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)

A new sulfated, cyclic depsipeptide, called cyanopeptolin S, from Microcystis sp. was isolated from a water bloom in the Auensee/Leipzig (Germany). The depsipeptide had a relative molecular mass of 925 and contained L-arginine, L-threonine, L-isoleucine, N-methyl-L-phenylalanine, a L-glutamic acid-delta-aldehyde ring system and a sulfated D-configurated glyceric acid as a side chain. The structure was elucidated by means of two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectroscopy. Fourier transformed infrared spectroscopy and combined gas-liquid chromatography/mass spectrometry. Cyanopeptolin S inhibited trypsin with an IC50 < or = 0.2 micrograms ml-1.
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PMID:Cyanopeptolin S, a sulfate-containing depsipeptide from a water bloom of Microcystis sp. 760 93

Contradictory findings have recently been reported regarding the (in)abilities of individual subunits of the Vibrio harveyi alpha beta dimeric luciferase to catalyze bioluminescence. We have produced individual alpha and beta subunits separately in Escherichia coli JM109 cells by recombinant DNA techniques. Both subunits were purified to more than 90% homogeneity and found to be catalytically active, with their general catalytic properties and the specific activities similar to those reported earlier (Sinclair, J. F., Waddle, J. J., Waddill, E. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044). Individual subunits were significantly distinct from the native luciferase with respect to inactivations by trypsin and N-ethylmaleimide, and the stability of the flavin 4a-hydroperoxide intermediate. The active species in isolated alpha and beta samples were each the predominant protein species, corresponding to a 42,000 M(r) alpha monomer and a 67,000 M(r) beta dimer, respectively. These findings clearly indicate that the activities of the individual subunits are not due to trace contaminations of the respective counter subunits. The much reduced specific activities of the individual subunits are, in part, a consequence of diminished abilities to oxidize the aldehyde substrate. Kinetic and equilibrium measurements indicate that alpha and beta 2 each contained a reduced flavin site, an aldehyde substrate site, and an aldehyde inhibitor site. The on and off rates of the decanal inhibitor binding were substantially slower than the bindings of decanal and reduced riboflavin 5'-phosphate substrates. These findings are consistent with a scheme that the aldehyde inhibitor blocks the binding of the reduced flavin substrate.
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PMID:Catalytically active forms of the individual subunits of Vibrio harveyi luciferase and their kinetic and binding properties. 762 95

To eliminate highly antigenic substances, bovine pericardium was washed in 5% sodium chloride (NaCl) for 24 hours, followed by incubation in trypsin for 40 minutes. To achieve adequate fixation, NaCl-trypsin-treated pericardium was preserved in glutaraldehyde (GA) solution with gradually increasing concentrations from 0.1% to 0.25%. To inactivate the free aldehyde groups and residual GA on the surface of the implant, NaCl-trypsin-GA-treated pericardial samples were posttreated separately with 1% lysine, 8% monosodium glutamate, and 4% chitosan. Fresh (untreated) and 0.1%, 0.2%, and 0.625% GA-treated and NaCl-trypsin-GA-treated pericardial specimens were prepared for comparative study. All samples were implanted subdermally in rats for 2, 4, 8, and 12 weeks for calcification studies. Morphologic and chemical analyses showed mild calcification in fresh pericardia (Ca, 10.5 +/- 1.25 micrograms/mg, von Kossa +) and in glutamate-posttreated pericardia (Ca, 11.5 +/- 3.45 micrograms/mg, von Kossa +). Calcium was practically undetectable in chitosan-posttreated implants (Ca, 1.1 +/- 0.27 micrograms/mg, von Kossa 0), whereas severe calcification was noticed in the rest of the samples (mean Ca greater than 200.0 micrograms/mg, von Kossa ) at 12 weeks. This study suggests that posttreatment with an amino compound such as chitosan would prevent the calcification of GA-treated bioprostheses at an early implantation stage, but elimination of antigenic factors and adequate GA fixation would prevent tissue degeneration, thus enabling the prosthesis to function over a long period.
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PMID:Prevention of calcification of heart valve bioprostheses: an experimental study in rat. 764 84

The regularities of directed polycondensation of proteins with opposite charges by glutaric aldehyde were studied using serum albumin and proteolytic enzymes (urokinase, trypsin, alpha-chymotrypsin) as models. The electrostatic interaction of oppositely charged protein macromonomers in the polycondensation process was shown to facilitate the selective synthesis of soluble heteroprotein conjugates with molecular mass from 2 x 10(5) to 9 x 10(5); these conjugates have controlled component composition, high enzyme content (up to 3 to 4 molecules of an enzyme per one albumin molecule) and completely retain the enzyme catalytic activity. The dependence of rate constants and activation parameters of thermal inactivation of the heteroconjugates upon their composition, molecular mass and the degree of modification of the protein amino groups was investigated. Heteroconjugates of electrically asymmetric proteins, in particular, trypsin and serum albumin, were found to be several hundred times more stable than the starting enzymes at 38-60 degrees C.
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PMID:[Effect of electrostatic interactions on formation and properties of soluble heteroprotein conjugates based on proteolytic enzymes]. 766 65

