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)

Rat basophilic leukemia cells were labeled either enzymically with 125I or biosynthetically by culture in the presence of 14C-glucosamine or 3H-amino-acids and then extracted with NP-40. IgE-anti-IgE precipitates insolubilized a radiolabeled macromolecule from these extracts largely or entirely absent in control IgG-anti-IgG percipitates. When specific precipitates were boiled in sodium dodecyl sulfate (SDS) and analyzed by polyacrylamide gel electrophoresis in the presence of SDS, most of the 14C or 125I radioactivity was in the area corresponding to an apparent m.w of 60,000 to 70,000 in 5.9% gels. In 10% and 12% gels, faster mobility was demonstrated indicating an atypical electrophoretic behavior often associated with glycoproteins and a presumptive m.w. of 50,000 or less. Since only IgE-containing precipitates localized label in this region and since such precipitates from cells saturated with IgE prior to surface iodination failed to show this band, the labeled macromolecule appears to be the IgE receptor itself. Analysis of the acid hydrolysates of precipitated 14C radioactivity demonstrated that label was entirely in hexosamines and sialic acid. 125I and 14C labels in the recepotr region were eliminated almost completely with pepsin and pronase and to a lesser extent with trypsin.
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PMID:The rat basophilic leukemia cell receptor for IgE. I. Characterization as a glycoprotein. 82 Aug 4

An acid stable protease inhibitor was isolated from human bronchial secretion. Two important stages of the purification procedure were affinity chromatography on trypsin bound to Affi-Gel 10 and ion-exchange chromatography on SP-Sephadex C-50. The isolated inhibitor appeared as a single band on analytical disc electrophoresis and eluted as a homogeneous protein peak on gel filtration on Sephadex G-75 corresponding to a molecular weight of about 10500. Amino acid analyses showed no tryptophan or histidine and as N-terminal amino acid tyrosine. No glucosamine or galactosamine was detected. The results of the analyses suggest that the purified inhibitor is identical to the low molecular weight trypsin-chymotrypsin inhibitor of human seminal plasma (HUSI-I).
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PMID:Isolation and partial characterization of a low molecular weight acid stable protease inhibitor from human bronchial secretion. 88 Nov 64

Three radioactive glycopeptides were isolated from human peripheral lymphocytes stimulated with Wistaria floribunda mitogen in the presence of D-[14C]glucosamine hydrochloride by mild trypsin digestion followed by gel filtration and preparative high-voltage paper electrophoresis. The carbohydrate compositions of these glycopeptides suggest that one has a sugar chain of the type found in serum glycoproteins, consisting of sialic acid, galactose, N-acetylglucosamine, mannose, and fucose in a molar ratio of 2:2:4:2:1, and the other two have sugar chains like those of mucins, consisting of sialic acid, galactose, and N-acetylgalactosamine in a molar ratio of 1 or 2:1:1. The results of enzymic degradation with purified glycosidases indicate that these sugar chains are similar in structure to their counterparts in human erythrocyte membranes.
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PMID:Isolation of glycopeptides from the lectin-stimulated human peripheral lymphocyte cell surface. 89 50

We have established conditions for the study of membrane glycoprotein synthesis and turnover in cultured human malignant melanoma cell lines using the labeled precursor [3H]glucosamine. Uptake of label increased parallel with cell growth, reaching a steady state in resting cultures. Fifteen to 30% of incorporated label can be released from the cells by trypsin treatment depending on the conditions of exposure to the enzyme, and about 50% of the incorporated label is spontaneously shed from the cells within 96 hr of incubation. Labeling in exhausted medium gave a 5- to 8-fold increase in uptake which was inhibited by addition of glucose (2 mg per ml) into the culture medium. The percentage of trypsin-releasable material was identical in fresh and exhausted medium; however, the percentage shed was less in cells initially labeled in exhausted medium. These data provide background information for further studies on the antigenic composition of the glycoproteins of cultured melanoma cells.
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PMID:Characterization of human malignant melanoma cell lines. VII. Glycoprotein synthesis and shedding as revealed by [3H]glucosamine labeling. 92 59

