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)

The maintenance of human osteoarthritic femoral head cartilage in long-term explant culture has been used to assess changes in newly synthesized and endogenous proteoglycans. Minced cartilage was radiolabeled with [3H]-leucine for 24 h. and the high density proteoglycans digested with tosyl-phenylalanine chloromethyl ketone-trypsin. Chondroitin-sulfate-rich peptides were separated from chondroitin-sulfate-poor peptides by DEAE-cellulose chromatography and hexuronic acid, protein, and galactosamine and glucosamine molar ratios determined. The incorporation of [3H]-leucine was highest in peptides enriched in chondroitin sulfate. when day 1 cultures were compared to those maintained for 20 days, several prominent changes were seen including, a reduction in the galactosamine to glucosamine ratio. Taken together with other data which showed a reduction in the hydrodynamic size of these proteoglycans with time in culture, these results showed that changes in the existing extracellular matrix pertinent to the osteoarthritic process can be assessed by maintaining cartilage in long-term organ-explant culture. A reduced hydrodynamic size of newly synthesized proteoglycans is consistent with a loss of chondroitin-sulfate and an increase in keratan sulfate in the high density proteoglycans.
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PMID:Changes in proteoglycans of human osteoarthritic cartilage maintained in explant culture: implications for understanding repair in osteoarthritis. 323 76

A preparation of peptidyl-tRNA from intact microsomes of mucin-synthesizing polysomes of sublingual salivary gland cells contained fatty-acylated galactosamine-free and galactosamine-enriched peptidyl-tRNA fractions, whereas trypsin-chymotrypsin treated microsomes yielded predominantly the acylated galactosamine-enriched peptidyl-tRNA complexes. Radioscanning and chemical analyses revealed that palmitate was substituted on all nascent peptides, except those shorter than 20 amino-acid residues. In contrast, the [35S]-methionine label was detected only on galactosamine-free peptides containing up to 70 amino acids. On SDS-polyacrylamide gel, the peptides released from galactosamine-enriched tRNA complexes separated into a multitude of bands ranging in size from 6000 to 60,000 dalton, whereas the total preparation afforded peptides ranging from 2000 to 60,000 dalton. Pulse-chase experiments, using radiolabelled methionine, palmitic acid and N-acetylgalactosamine, combined with chemical characterization of the radiolabelled fatty acids and carbohydrates from purified peptidyl-tRNA, confirmed that the N-terminal fatty acylation and the initial O-glycosylation with N-acetylgalactosamine are the co-translational processes taking place as soon as peptide is sufficiently large to be acylated, trimmed, and translocated to the luminal site of endoplasmic membrane.
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PMID:Co-translational processing and intracellular transport of rat salivary mucus glycoprotein. 325 86

The partial purification and characterization of a hepatocyte proliferation stimulatory factor (HPSF) isolated from the liver of D-galactosamine-treated rats are described. The HPSF was a heat-labile, acid-stable and trypsin-sensitive protein. The partially purified HPSF stimulated DNA synthesis and increased the labeling index of parenchymal hepatocytes at 5 micrograms/ml and maximally at 50 micrograms/ml. The effect of HPSF in stimulating DNA synthesis was synergistic with that of insulin plus epidermal growth factor (EGF). The HPSF was scarcely detected in normal rat liver. The results obtained indicate that this HPSF is distinct from insulin, multiplication-stimulating activity (MSA), EGF and other hepatocyte growth factors previously reported, and suggest a plausible role for HPSF in the regeneration of liver tissue following hepatotoxic damage.
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PMID:Partial purification and characterization of hepatocyte proliferation stimulatory factor from liver of rats treated with D-galactosamine. 327 7

Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].
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PMID:Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. 359 17

