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)

A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2 X 10(8) viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies.
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PMID:Culture of adult rat lung cells: benzo(a)pyrene metabolism and mutagenesis. 51 Dec 5

Incubation of 20,000 X g supernatant proteins of rat liver with benzo]a]pyrene (BP) and appropriate cofactors produced metabolites of BP covalently bound at the 4,5- position to protein. After being incubated, protein samples were freed from noncovalently bound metabolites and hydrolyzed chemically or both enzymatically and chemically. All steps were performed under minimum exposure to light and air; benzene extracts were prepared during hydrolysis. Chemical hydrolysis released the K-region metabolite BP-4,5-dihydrodiol (identified by UV and mass spectra) and BP. Hydrolysis with trypsin and pronase gave products with the characteristic UV spectrum of substituted chrysene. Chemical hydrolysis of these products released BP and BP-4,5-dihydrodiol.
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PMID:Covalent binding to protein of the K-region oxide of benzo(a)pyrene formed by microsome incubation. 100 7

The effects of photochemotherapy with the fluorescent fatty acid pyrenedodecanoic acid (P12) and long-wavelength ultraviolet (UVA) light on cells derived from human bladder carcinoma were studied. Exposure of these anchorage-dependent cells to P12 either in monolayers of adherent cells or in suspension resulted in a time-related uptake of P12 and its incorporation into the cells' neutral and phospholipids. The uptake and localization of P12 was visualized with fluorescence microscopy and the distribution of the cell population with respect to P12 uptake was analyzed by flow cytometry. Irradiation of P12-containing monolayers of bladder carcinoma cells with UVA light resulted in cell killing. But, on microscopic examination no apparent cell lysis was detected, and since digestion with trypsin did not result in the dispersion of the monolayers it was impossible to assess toxicity by cell count. Alternative procedures were therefore used, and the following cell parameters were determined: (a) cellular uptake or release of chromate; (b) ability of cells to re-adhere to the substratum; and (c) the long-range proliferation potential. The combined inhibitory effect of photoirradiation on cell adherence and on their proliferative potential was utilized for determining reductions of up to 7 log in cell viability. The results obtained with five independently established in vitro bladder carcinoma cell lines indicated that these cells are susceptible to P12-induced photosensitization, suggesting that bladder malignancies might be potential candidates for pyrene-induced photochemotherapy.
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PMID:Photosensitization of human bladder carcinoma cells by pyrene-dodecanoic acid: quantitative analysis of the cytotoxicity. 163 63

Cultured HeLa cells were incubated with pyrene-GM1/3H-radiolabeled GM1 ganglioside (1:4 M/M) mixtures for various times. The process of association of pyrene-GM1 with cells was qualitatively and quantitatively the same as that of 3H-GM1. The pyrene-GM1 and 3H-GM1 proportions in the various forms of association with cells were similar to that of the starting ganglioside mixture. After 2-h incubation, the association of ganglioside with cells was well established whereas almost no metabolic processing had occurred. During a 24-h incubation, pyrene- and 3H-GM1 underwent similar metabolic processing and gave rise to catabolic (GM2 and GM3) and anabolic (GD1a) derivatives. Fluorescence spectroscopy experiments carried out with the excimer formation technique on subcellular fractions containing plasma membranes showed that exogenous ganglioside was, in part, associated with the cells in a micellar form removable by trypsin treatment, and in part inserted in a seemingly molecular dispersion. Addition of Ca2+ salts caused aggregation of the ganglioside, as indicated by the increase of the excimer:monomer fluorescence ratio. The phenomenon was Ca2+ concentration dependent (maximum at 10 mM), and subsequent addition of EDTA had no effect. The saccharide portion of exogenously incorporated pyrene-GM1 was available to interact with external ligands, as shown by its ability to bind cholera toxin whose addition reduced the collision rate among the ganglioside lipid moieties.
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PMID:Association to HeLa cells and surface behavior of exogenous gangliosides studied with a fluorescent derivative of GM1. 211 Aug 27

