Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human alpha-1-antichymotrypsin has been purified to homogeneity by the following sequential steps--(a) ammonium sulfate fractionation; (b) chromatography on Cibacron Blue Sepharose at pH 7.0; and (c) chromatography on SP-Sephadex C-50 at pH 5.5. The inhibitor has a molecular weight near 68,000 and contains approximately 26% carbohydrate alpha-1-Antichymotrypsin has an amino-terminal arginine and a carboxy-terminal glycine. It also has some homology with alpha-1-PI based on amino-terminal sequence analysis of both proteins. Complexes of alpha-1-antichymotrypsin with human chymotrypsin and human leukocyte cathepsin G are stable in sodium dodecyl sulfate and have molecular weights near 90,000 suggesting 1:1 complex formation on a molar basis between inhibitor and enzyme.
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PMID:Human alpha-1-antichymotrypsin: purification and properties. 10 76

The presence of two distinct high-molecular-weight proteases with similar pH optima in the weakly alkaline region was shown in cytosol of the bovine brain cortex. They were separated by ammonium sulfate fractionation and each was further purified by DEAE-Sephacel, Sephacryl S-300, DEAE-Cibacron Blue 3GA-agarose, heparin-agarose, and Sepharose 6B chromatography. The larger enzyme (Mr 1,400 kDa), which precipitates at 0-38% ammonium sulfate saturation, seems to be active in ATP + ubiquitin (Ub)-dependent proteolysis; it has low basal caseinolytic activity that is stimulated 3-fold by ATP, and when Ub is present ATP causes a 4.5-fold stimulation. A second proteinase was also found to be present (Mr 700 kDa) that precipitates at 38-80% ammonium sulfate saturation, is composed of multiple subunits ranging in Mr from 18 to 30 kDa, and degrades both protein and peptide substrates, demonstrating trypsin-, chymotrypsin- and cucumisin-like activities. Catalytic, biochemical, and immunological characteristics of this proteinase indicate that it is a multicatalytic proteinase complex (MPC), whose enzyme activity, in contrast to that of MPC from bovine pituitaries (1-3), is stimulated 1.7-fold by addition of ATP in the absence of ubiquitin at the early steps of purification; this property is lost during the course of further purification. Both proteinases are present in the nerve cells, since the primary chicken embryonic telencephalon neuronal cell culture extracts contain both ATP + Ub-dependent proteinase and MPC activities.
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PMID:The presence of ATP + ubiquitin-dependent proteinase and multicatalytic proteinase complex in bovine brain. 255 23

The interaction between complement component factor B and the triazine dye ligand Cibacron Blue F3G-A coupled to a cross-linked agarose matrix (Blue Sepharose) was found to involve the Bb part of the molecule, and to be inhibited by benzamidine. Human, chicken and rainbow trout factor B which had bound to Blue Sepharose could, subsequently be eluted with benzamidine. Other serine proteases (C2, factor II, factor IX, trypsin, chymotrypsin, proteinase 3) also bound to Blue Sepharose but only those belonging to the trypsin family could be eluted with benzamidine. Trypsin treated with the active-site inhibitor phenylmethylsulfonyl fluoride did not bind to Blue Sepharose and pretreatment of Blue Sepharose with benzamidine did not influence binding of proteases. We conclude that trypsin-like serine proteases can be purified on Blue Sepharose and that the interaction of these serine proteases with Blue Sepharose involves the active site of the enzyme.
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PMID:Affinity chromatography of serine proteases on the triazine dye ligand Cibacron Blue F3G-A. 983 58

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.
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PMID:Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase. 1023 36

In the present study, we report the development of a cellulose-based affinity adsorbent and its application for the purification of proteases from fish by-products. The affinity adsorbent was synthesized using cellulose microfibers as the matrix, isolated from recycled newspapers using the acid precipitation method. As an affinity ligand, the triazine dye Cibacron Blue 3GA (CB3GA) was used and immobilized directly onto the cellulose microfibers. Absorption equilibrium studies and frontal affinity chromatography were employed to evaluate the chromatographic performance of the adsorbent using as model proteins bovine serum albumin (BSA) and lysozyme (LYS). Absorption equilibrium studies suggest that the adsorption of both proteins obeys the Langmuir isotherm model. The kinetics of adsorption obey the pseudo-second-order model. The affinity adsorbent was applied for the development of a purification procedure for proteases from Sparus aurata by-products (stomach and pancreas). A single-step purification protocol for trypsin and chymotrypsin was developed and optimized. The protocol afforded enzymes with high yields suitable for technical and industrial purposes.
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PMID:Reduce, Reuse and Recycle in Protein Chromatography: Development of an Affinity Adsorbent from Waste Paper and Its Application for the Purification of Proteases from Fish By-Products. 3247 Dec 69