In earlier studies from our laboratory, the intact sperm receptor was partially purified from Strongylocentrotus purpuratus crude egg membranes, but due to its insolubility, it was not possible to purify it to homogeneity. Nonetheless, this receptor preparation bound with species specificity to acrosome-reacted sperm, thereby inhibiting fertilization. Antibodies against the partially pure receptor inhibited fertilization in S. purpuratus (but not Arbacia punctulata) by coating the egg surface, indicating the presence of binding sites that can be species-specifically recognized by both sperm and antibody molecules. Recently we were able to further purify and characterize the receptor from S. purpuratus eggs. Chaotropic agent solubilization of the receptor prepared from crude egg membranes yielded a very high molecular weight glycoconjugate that had many of the properties of a proteoglycan. The receptor interacted with binding in an in vitro assay and bound with species specificity to acrosome-reacted sperm to inhibit fertilization. Unfortunately, this receptor preparation was soluble only in certain chaotropic agents. Exhaustive Pronase digestion of the intact receptor yielded a soluble high-molecular-weight (greater than 10(6)) polysaccharide that was virtually devoid of protein. This glycosaminoglycan-like fragment was highly sulfated, and contained fucose, galactosamine, and iduronic acid. The fragment inhibited fertilization, but did not do so with species specificity. Recently, soluble molecules with receptor activity were generated by treating intact dejellied eggs with trypsin. These proteolytically derived molecules contained (on a weight basis) approximately equal amounts of protein and carbohydrate. Importantly, they inhibited fertilization with species specificity. The results suggested that the binding activity was conferred by the polysaccharide component of the receptor and that the intact receptor and the tryptic fragments contained structural elements in the polypeptide chain necessary for species recognition.
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PMID:Characterization of the Strongylocentrotus purpuratus egg cell surface receptor for sperm. 382 83

We have determined the complete primary structure of human hemopexin, a plasma beta-glycoprotein that specifically binds one heme with high affinity and transports it to hepatocytes for salvage of the iron. Human hemopexin (Mr approximately equal to 63,000) consists of a single polypeptide chain containing 439 amino acid residues with six intrachain disulfide bridges. The amino-terminal threonine residue is blocked by an O-linked galactosamine oligosaccharide, and the protein has five glucosamine oligosaccharides N-linked to the acceptor sequence Asn-X-Ser/Thr. The 18 tryptophan residues are arranged in four clusters, and 12 of the tryptophans are conserved in homologous positions. Computer-assisted analysis of the internal homology in amino acid sequence indicates that hemopexin consists of two similar halves, thus suggesting duplication of an ancestral gene. Limited tryptic digestion cleaves apohemopexin after arginine-216 into two half-molecules, whereas heme-saturated hemopexin is cleaved after lysine-101. The half-molecules are connected by a histidine-rich hinge-like region that contains two glucosamine oligosaccharides. A structural model for human hemopexin is proposed that is based on these properties and on computer-assisted predictions of the secondary structure and the hydrophilic/hydrophobic character. In this model alpha-helices and beta-turns predominate, and the two halves are connected by an exposed connecting region in apohemopexin that becomes inaccessible to trypsin in hemesaturated hemopexin. Many segments of hemopexin are similar to sequences of other heme proteins, but no overall structural relationship of hemopexin to any other heme protein was identified.
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PMID:Complete amino acid sequence of human hemopexin, the heme-binding protein of serum. 385 50