The distribution and density of metachromatic cells (MCC) and mast cells containing chymase plus tryptase (MCTC) or tryptase alone (MCT) were studied in the nasal mucosa by dye-binding methods and immunohistochemical analysis. Biopsies were obtained from 17 subjects with birch pollen allergy before and during the peak season and from nine healthy controls. Six patients were treated with an intranasal glucocorticosteroid before and during the season in an open study. Hay fever patients, even when asymptomatic, showed signs of mast cell system activation, exhibiting an increased number of mast cells in the nasal epithelium. Basophils, lacking immunohistochemically detectable tryptase, were not a major component of the mast cell response. MCT, most conspicuous in the epithelium, were found to be the most frequent mast-cell type in the nasal mucosa of allergic, but not of normal, subjects. Only 33% of the epithelial, but 90% of the stromal, immunopositive cells in the atopic mucosa before as well as during the season were MCC. Intraepithelial MCT thus displayed a low capacity to stain metachromatically, indicating a relative deficiency of the glycosaminoglycan (heparin) component of the granules. Intraepithelial mast cells also appeared to be markedly sensitive to steroid treatment and aldehyde fixation. The findings suggest that the lack of chymase, the characteristic feature of MCT, may reflect a functional activation of the mast cells, rather than a stable phenotypic differentiation related to anatomic site.
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PMID:Proteinase content of mast cells of nasal mucosa; effects of natural allergen exposure and of local corticosteroid treatment. 774 Nov 84

The multicatalytic proteinase complex (MPC) or proteasome is a multimeric, high-molecular-weight (700,000), extralysosomal proteolytic enzyme found in eukaryotes and in archaebacteria. Its multiple catalytic sites grant it a broad cleavage specificity toward short peptides and protein substrates. The pH optima of the catalytic activities of MPC are in the neutral or slightly alkaline range. We present here evidence for cryptic catalytic components of MPC optimally active at an acidic pH. Studies with a hydrophobic fluorescent probe provide direct evidence for conformational changes brought about by exposing the complex to an acidic environment. One of the newly described components, designated "acidic chymotrypsin-like activity," cleaves the Leu-2-naphthylamide bond in the substrate Boc-Val-Glu-Ala-Leu-2-naphythylamide. Compared with the classical "neutral" chymotrypsin-like activity defined by cleavage of the Leu-p-nitroanilide bond in Z-Gly-Gly-Leu-p-nitroanilide, the newly described component is not inhibited by monovalent cations and is less sensitive to the peptidyl aldehyde Z-Gly-Gly-leucinal, an inhibitor of the neutral chymotrypsin-like activity. In addition, we describe the properties of a novel potent peptidyl aldehyde, Z-Ile-Glu(OtBu)-Ala-leucinal, which is an inhibitor of both the acidic and neutral chymotrypsin-like activities of MPC, with IC50 values of 0.25 and 6.5 microM, respectively. In the presence of 65 microM of the newly synthesized peptidyl aldehyde, other MPC components such as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities were decreased only by 14 and 9%, respectively. The hydrophobicity, potency, and specificity of Z-Ile-Glu(OtBu)-Ala-leucinal toward the chymotrypsin-like activities of the complex make it a valuable pharmacological tool with which to investigate the physiological roles of MPC.
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PMID:A novel chymotrypsin-like component of the multicatalytic proteinase complex optimally active at acidic pH. 787 5

An immunohistochemical double-labelling technique for the simultaneous identification of mast cells containing tryptase alone (MCT) or chymase together with tryptase (MCTC) was evaluated quantitatively using two monoclonal antibodies, mAb 1222A (antitryptase) and mAb 1254B (antichymase). Saturation conditions were established for the binding of the antibodies to the mast cell enzymes by counting labelled mast cells in consecutive sections of normal human intestine incubated with serial dilutions of the antibodies. When, under such conditions, the antitryptase was applied after saturation with mAb 1254B, the reproducibility of the double-labelling procedure was excellent. MCT were located preferentially in the intestinal mucosa but, in contrast to what has previously been reported, they were not the predominant type of mast cell at this site. The percentage of MCT of the total number of immunopositive mast cells varied considerably in the colonic mucosa (7-67%, average 30%), while this was not the case in the small intestinal mucosa (5-26%, average 10%). Mast cell chymase, unlike tryptase, was not recognized by the antichymase antibody after aldehyde fixation and a higher apparent fraction of MCT therefore occurred after double labelling. These findings suggest that the proteinase composition of human mast cells, unlike that of murine mast cells, should not be taken as evidence of phenotypic heterogeneity. Taken together with previous observations, they suggest instead that the lack of chymase may be related to functional activity or stage of maturation of the mast cells.
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PMID:The immunohistochemical demonstration of chymase and tryptase in human intestinal mast cells. 796 Sep 36

