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)

The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges.
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PMID:Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. A chemical approach for evaluating the role of disulfide bridges in protein folding and stability. 128 3

Synthesis of sulphated proteoglycans was compared in human erythroleukaemia (HEL) cells grown under control conditions and under stimulation by dimethyl sulphoxide (DMSO) and phorbol 12-myristate 13-acetate (PMA). Synthesis of [35S]sulphate-labelled proteoglycans by DMSO-treated cells was decreased by about 35% relative to controls, but synthesis of proteoglycans by PMA-treated cells increased 3-4-fold. Control and DMSO-treated cells secreted 65% of the newly synthesized proteoglycans, but PMA-treated cells secreted more than 90%. Sepharose CL-6B chromatography and SDS/PAGE suggested the presence of several proteoglycans in the cells and culture medium. The PMA-treated cells synthesized a low-Mr proteoglycan (Kav. 0.3( that was not present in controls and DMSO-treated cultures. The proteoglycans of the cells and medium from control, DMSO-treated and PMA-treated cultures could be separated into three fractions by octyl-Sepharose chromatography. The proteoglycans were resistant to trypsin but were degraded by Pronase and papain to fragments similar in size to the NaOH/NaBH4-generated glycosaminoglycans. The average chain length of the glycosaminoglycans (Kav. 0.20 on Sepharose CL-6B for controls) was decreased by DMSO (Kav. 0.25) and by PMA (Kav. 0.30-0.38). Chondroitin ABC lyase digestion of the proteoglycans from the medium of the control cultures produced two core proteins at Mr 31,000 and 36,000. The DMSO medium proteoglycans had only the 31,000-Mr core protein, and the PMA culture medium proteoglycans had core proteins of Mr 27,000, 31,000 and 36,000. Changes in synthesis of proteoglycans induced by DMSO or PMA may have relevance for the maturation of haematopoietic cells.
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PMID:Proteoglycan synthesis in human erythroleukaemia (HEL) cells. 137 1

Three-dimensional alteration of fibrillar matrix in the rat mandibular condylar cartilage was investigated with a high-resolution scanning electron microscope (SEM) and it was determined whether alterations correlate with developing occlusion and advancing age. Two important SEM techniques of DMSO freeze-cracking and treatment with trypsin and hyaluronidase were employed to remove interfibrillar proteoglycans and disclose fibril arrangement. Our SEM investigation demonstrated that collagen fibrils in the fibrous zone covering hyaline-cartilaginous area in the condyle are thicker (50 to 80 nm in diameter) than the fibrils (30 to 50 nm in diameter) that predominantly constituted an interterritorial fibrillar matrix (IFM) in the area. While the thick fibrils had a distinct striation of about 55 nm periodicity, the thin fibrils had no distinguishable striation. The thick fibrils having a periodic striation of about 60 nm was found along with the thin fibrils, also in the IFM in the aged rats and in the deep IFM, but were considerably less than the thin fibrils. The fibrils in the fibrous zone and IFM were disorderly arranged at 19-day-insemination age. In 1-week-old rats whose incisors erupted, the fibrils constituting the fibrous zone altered from disordered to ordered arrangement. The IFM in these rats took the form of a network. Incorporation of small fibrillar bundles into the fibrillar network was seen in 2-week-old rats whose upper and lower first molars erupted. In 8-week-old rats whose molars had erupted completely, the IFM completely occupied by regularly oriented fibrils appeared additionally.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ultrastructural alteration of cartilaginous fibril arrangement in the rat mandibular condyle as revealed by high-resolution scanning electron microscopy. 145 52

We have developed monoclonal antibodies (MoAbs) to the storage mite Lepidoglyphus destructor (Ld). Employing these anti-Ld MoAbs Ld-MoAbs) in ELISA and ELISA inhibition techniques we have analysed the reaction pattern of Ld-MoAbs to both non-pyroglyphid and pyroglyphid mites. The storage mite Glycyphagus domesticus (Gd) exhibited most efficient inhibition, followed by Acarus siro (As), Tyrophagus putrescentiae (Tp), Dermatophagoides pteronyssinus (Dpt) and Euroglyphus maynei (Em). Of the two pyroglyphid species, Dpt showed at least 1000 times less inhibition than Gd. Two of the MoAbs immunoprecipitated a band of 39 kD whereas the third reacted weakly, with a high-molecular band of approximately 110 kD. The Ld extract was also subjected to various reagents and conditions and the antigen was heat stable, it was not affected by low pH, or sensitive to dimethyl sulphoxide (DMSO) or paraformaldehyde. After exposure of the extract to various reagents, such as protease trypsin and periodate, we conclude that the epitopes recognized by Ld-MoAbs were of carbohydrate rather than of protein nature. It would thus seem that MoAbs recognize the carbohydrate part of a glycoprotein.
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PMID:Monoclonal antibodies to Lepidoglyphus destructor: delineation of crossreactivity between storage mites and house dust mites. 146 29

The surface glycoproteins of influenza A viruses are the viral components first recognized by the immune system of the infected host, and they are the viral proteins first to contact the infecting cell. Cleavage of the hemagglutinin (HA) is the presupposition for the uptake and fusion between viral and endosomal membranes at a relatively low pH. If this cleavage does not occur during synthesis and migration within the cell, an external trypsin-like protease has to activate the virus with a non-cleaved HA. This latter property is presumably the reason, why such a large reservoir of non-pathogenic influenza A viruses could be built up in water fowl. Especially feral ducks can disseminate influenza viruses along their flight routes all over the world. The role of the neuraminidase (NA) in the infectious process is not so clear. Its main task in the natural infection seems to be removal of mucoids at the site of entry and in this way to start the primary infection. The synthesis of the viral proteins is a highly regulated process. There is not only a transcriptional but also a translational control. The viral glycoproteins belong to the late proteins. Specifically their synthesis can be inhibited by compounds acting in completely different ways like a specific methylase inhibitor (3DA-Ado), a protein phosphokinase C inhibitor (H7), or a lipid solvent (DMSO). It remains to be determined whether the underlining mechanism is in all these cases the same, namely posttranscriptional modification of viral mRNA. All these viral components do not act separately but they cooperate in their functions and sometimes interfere with each other.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and function of influenza A virus glycoproteins. 193 Jan 3