Binding region and link protein were prepared from pig laryngeal cartilage proteoglycans after chondroitinase ABC and trypsin digestion. Experiments on gel chromatography showed the purified binding region to interact reversibly with hyaluronate (HA), and this binding was also shown to be stabilized by native link protein. The trypsin-prepared link protein showed properties of self-association in solution that were partially inhibited by oligosaccharides (HA10-16) and abolished by modification of free amino groups (lysine residues) with 2-methylmaleic anhydride. The Mr (sedimentation equilibrium) of the modified link protein was 41 700. Analysis of binding region showed it to contain 25% (w/w) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulphate, as digestion with endo-beta-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the Mr (sedimentation equilibrium) decreased from 66 500 to 60 800. After keratanase digestion the interaction with polyclonal antibodies specific for binding region was unaffected, but the response in a radioimmunoassay with a monoclonal antibody to keratan sulphate was decreased by 47%. Preparation of a complex between binding region, link protein and HA approximately 34 showed a single component (5.5S) of Mr (sedimentation equilibrium) 133 500. In this complex the antigenic determinants of link protein appeared masked, as previously found with proteoglycan aggregates. The isolated binding region and link protein were thus shown to retain properties comparable with those involved in the structure and organization of proteoglycan aggregates.
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PMID:Structure and interactions of cartilage proteoglycan binding region and link protein. 400 17

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.
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PMID:Purification and properties of rabbit liver cathepsin M and cathepsin B. 406 7

The Streptococcus mutans group b antigen of strain FA1 has been defined as to chemical composition and immunological specificity. The antigen in cold trichloroacetic acid extracts was fractionated on diethylaminoethyl-Sephadex A-25 at pH 8.5. Two forms were isolated: a polysaccharide and a mucoprotein. The two polymers reacted as a single substance in agar gel diffusion against specific adsorbed FA1 rabbit antisera but were separated by gel immunoelectrophoresis. No reaction with any other S. mutans or streptococcal group sera occurred. Galactose composed about one-third and galactosamine about 3% of the total weight of each polymer. Rhamnose was a major component of the polysaccharide (47%) but was present only in traces in the mucoprotein. The protein content of the latter was about 40%. No significant quantities of glycerol, phosphorus, or muramic acid were present in either case. Pepsin and trypsin had no effect on the serological specificity of the mucoprotein. d-Galactose and d-galactosamine were strong inhibitors (70%) of the precipitin reaction, whereas d-glucose, d-glucosamine, and N-acetyl-d-glucosamine inhibited between 25 and 35%. The results indicate that the antigen is a major antigenic component of the cell wall and that the specificity of the antigen resides in binding sites which contain both d-galactose and d-galactosamine. Agglutination of whole cells by specific group b antiserum indicates the antibody receptor sites of the polysaccharide antigen are at the surface of the streptococcal cell. The mucoprotein, but not the polysaccharide, was released from the cell by lysozyme. Lysis did not occur. The immunological specificity and other characteristics of the antigen establishes it as the identifying antigen of S. mutans group b.
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PMID:Structure and immunological specificity of the Streptococcus mutans group b cell wall antigen. 412 3

In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the alpha1(I) and alpha2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 muM and 55 muM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 muM alpha1(I) and 160 muM alpha2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen. Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. One class of higher molecular weight contained sialic acid, glucosamine, galactosamine, fucose, mannose, galactose, and glucose, and the other of lower molecular weight consisted primarily of a mixture of galactose and galactosyl-glucose units O-glycosidically linked to hydroxylysine-containing peptides. We found that, after the residual tryptic activity contaminating the higher molecular weight fraction was inhibited, neither of the glycopeptide classes produced nor inhibited native human skin insoluble collagen-mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet-rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosylglucose (Hyl-Gal and Hyl-Gal-Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet-rich plasma showed that, at concentrations greater than 50 nM, Hyl-Gal gives apparent binding to platelets, but there was no evidence of Hyl-Gal-Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl-Gal (at 29 muM) nor Hyl-Gal-Glc (at 18 muM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 muM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Gal-Glc-containing 36 residue rat skin soluble collagen alpha1(I)cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Modification of at least 90% of the rat skin soluble collagen carbohydrate by mild periodate oxidation had no effect on the platelet aggregating activity. Human skin insoluble collagen was reacted with periodate under the same conditions, and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation.Thus, collagen-platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple-helical fold or perhaps even for a particular amino acid sequence in the triple helix.
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PMID:Collagen-mediated platelet aggregation. Effects of collagen modification involving the protein and carbohydrate moieties. 435


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