The alpha subunit of human chorionic gonadotrophin was reduced with dithiothreitol followed by carboxymethylation with iodoacetic acid. The modified glycoprotein was hydrolysed with trypsin to give various peptides, the identities of which were established, and glycopeptides. The glycopeptides were separated by gel filtration and ion-exchange chromatography; they were subjected to component analysis and were found to represent the two carbohydrate moieties in the parent glycoprotein. Sequential removal with glycoside hydrolases of monosaccharide units from the glycopeptides demonstrated (1) that galactose, mannose, glucosamine (2-amino-2-deoxyglucose) and neuraminic acid (5-amino-3,5-dideoxy-glycero-galacto-2-nonulosonic acid) residues possess the D configurations, (2) that the glucosamine units are N-acetylated and (3) the order of the monosaccharide units in the chain, the neuraminic acid units being furthest from the peptide backbone of the subunit and substituting the D-galactose units. Methylation analysis of the glycopeptides by adaptation of the Hakomori technique demonstrated that: (4) D-galactose, D-mannose and N-acetylglucosamine (2-acetamido-2-deoxy-D-glucose) units exist in the pyranose forms; (5) the D-galactopyranose units are linked in the 1 and 6 positions; (6) the D-mannopyranose units exist in several forms, one in a terminal non-reducing position, one as 1,2-linked residues and some as 1,6-linked branch points; (7) the N-acetylglucosamine units are 1,6-linked. On the basis of the results of methylation and enzymic analysis, structures are proposed for the carbohydrate moieties and the assignments are compared with other data previously obtained by periodate-oxidation studies [Kennedy et al. (1974) Carbohydr. Res. 36, 369-377].
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PMID:The structures of the carbohydrate moieties of the alpha subunit of human chorionic gonadotrophin. 93 81

The cell surface of embryonic chick liver cells contains transferases for mannose, fucose, galactose, N-acetyl-glucosamine and N-acetyl-neuraminic acid. Liver cells obtained by trypsin-dissociation of the tissue use the corresponding exogenous sugar nucleotides as substrates. The activities of the enzymes tested do not depend neither no the dissociation procedure nor on de novo protein synthesis. They vary considerably during development of the embryos, reaching maximal values at the 8th+/-1 day and at the 12th+/-1 day. Glycoproteins are the final stable endogenous acceptors for all sugars. Mannose transfer proceeds via a two or multistep reaction sequence. In a first step labile lipophilic intermediates are formed. Mannose can be liberated by treating the intermediates with 0.1 N HCl at 100 degrees C. In a second reaction step mannose becomes attached to glycoproteins. From embryonic chick liver cells a glycopeptide fraction has been obtained by pronase digestion followed by several purification steps. The purified glycopeptides inhibit all transferase systems and act as exogenous acceptors for mannose transfered from exogenous GDP-mannose.
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PMID:Cell surface glycosyl transferase activities in liver cells of developing chicken embryos. 94 93

Experiments were performed to determine if animal cells in culture possess specific mechanisms to repair surface molecules damaged by enzymes. The surface membranes of a primary cell culture, chick fibroblasts, a permanent hamster cell line, BHK21/C13, and its virally transformed counterpart, C13/B4 were damaged by exposure to trypsin or to neuraminidase. Following digestion with trypsin, the incorporation of radioactive amino acids or sugars into purified surface membrane of cells was monitored. No differences were noted in rates of incorporation when control and trypsin-damaged cells were compared. Neuraminidase damage to the surface of BHK21/C13 and C13/B4 cells was evidenced by altered gel filtration profiles of surface glycopeptides, i.e., delayed elution because of reduction in size. By labelling cells with 14C-L-fucose prior to neuraminidase treatment and following the incorporation of 3H-L-fucose into cell surface glycopeptides after neuraminidase digestion, we were able to monitor the synthesis and turnover of fucose-containing glycopeptides in the same cells. Gel filtration profiles indicated that little or no desialylated glycoproteins were resialylated (repaired) by specific replacement of sialic acid. Comparing neuraminidase-digested and control cells we observed no difference in rates of 3H-L-fucose incorporation or of 14C-L-fucose loss from these cells; nor did we find differences in the rate of incorporation of isotopic glucosamine into sialic acid. Neuraminidase treatment failed to alter the rate of cell growth or the pattern of isotopic incorporation into various cell surface components. These results support the suggestion that return of sialic acid (repair) was effected by turnover which serves as a non-specific repair mechanism to replace damaged cell surface molecules (Warren and Glick '68; Warren, '69).
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PMID:The repair of the surface structure of animal cells. 97 62

