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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo-complex and high-mannose) oligosaccharides released from glycoproteins with peptide N-glycosidase F. endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion-exchange column HPIC-AS6/CarboPac PA-1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1-0.3 microA output with suitable sensitivity (less than 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients. Oligosaccharides released from heat-denatured and trypsin-treated glycoproteins were purified initially from large-scale digestion (greater than 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ion-exchange chromatography of the acqueous phase. Oligosaccharides isolated from the peptide N-glycosidase digests of bovine fetuin, human transferrin and alpha 1-acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono-, di-, tri- and tetra-sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high-mannose and complex types were poorly resolved. The high-mannose peaks were eliminated specifically from complex oligosaccharides by digesting with alpha-mannosidase. Treatment with beta-galactosidase, beta-N-acetylglucosaminidase and alpha-mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with alpha-L-fucosidase, endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue. Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endo- and exo-glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.
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PMID:Rapid characterization of asparagine-linked oligosaccharides isolated from glycoproteins using a carbohydrate analyzer. 199 74

A soluble tryptic fragment of the human transferrin receptor (residues 121 to 760) has been crystallized from 2.8 M-KCl (pH 6.2) and polyethylene glycol 8000. This fragment retains the transferrin-binding activity of intact transferrin receptor. Although the trypsin treatment removes the intermolecular disulfide bonds, the receptor fragment is dimeric both under physiological conditions and at the high salt concentrations used for crystallization. The receptor fragment crystallizes in the orthorhombic space group P2(1)2(1)2(1), a = 105.5 A, b = 224.5 A, c = 363.5 A. The crystals are extremely radiation sensitive. Their diffraction extends to 3.8 A, and there is some diffuse scatter with helical characteristics. Analysis of these diffraction patterns indicates that the transferrin receptor fragments are arranged in continuous 8-fold symmetric helical columns parallel to the c axis, with a total of 32 receptor fragment monomers in the unit cell. A structure determination is in progress.
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PMID:Crystallization and X-ray diffraction studies of a soluble form of the human transferrin receptor. 202 43

Human Sertoli cells in vitro secrete a factor that stimulates steroid biosynthesis in purified human and rat Leydig cells as well as in the MA-10 mouse tumor Leydig cell line. MA-10 cells were used as a bioassay system to follow the characterization and purification of the active principle in the conditioned medium of human Sertoli cells. The Leydig cell stimulatory factor is a thermo-labile and trypsin-sensitive protein retained onto 10,000 mol wt (MW) cut-off filters. The following scheme was used to purify the active protein: concentration by ammonium sulfate (80%) precipitation, followed by dialysis using molecularporous membrane tubing of MW cut-off 12,000-14,000, heparin-agarose, Concanavalin-A-Sepharose, and immobilized reactive textile dye affinity chromatography. Yellow 86 and green 19 dyes immobilized on agarose matrix were used. This procedure resulted in the rapid (less than 24-h) purification of a 79,000 +/- 6,082 (n = 3; under denaturating conditions) MW protein which stimulated Leydig cell steroid biosynthesis 25-fold at picomolar concentrations. The MW of the biologically active protein was further confirmed to be around 80,000 by gel filtration chromatography. This 80,000 MW human Sertoli cell-secreted protein (hSCSP-80) was shown to be different from human transferrin, human serum albumin, and rat testibumin. hSCSP-80, by modulating Leydig cell steroid biosynthesis, may play a significant role in the regulation of testicular function.
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PMID:Identification and purification of a human Sertoli cell-secreted protein (hSCSP-80) stimulating Leydig cell steroid biosynthesis. 202 54

Doxorubicin is shown to be present in albumin microspheres (10-40 microns) in two forms: the native drug and a fraction of drug covalently coupled to the protein matrix probably via glutaraldehyde. Upon trypsin digestion the fraction covalently coupled is released and can be resolved from native doxorubicin by high performance liquid chromatography and quantitated either by using 14C-labelled doxorubicin or by measuring the absorption of the doxorubicin chromophore at 480 nm. Albumin microspheres contained 6.9 micrograms/mg protein covalently bound drug versus 11.1 micrograms/mg native drug when 1% glutaraldehyde was used in microsphere preparation. The covalently bound fraction increased significantly with 2% glutaraldehyde. Albumin/polyaspartic acid microspheres lacked a covalently bound fraction when prepared under the same conditions as pure albumin microspheres (35 micrograms/mg native drug, 1% glutaraldehyde) but transferrin microspheres contained similar amounts of bound and native albumin. In vivo, albumin microspheres altered the disposition of doxorubicin in a rat mammary carcinoma (Sp107) compared to albumin/polyaspartic acid microspheres by reducing the rate of parent drug elimination from the tumour and by reducing its biotransformation to 7-deoxyaglycone metabolites. These data indicate that covalent coupling is a key component in the way doxorubicin is handled in tumours after administration of protein microspheres.
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PMID:Covalent coupling of doxorubicin in protein microspheres is a major determinant of tumour drug disposition. 203 40

