original polypeptide. Primary amino acid sequencing of the 65-kDa species gave a defined sequence for the first 4 N-terminal residues, whereas sequencing through the first 4 residues of a fully carbamylated species gave no dabsylated derivative for the first residue but identical residues in position 2-4 as for the noncarbamylated species and loss of radioactive derivative. Digests of 14C-carbamylated ternary complex with alpha-chymotrypsin resulted in a limit 14C-carbamylated 55-kDa species which contained greater than 85% of the radiolabel originally in the 65-kDa peptide. Similarly, trypsin generated two radiolabeled species, 60 and 58 kDa. These limit digest peptides (55, 60, 58 kDa) all contained the 4.5-kDa N-terminal tryptic peptide. Thus peptides removed from the 65-kDa peptide digestion with either alpha-chymotrypsin or trypsin were on the carboxyl end of the molecule.
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PMID:N-terminal carbamylation of the hyaluronic acid-binding region and the link protein from the chondrosarcoma proteoglycan aggregate. 353 3

Serine residues, which are the sites of phosphorylation in proteoglycans, were demonstrated to be located on chondroitin sulfate-containing peptides. These peptides appeared to be derived primarily from the chondroitin sulfate-rich region of the proteoglycan core protein. The localization of phosphate moieties in the chondroitin sulfate-containing peptides was observed in all experiments. The phosphate moieties were retained on chondroitin sulfate-containing peptides after the protein core was treated with either papain or trypsin. Two phosphopeptide preparations, derived from chondroitin sulfate-containing peptides of proteoglycan subunits, were extensively purified. These 2 phosphopeptide preparations were shown by carbohydrate analysis to be free of keratan sulfate-containing peptides or peptides from the hyaluronic acid binding region of the core protein. One of the phosphopeptide preparations had a phosphate: serine molar ratio of 0.40. This indicated that nearly one-half of the serine residues were phosphorylated rather than glycosylated. Peptides derived from the core protein that contained keratan sulfate had no phosphate moieties.
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PMID:Phosphorylation of proteoglycans. Identification of phosphorylation sites in chondroitin sulfate-rich region of core protein. 392 58

In the course of a study of UDP-xylose:proteoglycan core protein xylosyltransferase (EC 2.4.2.26), another xylosyltransferase was discovered in the soluble fraction of a rat kidney homogenate. The latter enzyme catalyzed [3H]xylosyl transfer from UDP-[3H]xylose to an endogenous acceptor and yielded a product in which the xylose was bound by an alkali-stable linkage. It was therefore concluded that the acceptor was not the core protein of one of the proteoglycans containing a xylose-->serine linkage, since this linkage is cleaved by alkali. The [3H]xylose-labeled product emerged with the void volume when chromatographed on Sephadex G-50, it was precipitated by trichloroacetic acid, and it had a mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of about 32,000 Da. Digestion with trypsin or alpha-amylase degraded the labeled product to small fragments, as determined by gel chromatography, suggesting that it was a glycoprotein related to glycogen. A product of similar characteristics was formed when UDP-[3H]glucose was substituted for UDP-[3H]xylose as the glycosyl donor, and the two nucleotide sugars were mutually competitive in the respective transfer reactions, indicating that they were substrates for the same enzyme. On the basis of these findings, it was tentatively concluded that the xylosyltransferase and its acceptor were the renal form of glycogenin.
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PMID:Xylosyl transfer to an endogenous renal acceptor. Characteristics of the reaction and properties of the product. 815 79

LAMA-84, a human leucocytic cell line, which upon establishment was described as having megakaryocytic, erythroid and granulocytic characteristics, was analysed for expression of various differentiation markers. In addition to some of the previously described phenotypic characteristics, this cell line was found to express mRNA for several proteins characteristic for basophilic leucocytes and mast cells. The authors show that LAMA-84 cells express mRNA for the mast cell tryptase, the proteoglycan core protein, carboxypeptidase A and the alpha and beta chains of the high affinity IgE receptor (Fc epsilon RI). The authors examined the potential of LAMA-84 to differentiate in serum-free medium or after DMSO or PMA treatment. Depending on the inducing factor, surface expression of the Fc epsilon RI alpha-chain was increased from 20% to 35-50% of the cells and mRNA levels for tryptase were increased in serum-free medium and after DMSO treatment. LAMA-84 was found to express CD13, CDw17, CD29, CD33, CD40, CD45 and CD117. Furthermore, mRNA for the eosinophil/basophil markers Charcot-Leyden crystal (CLC) protein and the major basic protein (MBP), as well as the erythrocyte differentiation marker alpha-globin, was detected. However, the authors observed only trace amounts of mRNA for another erythroid differentiation marker (glycophorin), trace amounts of the megakaryocytic marker GPIIIa, and no detectable level of GPIb alpha. By comparing the expression pattern of a panel of differentiation markers in LAMA-84, and a second human cell line (KU812) expressing a basophil phenotype, it is evident that these cell lines, which presently are the only two cell lines identified with basophilic characteristics, share a large number of phenotypic characteristics.
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PMID:Characterization of a human basophil-like cell line (LAMA-84). 869 92

Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC, XT) initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein. In this study, we report the first isolation of the XT and present the first partial amino acid sequence of this enzyme. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcinoma cell culture supernatant. The isolation procedure included a combination of ammonium sulfate precipitation, heparin affinity chromatography, ion exchange chromatography, and protamine affinity chromatography. Among other proteins an unknown protein was detected by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman degradation. Searches with the amino acid sequences in protein and EST data bases showed no homology to known sequences. XT was enriched by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was specifically eluted with the antigen. In addition, XT was purified alternatively with an aprotinin affinity chromatography and was detected by Western blot analysis in the enzyme-containing fraction.
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PMID:First isolation of human UDP-D-xylose: proteoglycan core protein beta-D-xylosyltransferase secreted from cultured JAR choriocarcinoma cells. 1108 29