A procedure has been developed for the purification of Z-type alpha-1-antitrypsin (alpha-1-AT) which is rapid, gentle, and results in good yields. From 4 units (750 ml) of fresh human plasma, obtained from two individuals possessing the Pizz phenotype, 53 mg of pure Z-type alpha-1-AT was obtained. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis, both in the presence and absence of sodium dodecyl sulfate, and by analytical ultracentrifugation. When compared to pure alpha-1-AT from plasma of individuals possessing the normal PiMM phenotype, the two proteins were indistinguishable with respect to amino acid composition, sedimentation coefficient (s20w of 3.33 for both M and Z), molecular weight (51,000 by sodium dodecyl sulfate gel electrophoresis and 47,000 by sedimentation equilibrium for both M and Z), and trypsin-combining ratio (0.91 for Z and 0.99 for M). The only difference which was observed between the variant forms of alpha-1-AT was in the carbohydrate composition. The Z-type alpha1-AT contains between 20 and 25% less carbohydrate than the M-type alpha-1-AT. Specifically, the Z-type alpha-1-AT is deficient in 1 glucosamine residue, 3 neutral sugar residues (1 mannose and 2 galactose), and 2 sialic acid residues. Although the Z-variant is deficient in sialic acid, its survival time in the serum of a rabbit was not significantly different from that of M-type alpha-1-AT.
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PMID:Comparison of the chemical, physical, and survival properties of normal and Z-variant alpha-1-antitrypsins. 108 86

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-14C]glucosamine and L-[4,5-3H]leucine were obtained by electrophoretic separation. Envelopes were isolated from cells labelled with D-[1-14C]glucosamine--HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. The glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-14C2]pimelic acid, while orth[32P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAE-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.
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PMID:Glucosamine-labelled envelope proteins of Escherichia coli K-12. I. Electrophoretic studies and partial fractionation of phenol-soluble proteins. 110 Jan 12

1. Radioactivity from [3H]glucosamine is rapidly incorporated into cellular fractions of lens epithelial cells cultured in vitro. The incorporated isotope appears largely in glycoproteins of the cell surface that are exposed to trypsin and are released into a soluble form by proteolysis of intact cells. Glycoproteins are also secreted by cultured cells and can be recovered in the culture fluids. Sodium dodecysulphate-polyacrylamide gell electrophoresis shows that a range of glycoproteins with apparent molecular weights from approximately 14000 to 120000 are present. The relationships of these glycoproteins to collagen and the non-collagenous glycoproteins of lens basement membranes are discussed. 2. A plasma membrane fraction obtained from non-trypsinised lens epithelial cells contains one major glycoprotein of apparent molecular weight 120000. A major non-glycosylated polypeptide of molecular weight about 38000 detectable by Bloemendal et al. (1972) in plasma membranes of differentiated lens fibre cells was not prominent in lens epithelial cell membranes. 3. Examination of lens basement membranes extracted in various ways failed to reveal major glycoproteins of low molecular weight. Higher molecular weight glycoproteins, some of them related to collagen, were present.
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PMID:Lens glycoproteins: biosynthesis in cultured epithelial cells of bovine lens. 116 11


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