HeLa cells incubated in serum-free medium accumulated 59Fe ("non-transferrin iron") when incubated with either 59Fe-citrate, 59Fe-nitrilotriacetate, or 59Fe dissolved in Tricine ascorbate. Accumulation of iron was time-, concentration-, and Ca2+-dependent and was saturable. Uptake of non-transferrin (non-Tf) iron was transferrin-independent because of the fact that uptake occurred at pH 5.5, a pH at which transferrin binds iron poorly and at which transferrin is not internalized by cells. Uptake of non-Tf iron was less affected than uptake of transferrin iron by 1) exposure of cells to trypsin, a maneuver that cleaves Tf receptors, or 2) incubation of cells with phenylarsine oxide, an agent that inhibits both fluid- and receptor-mediated internalization. After exposure of cells to non-Tf iron at 37 degrees C, most of the cell-associated radioactivity was recovered in heme and ferritin, demonstrating that iron gained access to intracellular compartments and was not simply adsorbed to the cell surface. Uptake of non-Tf iron could be partially blocked by Cu2+ in a dose-dependent manner, while the accumulation of transferrin-bound iron was unaffected by Cu2+. Other transition metals, such as Zn2+, Cd2+, and Mn2+ were able to inhibit the uptake of non-Tf iron to different degrees. The accumulation of 109Cd was inhibited by incubation of cells with non-Tf iron, Cu2+, or Mn2+. The extent of inhibition was concentration- and metal-dependent. A number of cultured cell lines including HeLa, human skin fibroblasts, and Chinese hamster ovary cells demonstrated uptake of non-Tf iron and 109Cd. Additionally, an endosome acidification mutant of Chinese hamster ovary cells, which exhibited an increase in non-Tf iron uptake, also exhibited an increase in the uptake of Cd2+. These observations suggest that the characteristics of the non-Tf iron transport system in HeLa cells are similar if not identical to those reported for perfused rat liver (Wright, T. L., Brissot, P., Ma, W.-L., and Weisiger, P. A. (1986) J. Biol. Chem. 261, 10909-10914) and suggest the existence of a family of transition metal transport systems, each with a different metal specificity.
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PMID:Characterization of a transferrin-independent uptake system for iron in HeLa cells. 210 43

The specificity by which Haemophilus species acquired iron from transferrin (TF) was investigated. In a plate bioassay H. influenzae used iron bound to human, bovine and rabbit TFs but not mouse, rat, dog, horse, guinea-pig, pig or ovo- TFs or human and bovine lactoferrins. In contrast, H. pleuropneumoniae used iron only from pig TF whilst H. parainfluenzae was unable to utilize iron bound to any of the human or animal TFs tested. The inhibition of growth imposed on H. influenzae type b strain Eagan by the addition of the synthetic iron chelator EDDA to the culture medium was reversed by 30% iron-saturated human TF added directly to the medium but not when the TF was contained inside a dialysis bag. Dot-blotting of whole cells revealed that human TF bound to the surface of bacteria cultured in iron-restricted but not in iron-plentiful media. Incubation of whole bacterial cells in the presence of the proteolytic enzyme trypsin also abolished TF-binding activity, suggesting that the TF receptor was a protein. In competition dot blotting experiments, human and bovine but not rabbit, dog, mouse or guinea-pig TFs blocked the binding of a horseradish peroxidase--human TF conjugate. SDS-PAGE and Western blotting of outer membranes revealed the presence of a TF-binding protein of approximately 72 kDa. These results suggest that the acquisition of TF-bound iron by H. influenzae type b probably involves a direct interaction with an outer-membrane protein which shows some TF-species specificity.
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PMID:Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b. 214 16

