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)

Two clones were isolated by screening a shrimp hepatopancreas cDNA library with a DNA fragment obtained by PCR amplification using two oligonucleotides based on the partial protein sequence of Penaeus vanameii chymotrypsin purified earlier. One of these clones, PVC 7 contains a complete cDNA coding for a serine protease. The deduced amino acid sequence shows the existence of a 270 residue-long preproenzyme containing a highly hydrophobic signal peptide of 14 amino acids. This suggests the existence of a putative zymogen form of the enzyme containing a 30 amino acid-long peptide which is cleaved to give a mature protein of 226 residues. A highly preferred codon usage is observed for this protein. The other obtained cDNA was found to encode the less predominant variant of the protein. Sequence alignments show that shrimp chymotrypsin is highly homologous with crab collagenase (77% homology taking into account the same amino acid at the same position, and 83% homology taking into account amino acids with conserved function) and that it is more similar to mouse trypsin (41% homology of strictly conserved amino acids) than to hornet chymotrypsin (35% homology).
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PMID:Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda). 151 90

Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (alpha'2 in cones and alphabeta in rods) with inhibitory subunits (gamma) that regulate their activity. In addition, a 17-kDa protein referred to as the delta subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod delta subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest delta subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod delta subunit anti-peptide antibodies label rod but not cone outer segments. delta subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the delta subunit to the phosphodiesterase holoenzyme.
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PMID:Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant delta subunit. 879 40

Leukotriene A(4) (LTA(4)) is a chemically unstable triene epoxide product of 5-lipoxygenase metabolism of arachidonic acid. Despite this chemical reactivity and its synthesis at the perinuclear membrane, LTA(4) is enzymatically converted into the cysteinyl leukotrienes and leukotriene B(4). Furthermore, LTA(4) participates in transcellular biosynthesis and is thus transferred between cells as an intact molecule. A cytosolic fatty acid-binding protein present in the rat basophilic leukemia cells was identified using mass spectrometry. This protein was determined to be the stabilizing factor present in the cell cytosol responsible for increasing the effective chemical half-life of LTA(4). Rat epithelial fatty acid-binding protein (E-FABP) was isolated using partial protein purification and immunoprecipitation. In-gel digestion with trypsin followed by peptide fingerprint analysis using matrix-assisted laser desorption ionization mass spectrometry and sequencing the major tryptic peptide obtained from liquid chromatography/mass spectrometry/mass spectrometry analysis identified E-FABP in the active fraction. Semi-quantitative Western blot analysis indicated that E-FABP in the cytosolic fraction of RBL-1 cells was present at approximately 1-3 pmol/10(6) cells. E-FABP (9 microm) was tested for its ability to stabilize LTA(4), and at 37 degrees C E-FABP was able to increase the half-life of LTA(4) from the previously reported half-life less than 3 s to a half-life of approximately 7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA(4) to be available for further enzymatic processing in various cellular regions.
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PMID:Stabilization of leukotriene A4 by epithelial fatty acid-binding protein in the rat basophilic leukemia cell. 1467 86

Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD65 and GAD67, were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD65 and GAD67 exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the Km of GAD67 and increasing the Vmax of both isoforms. Of the 15 cysteines in GAD65, the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD67, cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD65 and GAD67 are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD65 suggests that alteration of normal redox conditions could affect GAD targeting.
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PMID:Glutamate decarboxylase: loss of N-terminal segment does not affect homodimerization and determination of the oxidation state of cysteine residues. 1625 48

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A.
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PMID:Purification, characterization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds. 1871 90

One of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates, which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution. The molecular basis for this widespread problem remains hazy. To investigate the mechanism of inhibition at a molecular level, we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogen-deuterium exchange mass spectrometry. For AmpC beta-lactamase, binding to aggregates of the small molecule rottlerin increased the deuterium exchange of all 10 reproducibly detectable peptides, which covered 41% of the sequence of beta-lactamase. This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation. We then investigated whether enzyme-aggregate complexes were more susceptible to proteolysis than uninhibited enzyme. For five aggregators, trypsin degradation of beta-lactamase increased substantially when beta-lactamase was inhibited by aggregates, whereas uninhibited enzyme was generally stable to digestion. Combined, these results suggest that the mechanism of action of aggregate-based inhibitors proceeds via partial protein unfolding when bound to an aggregate particle.
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PMID:Promiscuous aggregate-based inhibitors promote enzyme unfolding. 1928 Dec 22

The protein inhibitor of cysteine proteases was isolated from an important zoophilic dermatophyte species Trichophyton mentagrophytes (T. mentagrophytes) and partially characterized. The isolation process involved affinity chromatography, followed by ion-exchange chromatography and reverse phase high performance liquid chromatography. The fungal inhibitor appears to exist in a high (24 kDa) and low (12 kDa) molecular mass form. It inhibits proteolytic activity of papain, cathepsins B and L but not of cathepsin H or trypsin. Results of immunoblotting procedures indicate that sera of T. mentagrophytes infected rabbits contain antibodies against higher molecular mass forms of the inhibitor. Since no sequence homology has been found between partial protein sequences of T. mentagrophytes inhibitor and other known cysteine protease inhibitors so far, we can speculate that this inhibitor has some structurally unique characteristics. The T. mentagrophytes inhibitor shares some biochemical similarities (molecular mass, high and low molecular mass forms, inhibitory profiles) with clitocypin from Clitocybe nebularis and macrocypins from Macrolepiota procera.
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PMID:Dermatophyte Trichophyton mentagrophytes Produces Cysteine Protease Inhibitor. 2406 40