This study was an investigation of the role of cholecystokinin (CCK) in the stimulatory action of cholestyramine on rat exocrine pancreas. Postprandial CCK release was significantly enhanced by acute administration of cholestyramine (12.7 +/- 1.8 vs 3.7 +/- 0.5 pmol/L in controls). Over four weeks, rats were fed either regular diet or diet containing 6% cholestyramine, and were treated with the specific CCK receptor antagonist L-364,718 (2 x 0.5 mg/kg body weight/day s.c.) or DMSO (vehicle for the antagonist). Cholestyramine significantly increased pancreatic weight and trypsin and chymotrypsin contents. L-364,718 abolished these effects. Concomitant administration of antagonist and cholestyramine elevated amylase content, compared to controls. CCK levels in fasted animals did not differ between the four groups. The effect of the same dose of L-364,718 on pancreatic enzyme depletion, induced by the protease inhibitor camostate, was studied in a control experiment. A single dose of camostate (200 mg/kg) caused a 44-68% decrease in enzyme content. L-364,718 reversed this effect for all enzymes. We conclude that CCK is the mediator of cholestyramine-induced pancreatic hypertrophy and increase in content of proteases. After long-term administration, the CCK receptor antagonist, in combination with cholestyramine revealed an agonistic effect on individual, pancreatic enzyme content.
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PMID:Role of cholecystokinin in cholestyramine-induced changes of the exocrine pancreas. 194 14

Our previous work has shown that retinoic acid (RA) enhances fibroblast cell attachment to plastic and to laminin. The treatment of NIH-3T3 cells with RA for 2 days also caused a reproducible increase in the binding of the lectin Phaseolus vulgaris leukoagglutinin (PHA-L) to a glycoprotein of molecular weight 130,000 (gp130) as judged by SDS-PAGE analysis. This finding is consistent with an increased number of beta-1,6-linked N-acetylglucosaminyl residues on gp130. Of the 11 additional lectins tested Ricinus communis agglutinin I (RCA), Phaseolus vulgaris erythroagglutinin (PHA-E), soybean agglutinin (SBA), and succinylated wheat germ agglutinin (sWGA) showed a significant increase in binding specifically to gp130. Similar to RA, 13-cis-RA and 3,5-di-tert-butyl-4-chalcone carboxylic acid, a synthetic retinoid, also increased PHA-L binding to gp130; they also enhanced cell adhesiveness and inhibited cell growth. N-(4-Hydroxyphenyl)-all-trans-retinamide and thyroxine failed to influence adhesion and did not increase PHA-L binding to gp130. Moreover these compounds also failed to inhibit cell growth and to alter the morphology of the cultured cells. Since trypsin is utilized to remove cells from the culture dishes before they are used in the attachment assay to laminin, we studied the effect of this trypsinization step on PHA-L binding to gp130. Trypsin reduced PHA-L binding thus suggesting cell surface localization of gp130. After trypsin treatment RA-treated cells still showed enhanced PHA-L binding compared to dimethyl sulfoxide (DMSO) control. In conclusion RA-induced cell adhesiveness and growth inhibition are accompanied by an increase in the PHA-L, PHA-E, SBA, RCA, and sWGA binding to gp130. The sensitivity of gp130 to trypsin suggests that it is a cell surface glycoprotein.
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PMID:Retinoids enhance lectin binding to gp130, a glycoprotein of NIH-3T3 cells: correlation with cell growth and adhesion. 198 85

Pure DMSO (instead of water) is used as the reaction medium for protein separations. It is shown that common extracellular proteins (i) have high solubility in DMSO (1-50 mg/ml), (ii) do not irreversibly inactivate in this solvent, and (iii) can adsorb onto carboxymethyl cellulose in DMSO and be subsequently fully desorbed in this solvent by inorganic salts. Ion-exchange chromatography on this resin in DMSO has been used to purify bovine pancreatic trypsin and to separate it from hen egg-white lysozyme in their mixture. Another approach to protein separation in DMSO, fractional precipitation with ethyl acetate (which does not dissolve proteins), has been verified with a mixture of bovine pancreatic chymotrypsinogen and chicken egg ovalbumin.
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PMID:Protein separation and purification in neat dimethyl sulfoxide. 203 25

Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro. DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro. By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro. Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury. DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes. Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells. In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO. Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions. In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers. Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.
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PMID:Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process. 254 52

Human promyelocytic cells (HL-60) were labeled with 35S-sulfate and either 3H-glucosamine or 3H-serine as precursors. Accumulation of 35S-labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/10(5) viable cells reached a plateau level after 24 h. Virtually none of the cell-associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE-Sephacel chromatography, isopyknic CsCl gradient centrifugation, and gel filtration chromatography. HL-60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL-4B and Kav = 0.32 on Sepharose CL-6B, recovered primarily from the high-density fractions of a dissociative CsCl gradient (rho greater than 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4-sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL-60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S-sulfate incorporation to 45% of control values or 32%, expressed as activity/10(5) cells. Proteoglycans synthesized by DMSO-treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.
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PMID:Biosynthesis of proteochondroitin sulfate by HL-60 human promyelocytic cells. 291 Oct 20


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