Using the dynamic fluorescence quenching method, it was shown that very low density (VLDL) apoproteins (apo B, E and C) tryptophanyls exhibit a lower accessibility towards water-soluble quenchers as compared to apo B LDL chromophores. The efficiency of proteolytic degradation by trypsin of VLDL-associated apo E and apo C was much lower than that of apo B. These results may be due to the cluster arrangement of amphipatic apo E and apo C on the VLDL surface and/or to their partial shielding by apo B. Treatment of VLDL particles with sub-lytic concentrations of the detergent, Tween-20, did not change the relaxation characteristics of amphipatic apoprotein tryptophanyl microenvironment, but resulted in a reversible structural transition registered by a "red" shift of the emission spectrum maximum as well as by change of the iodine quenching pattern. The detergent-induced increase of the VLDL tryptophanyl accessibility to acrylamide and the decrease of the quenching constant at the partial and complete particle solubilization were related to a change of the apo B molecular package. Treatment of VLDL with Tween-20 or cow milk lipoprotein lipase resulted in the appearance of tryptophanyl population that was not involved in the resonance energy transfer to the lipid phase-localized fluorescent probe pyrene, which is indicative of the protein dissociation. Treatment of VLDL particles with sub-lytic concentrations of Tween-20 revealed a lower (compared to apo C) relative affinity of apo E for the VLDL lipid surface. Inhibition of the lipoprotein lipase activity by apoprotein C-III was found to be non-competitive. It was concluded that lipolysis is a self-regulatory process which involves changes in the effector apoprotein concentration on the surface of triglyceride-rich particles.
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PMID:[Dynamic behavior of apoproteins of human plasma very low density lipoproteins and lipolysis regulation]. 216 Aug 40

Evidence is accumulating that the levels of covalent carcinogen-macromolecule adducts, including adducts with hemoglobin, reflect biologically effective levels of carcinogen exposure. The purposes of the present study were (a) to establish a cellular system for obtaining adducts between intracellular human hemoglobin and metabolites of polycyclic aromatic hydrocarbons (PAH), and (b) to evaluate techniques for chromatographic characterization of the adducts. We showed that hemoglobin-benzo[a]pyrene adducts were formed when human erythrocytes were treated with [3H]benzo[a]pyrene (BP) in the presence of hamster embryo fibroblasts, which are known to be effective for BP metabolism. After lysis of the erythrocytes, noncovalently bound BP and its metabolites were effectively removed from hemoglobin under mild conditions by using hydrophobic interaction and size-exclusion liquid chromatography. Three to five distinct adducts were resolved by reversed-phase and ion-exchange liquid chromatography. As determined by a two-step, reversed-phase liquid chromatographic procedure, trypsin treatment of globin from the cellular system yielded at least three of the four 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy BP tetrols known to arise from mammalian metabolism of BP. This observation is consistent with both (a) the recently described formation of labile carboxyl esters via reaction of BP-7,8-dihydrodiol-9,10-epoxide (BPDE) with hemoglobin and (b) the known formation of both anti- and syn-BPDE in hamster embryo fibroblasts. In addition, high-performance liquid chromatographic analysis demonstrated the presence of other products presumed to be BP-peptide adducts because of their susceptibility to thermolysin treatment.
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PMID:Formation of hemoglobin-benzo[a]pyrene adducts in human erythrocytes incubated with benzo[a]pyrene and hamster embryo cells. 227 62

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.
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PMID:Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays. 233 86