Evidence indicates that a component of the multicatalytic proteinase complex (MPC) that preferentially cleaves bonds after branched chain amino acids (BrAAP) is a major factor responsible for the protein-degrading activity of the MPC. We report here the synthesis of substrate-related peptidyl aldehydes that inhibit the activity of this component toward both synthetic peptide substrates and proteins. The most potent of the inhibitors, Cbz-Gly-Pro-Phe-leucinal (Cbz-GPFL-CHO) inhibits competitively with a Ki of 1.5 microM. The peptidyl aldehydes also inhibit the small neutral amino acid preferring and the peptidylglutamyl-peptide hydrolyzing activities of the MPC. The chymotrypsin-like activity is only weakly inhibited, and the trypsin-like activity is moderately activated. The importance of a Pro residue in the P3 position and a leucinal residue in the P1 position for inhibition of the BrAAP component is indicated by the finding that replacement of these residues by a glycine or phenylalaninal, respectively, markedly increases the Ki. Cbz-GPFL-CHO inhibited the BrAAP activity with the same Ki both before and after activation of this component by exposure of the MPC to 3,4-dichloroisocoumarin, suggesting that the peptidyl aldehyde is an effective inhibitor of both the overt and latent proteolytic activities of the MPC. Incubation of a human breast cancer cell line (MCF-7) in culture with the inhibitors of the BrAAP component led to an accumulation of ubiquitin-protein conjugates, indicating inhibition of the ubiquitin-dependent proteolytic pathway.
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PMID:Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes. 796 80

Polyacrolein microspheres covalently coupled onto solid surfaces, e.g., glass, silicon crystals, and polystyrene, have been prepared and characterized. These surfaces were synthesized by derivatization of the substrate surfaces with SiCl3(CH2)3CN or Si(OEt)3-(CH2)4NH2. The terminal nitrile groups were then reduced to primary amine groups. Polyacrolein microspheres of various diameters (0.08 and 0.4 microns) were then covalently bound in a monolayer structure to the modified surfaces. This binding of the microspheres to the derivatized surfaces is achieved via polyvalent Schiff-base bonds formed by the interaction between aldehyde groups of the microspheres and omega-primary amine groups of the modified surfaces. Residual amine groups were blocked with acetic acid N-hydroxysuccinimide ester. The residual aldehyde groups of the immobilized microspheres can then be used for covalent binding of amino ligands, e.g., proteins, in a single step and at physiological pH (or any other desired pH). The potential use of the immobilized polyacrolein microsphere surfaces for diagnostics has been demonstrated by determination of alpha 1-antitrypsin in human serum with trypsin bound to the immobilized microspheres. The comparison between this new method for the determination of alpha 1-antitrypsin and the routine methods is discussed.
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PMID:Synthesis, characterization, and use of immobilized polyacrolein microspheres in diagnostics: a model determination of alpha 1-antitrypsin in human serum. 797 67

Acetaldehyde readily reacted with red blood cells and isolated haemoglobin to form adducts. We examined acetaldehyde-haemoglobin reaction products, isolated from red blood cells incubated with acetaldehyde (AcH), for the presence of stable peptide-specific acetaldehyde adducts. Red blood cell incubations were with 20, 50, and 200 mM acetaldehyde for 3, 24 and 48 hr. Peptide-specific [14C]acetaldehyde modifications of Hb from each incubation were identified by tryptic peptide mapping procedures. Nine peptides had modifications and six were found in incubations with 20 mM acetaldehyde. Two of the peptides were acetaldehyde modifications of the Hb alpha- and beta-chain N-termini. Stability studies indicated that the remaining seven [14C]AcH-modified peptides did not result, as can occur under certain conditions, from the transfer of AcH on the N-termini of Hb to N-termini formed after trypsin digestion. An analysis of the relative amounts of [14C]AcH-peptide adducts indicated that at least two of the seven peptides had reactivities for AcH different than the N-termini of Hb; that is, at least two modified peptides differ from imidazolidine-type adducts formed on the N-termini. The presence of multiple modifications with different sensitivities for AcH adduct formation may be useful in developing markers of alcohol consumption.
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PMID:Evidence for the formation of multiple types of acetaldehyde-haemoglobin adducts. 800 14


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