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 aim of the present study was to develop a chemical treatment to eliminate highly antigenic substances, to standardize the glutaraldehyde fixation procedure, to determine the dominant factors contributing to the calcification process and to understand the role of macromolecules like chitosan in the prevention of calcification of bioprosthetic heart valves. Bovine pericardium treated with 5% sodium chloride-trypsin-glutaraldehyde (GA)-chitosan did not calcify at 12 wk in the rat (calcium, 1.1 +/- 0.27 mg/g; von Kossa, 0). Slow release of residual GA from the bioprosthesis and free aldehyde groups on that are still considered the dominant factors for enhancing calcification of GA-treated bioprostheses.
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PMID:Anticalcification treatment of pericardial prostheses. 808 Sep 38

Tripeptide aldehydes such as Boc-D-Phe-Pro-Arg-H (51) exhibit potent direct inhibition of thrombin. This distinction offers important insight for the design of more potent and selective serine protease inhibitors which may be useful pharmacological tools and hold promise for development of clinically useful agents. The structure-activity relationships (SAR) on a series of anticoagulant peptides with high selectivity for the enzyme thrombin are discussed. The SAR is centered on a series of di- and tripeptide arginine aldehydes based on the structure of 51. The structural and conformational role of the amino acid residue in position 1 was investigated by substitution with conformationally restricted aromatic amino acids, aromatic acids, and a dipeptide isostere containing the psi[CH2N] amide bond replacement. Many of these peptides demonstrate potent antithrombotic activity along with selectivity toward thrombin, determined by comparison of in vitro inhibitory effects on trypsin, plasmin, factor Xa, and tissue plasminogen activator. Compound 5f, D-1-Tiq-Pro-Arg-H.sulfate is highly active and the most selective tripeptide aldehyde inhibitor of thrombin reported to date.
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PMID:Highly selective tripeptide thrombin inhibitors. 842 61

Acetaldehyde, the first product of alcohol metabolism, is highly reactive. Several proteins have been shown to be covalently modified by acetaldehyde in vivo. We have previously reported the detection of a cytosolic 37-kd protein-acetaldehyde adduct (-AA) in the liver of alcohol-fed rats. The liver extract from an alcohol-fed rat was subjected to 2-dimensional (2D) sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and the 37-kd protein-AA spot was digested with trypsin and sequenced for amino acids. Degenerate oligonucleotides corresponding to a peptide sequence of the protein-AA were used as the probe to screen a lambda gt11 rat liver complementary DNA (cDNA) library. A clone that extended to a potential ATG start codon was identified. The open reading frame was 978 nucleotides long, encoding 326 amino acid residues. The sequence matched that of rat liver delta 4-3-ketosteroid 5 beta-reductase. The cloned cDNA was expressed in Escherichia coli using pGEX-KG as the vector. The expressed protein was found to be of correct molecular weight. It reacted with an antibody that recognized the unmodified liver 37-kd protein by Western blotting. Peptide profiles of tryptic-digested recombinant protein and the purified rat liver 37-kd protein were similar and yielded the same peptide sequence. delta 4-3-ketosteroid 5 beta-reductase catalyzes the reduction of key intermediates during bile acid biosynthesis. Whether modification of the 5 beta-reductase by acetaldehyde affects the enzyme activity and bile acid synthesis remains to be studied.
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PMID:Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as delta 4-3-ketosteroid 5 beta-reductase. 855 30

Hepatic fibrosis often occurs in alcoholic liver diseases without accompanying tissue necrosis or inflammation. However, the precise mechanism of this fibrosis has not been fully clarified. In the present study, using the hepatoblastoma cell line HepG2 as a model for hepatocytes, we identified a factor that stimulates collagen synthesis of fibroblasts in a conditioned medium of HepG2 cells after treatment with ethanol. Type 1 procollagen peptide (PIC) in a culture of human fibroblast IMR-90 markedly increased after incubation with the conditioned medium of ethanol-treated HepG2 cells. The stimulating activity on the production of PIC by IMR-90 remained after the dialysis and evaporation of the conditioned medium of HepG2 cells, indicating this factor was not as volatile from low molecular substances such as acetaldehyde, acetate, or lactate. The activity of this factor diminished with heat or trypsin treatment. A gel chromatographic analysis disclosed that the molecular weight of this factor was approximately 8000 Da. These results suggest that a polypeptide factor secreted from HepG2 cells by treatment with ethanol stimulates collagen synthesis of fibroblasts.
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PMID:Detection of activity in the conditioned medium of ethanol-treated HepG2 cells which stimulates collagen synthesis in IMR-90 cells. 865 93

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities, and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. To determine the possible role that proteasomes may play in controlling the life cycle of African trypanosomes, we have isolated proteasomes from the bloodstream and the insect (procyclic) forms of Trypanosoma brucei by DEAE-cellulose chromatography and glycerol gradient fractionation in the presence of ATP. No 26 S proteasome homologs was identified in T. brucei under these experimental conditions. The proteasomes isolated from these two forms of T. brucei are very similar to the rat blood cell 20 S proteasome in their general appearance under the electron microscope. The profile of trypanosome proteasome subunits in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has eight visible protein bands with molecular weights ranging from 23 to 34 kDa, and cross-reacted very poorly with the anti-human 20 S proteasome antibodies on immunoblots. Two-dimensional gel electrophoresis of the parasite proteasomes shows a similar number of major subunits with pI's ranging from 4.5 to 7. Using a variety of fluorogenic peptides as substrates, the trypanosome proteasomes exhibited unusually high trypsin-like, but somewhat lower chymotrypsin-like activities, as compared to the rat 20 S proteasome. These proteolytic activities were, however, insensitive to phenylmethylsulfonyl fluoride (PMSF), tosyl-phenylalanine chloromethylketone (TPCK), tosyl-lysine chloromethylketone (TLCK) and trans-epoxy succinyl-L-leucylamido-(4 guanidino) butane (E-64), but the trypsin-like activity of trypanosome proteasomes was inhibited by leupeptin, an aldehyde known to inhibit the trypsin-like activity of mammalian proteasomes, thus ruling out possible contamination by other serine or cysteine proteases. Some quantitative differences in the substrate specificities between the proteasomes from bloodstream and procyclic forms were indicated, which may play a role in determining the differential protein turnovers at two different stages of development of T. brucei.
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PMID:Purification and characterization of proteasomes from Trypanosoma brucei. 881 75

Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57.
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PMID:Two crystal structures of the leupeptin-trypsin complex. 884 65

African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolysing activities determined by fluorogenic peptides, Z-Gly-Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively. The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z-Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxidized proteins. In addition, the chymotryptic and trypsin-like activities were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.
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PMID:Characterization of a multicatalytic proteinase complex (20S proteasome) from Trypanosoma brucei brucei. 922 59

Mucosal mast cells (MCs) are normally found in the connective tissue stroma but are redistributed into the epithelium in conditions associated with immunoglobulin E responses, such as allergic inflammation and nematode infections, as well as in interstitial cystitis, a condition of unknown etiology. The potential role of epithelium-derived factors in this response prompted this inquiry into growth and differentiation signaling in normal tissue as well as in tissues from five different metaplastic conditions of the urothelium (cystitic cystica, cystitis glandularis, colonic metaplasia, squamous cell metaplasia, and nephrogenic metaplasia). Expression of the two major human MC growth factors, stem cell factor (or kit ligand) and interleukin 6, was detected using immunohistochemistry. In the case of interleukin 6, its mRNA expression was also detected using in situ reverse transcription-polymerase chain reaction. Among the different metaplastic lesions, nephrogenic metaplasia was the only one associated with an abundance of MCs, which were distributed within or in close relationship to the epithelium. Unlike in the other types of metaplasia, the epithelium strongly co-expressed interleukin 6 and stem cell factor. The MCs expressed the stem cell factor receptor CD117 and exhibited a variable tryptase immunoreactivity, but lacked chymase. They also displayed a relative deficiency of granular glycosaminoglycan, as indicated by a lack of metachromasia, and were sensitive to strong aldehyde fixation. The findings suggest that the MC response in nephrogenic metaplasia may be the result of local epithelial stem cell factor/interleukin 6 expression.
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PMID:Metaplastic transformation of urinary bladder epithelium: effect on mast cell recruitment, distribution, and phenotype expression. 966 75

Purified membrane fractions have been widely used for the study of the factors regulating the functions of Rho small GTP-binding proteins. Using brush border membranes from the rat kidney as a model, we observed that in vitro incubation of these membranes resulted in time- and temperature-dependent proteolytic degradation of Cdc42 and RhoA. Treatment of kidney brush border membranes with various nucleotides showed that GDP and GTP weakly protected Cdc42 but not RhoA and that their nonhydrolyzable counterparts, guanosine 5'-O-[beta-thio] diphosphate (GDP beta S) and guanosine 5'-O-[gamma-thio]triphosphate (GTP gamma S), were highly efficient in protecting both proteins from endogenous proteolytic activity whereas ADP and ATP were without effect. GTP gamma S also protected Cdc42 and RhoA from proteolytic degradation in crude cell membranes from several rat tissues including intestine, kidney, liver, and testis. In addition, Cdc42 and RhoA associated with brush border membranes were largely resistant to increased proteolytic degradation induced by membrane treatment with the denaturing reagent urea as well as to added trypsin when incubated in the presence of GTP gamma S. In brush border membranes, the resistance to endo- and exo-genous proteolytic activity conferred by GTP gamma S was usually lower for RhoA than for Cdc42. GTP gamma S also protected recombinant Cdc42 and RhoA from the action of proteases associated with brush border membranes. The only protease inhibitor protecting Cdc42 but not RhoA from proteolytic degradation in brush border membranes was the synthetic peptide acetyl-Tyr-Val-Ala-Asp-aldehyde, a selective inhibitor of interleukin-1 beta-converting enzyme. This latter result showed that different proteases cleaved the two Rho proteins. Taken together, these results suggest that the GTP gamma S-bound forms of Cdc42 and RhoA are maintained in a conformation that protects them from proteases found in many cell membranes.
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PMID:Guanine nucleotides protect Rho proteins from endogenous proteolytic degradation in renal membranes. 966 7

The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined and refined to R values 0.152-0. 170 at 1.8-2.1 A resolution. Since the selectivity of two of the inhibitors was >1600 with respect to trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also determined (R = 0.142-0.157 at 1.9-2.1 A resolution). The selectivity appears to reside in the inability of a benzenesulfonamide group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. Differences in the binding constants of these inhibitors are correlated with their interactions with thrombin and trypsin. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism. The structures of both thrombin and trypsin complexes show similar well-defined transition states in the S1 site and at the electrophilic carbon atom and Ser195OG. The trypsin structures, however, suggest that the first step in a two-step kinetic mechanism may involve formation of a weak transition-state complex, rather than binding dominated by the P2-P4 positions.
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PMID:Highly selective mechanism-based thrombin inhibitors: structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. 972 21


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