Enzyme I is the first protein of the phospho transfer sequence in the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This protein exhibits a temperature-dependent monomer/dimer equilibrium. The nucleotide sequence of Escherichia coli ptsI indicates four -SH residues per subunit (Saffen, D. W., Presper, K. A., Doering, T. L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253). In the present experiments, the sulfhydryl groups of the E. coli enzyme were studied with various -SH-specific reagents. Titration of Enzyme I with 5,5'-dithiobis-2-nitrobenzoic acid also revealed four reacting -SH groups. The kinetics of the 5,5'-dithiobis-2-nitrobenzoic acid reaction with Enzyme I exhibit biphasic character, with pseudo-first order rate constants of 2.3 x 10(-2)/s and 2.3 x 10(-3)/s at pH 7.5, at room temperature. Fractional amplitudes associated with the rate constants were 25 +/- 5% for the fast and 75 +/- 5% for the slow rate. The "slow" rate was influenced by ligands that react with Enzyme I (the protein HPr, Mg2+, Mg2+ plus P-enolpyruvate), and also by temperature (at the temperature range where the monomer/dimer association occurs). The fractional ratio of the two rates remained at 1:3 under these conditions. Thus, under all conditions tested, two classes of -SH groups were detected, one reacting more rapidly than the other three -SH groups. Modification of the "fast" -SH group results in an active enzyme capable of forming dimer, whereas modification of the slow -SH groups results in inactive and monomeric Enzyme I. The enzyme was labeled with pyrene maleimide under conditions where only the more reactive sulfhydryl group was derivatized. Hydrolysis by trypsin followed by reverse-phase high performance liquid chromatography analysis of the peptide mixture resulted in only one fluorescent peak. This peak was not observed when the more reactive sulfhydryl residue was protected prior to pyrene maleimide labeling. Amino acid sequencing of the fluorescent peak indicated that the more reactive residue is the C-terminal amino acid residue, cysteine 575. The results provide a means for selectively labeling Enzyme I with a fluorophore at a single site while retaining full catalytic activity.
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PMID:Sugar transport by the bacterial phosphotransferase system. Characterization of the sulfhydryl groups and site-specific labeling of enzyme I. 240 75

Pyrenebutylmethylphosphonofluoridate reacts with trypsin and elastase to yield a conjugate with a stoichiometry of one fluorescent label per enzyme molecule as already observed with chymotrypsin. The kinetics of inactivation indicate that the serine active center of the proteases is involved in the labeling reaction. The binding of the proteases to alpha 2-macroglobulin does not modify the specificity of the reaction but drastically diminishes the labeling rate which also depends upon alpha 2-macroglobulin protease binding ratio. Dynamic quenching of the conjugated pyrene moiety by acrylamide, and iodide ions is markedly reduced upon reaction of the protease with alpha 2-macroglobulin, indicating a reduced accessibility of the protease active center in the complex. Singlet--singlet energy transfer measurements from the donor pyrene labeled active center of the proteases to the alpha 2-macroglobulin acceptor labeled thiol groups which are liberated upon protease fixation, gave a rough estimate of the distance (about 25 A) between the active center of the two alpha 2-macroglobulin bound protease molecules.
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PMID:Specific fluorescent labeling of alpha 2-macroglobulin-bound proteases: accessibility and localization of their active site within the complex. 243 Jun 27

1. Two forms of fatty acid-binding proteins (FABPs) were isolated from human, pig and rat liver cytosols by gelfiltration and anion-exchange chromatography. 2. Both forms did not show physicochemical or chemical differences. They had an Mr of about 14.5 kDa for all species. pI Values were 5.8 for both forms of human and pig liver FABP and 6.4 for both forms of rat liver FABP. In contrast to heart FABPs no tryptophan was present in liver FABPs. 3. Liver FABPs show a much higher enhancement of fluorescence at binding of 11-dansylaminoundecanoic acid, 16-anthroyloxy-palmitic acid and 1-pyrene-dodecanoic acid than heart FABPs and additionally a blue shift in excitation and emission wavelengths with the first fatty acid. 4. The bulky side-chain did not affect fatty acid binding since binding constants of liver FABPs were comparable for these fluorescent fatty acids and oleic acid (0.3-0.7 microM). 5. A 1:1 binding stoichiometry was obtained for oleic acid binding with heart and liver FABPs. 6. Liver FABPs have a high binding affinity for C16-C22 saturated and unsaturated fatty acids, palmitoyl-CoA, bromo-substituted fatty acids, POCA, tetradecylglycidic acid and flavaspidic acid. 7. Fatty acid binding could be reduced to less than 50% by arginine modification with 2,3-butadione or by enzymatic degradation of FABPs with trypsin or pronase.
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PMID:The binding affinity of fatty acid-binding proteins from human, pig and rat liver for different fluorescent fatty acids and other ligands. 274 9


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