Conditions were established to stimulate human gingival fibroblast explant cultures to synthesize milligram quantities of the metalloproteinase proenzymes, prostromelysin and procollagenase. To stimulate enzyme production, cells were treated with 1 nM recombinant human IL-1 beta for approximately 7 days under serum free conditions. Using a combination of rapid column chromatography steps, approximately 10 milligrams of prostromelysin and 5 milligrams of procollagenase were purified from 1 liter of conditioned media. Prostromelysin electrophoresed as a doublet with molecular weights of 55,57 kD, whereas, procollagenase migrated with slightly lower molecular weights of 52, 54 kD. Both proenzymes were treated with trypsin or aminophenylmercuric acetate to generate active species. The molecular weights of the active enzymes were approximately 10 kD smaller than the proenzymes. Active enzymes were inhibited by metal chelators and the natural metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP), but not by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). Activated stromelysin degraded a number of substrates including transferrin, proteoglycan monomer, proteoglycan aggregated with hyaluronic acid, and substance P. By contrast, collagenase degraded interstitial type I collagen and the peptide thioester, Ac-Pro-Leu-Gly-SCH(iBu)Co-Leu-GlyOEt. Identity of both enzymes were confirmed by amino-terminal protein sequence analysis as well as by immunoblot analysis using monoclonal antibodies.
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PMID:Production and purification of prostromelysin and procollagenase from IL-1 beta-stimulated human gingival fibroblasts. 217 90

A transferrin binding protein was isolated from normal rat placenta and from iron-deficient rat plasma using a human transferrin affinity column. The yield of the isolated pure protein from iron-deficient rat plasma was about 0.5 micrograms/ml plasma. The major protein had a molecular mass of 85 kDa and contained carbohydrate. Reduction with mercaptoethanol did not change the molecular mass of the plasma transferrin binding protein whereas the native placental transferrin receptor of 180 kDa was reduced to 90 kDa. The transferrin binding protein reacted with both monoclonal and polyclonal antibodies raised against rat transferrin receptor. Immunoblotting of both normal and iron deficient rat plasma showed that the transferrin binding protein had a molecular mass of 85 kDa. In vitro digestion of purified rat placental transferrin receptor and red blood cells with trypsin provided an identical peptide profile, suggesting that the transferrin binding protein in rat plasma is derived from proteolysis of the extracellular portion of the transferrin receptor of the erythroid tissues.
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PMID:Isolation and characterization of a transferrin binding protein from rat plasma. 220 26

Various electrophoretic techniques, immunoblotting and inhibitions of trypsin and chymotrypsin were used to study the variability of serum proteins in farmed red deer, Cervus elaphus L., of Czechoslovakian origin. Easily interpretable polymorphisms were observed in transferrin (variants A, B1, B2, C) and vitamin D binding protein, GC (variants D, F, I, S). Great variability was observed in the protease inhibitors PI2, PI3, PI4, PI5, and PI8 and in unidentified zones in the vicinity of albumin, but no genetical or physiological interpretation for this variability is yet available. Haemopexin, alpha 1 glycoprotein, protease inhibitors PI1, PI6 and PI7 were monomorphic.
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PMID:Variation of some serum proteins in red deer, Cervus elaphus L. 226 75

In vitro binding, internalization and release of the plasma protein ceruloplasmin was investigated using primary cultures of human placental trophoblast cells. Binding of 125I-labelled ceruloplasmin at 4 degrees C reached equilibrium by 5-6 h; binding was linear throughout all concentrations tested (1 nM-3.3 microM). Addition of greater than 5-10 microM unlabelled ceruloplasmin or a variety of other proteins (albumin, transferrin, IgG) were equally effective in displacing bound ceruloplasmin in a concentration-dependent manner. When cells were incubated at 37 degrees C, the majority of surface-bound 125I-labelled ceruloplasmin was released directly to the extracellular medium. Trypsin-resistant radioactivity increased to 18 per cent of initially bound ceruloplasmin within 1 min, declining to 5 per cent by 2 h. The acquisition of trypsin-resistant radioactivity was unaffected by the addition of a variety of metabolic inhibitors and no evidence of intracellular degradation of ceruloplasmin was found. In summary, our results suggest that the majority of ceruloplasmin binding to trophoblast cells is nonspecific, of low affinity, and easily dissociable at 4 degrees C. Only a small amount of ceruloplasmin appeared to be internalized, by an as yet unknown mechanism.
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PMID:Interaction of 125I-labelled ceruloplasmin with human trophoblast cells in vitro. 229 Aug 3


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