Protease trypsin

Meanwhile we have shown that the excision activation of ICOR channels is due to disinhibition [72]. The respective inhibitor, operationally named cytosolic inhibitor (Cl), is present in the cytosol of placenta trophoblast cells HT29- and Tg4-colonic carcinoma cells and RE cells of normal and CF patients. The molecule has an apparent molecular weight of 700-1 500 Da it is amphiphilic heat stable and not digested by trypsin, proteases, nucleotidases, lipases or amylase [72]. Burc-khardt, Fromter and their collaborators [114] have confirmed our results and extracted a similar or identical Cl from kidney cortex. 

NS3 is a 631 amino acid protein, and its first 180 amino acids encode a serine protease of the chymotrypsin family (Figure 2.2A). It has a typical chymotrypsin-family fold consisting of two jS-barrels, with catalytic triad residues at the interface. His-57 and Asp-81 are contributed by the N-terminal jS-barrel and Ser-139 from the C-terminal jS-barrel. NS3 and closely related viral proteases are significantly smaller than other members of the chymotrypsin family, and many of the loops normally found between adjacent jS-strands in trypsin proteases are truncated in NS3 [31]. Probably... 

Trypsin and factor Xa (fXa) are two members of the chymotrypsin family that have 38% sequence identity on the amino acid level and have distinguishable substrate specificities. Recently, the N-terminal 13-barrel of fXa and the C-terminal /3-barrel of trypsin were fused at a rationally designed site in the linker region between the two domains in order to create a hybrid fXa-trypsin protease (Hopfner et al., 1998). The fXa-trypsin hybrid was highly active and more active than either parent on three of the ten substrates assayed, as determined by k /Km. For most substrates, the activity of fXa-trypsin was an admixture of the two parents, probably because trypsin had higher activity than fXa for all the substrates tested. 

Dissolution and Separation by Special Applications Enzymes and Microextraction Several enzymes such as trypsin, protease type XIV, lipase and/or cellulase, are used for enzymatic hydrolysis. For the determination of Hg2+ and Me-Hg in fish... 

Chymo trypsin protease bovine or porcine pancreatic extract none 3.4.21.1... 

Horseradish peroxidase Trypsin (protease) Subtilisin (protease) Phenol polymerization Transpeptidation Ester hydrolysis Ethylacetate Butan-l,4-diol Dioxane, chloroform, etc. 

Enzymes (trypsin, proteases) Used in techniques to amplify and retrieve antigens for immunohistochemistr)r, enclosed, automated used in epidermal separation techniques N/N Many... 

Figure 37.6 Effect of flavin cofactor binding on the stability of the human electron-transfer flavoprotein (ETF) mutant variant Aspl28Asn. (A) Activity of the protein is affected by incubation at 39 °C (open circles) however, in the presence of 2.5-fold excess FAD the activity is preserved (black circles). (B) The stability of ETF Aspl28Asn to urea-induced chemical denaturation is higher when the flavin is bound to the protein (black circles) than in flavin-depleted ETF (open circles). (C) The presence of flavin cofactor affects the proteolytic susceptibility of ETF Aspl28Asn. Upon incubation with trypsin protease ETF Aspl28Asn is rapidly degraded (top panel), whereas in the presence of excess flavin, the protein is more resistance to proteolysis.

Glawe GA, Zavala JA, Kessler A, Van Dam NM, Baldwin IT (2003) Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attemiata. Ecology 84 79-90... 

Liu et alP proposed a simple approach for the construction of a trypsin-FITC( MOF bioreactor by a 30-min vortex-assisted host-guest interaction to immobilize the trypsin protease in mesoporous CYCU-4... 

Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

Prior to the bating process, the hides are delimed with ammonium sulfate and/or ammonium chloride. Proteases are then appUed. The early preparation proposed by Rn hm was pancreatic trypsin. The use of a bating enzyme makes the hides soft and supple to prepare them for tanning. A new microbial protease, Pyrase 250 MP (82) (Novo Nordisk A/S) has been found to be a promising substitute for pancreatic trypsin [9002-07-7] which is more expensive because it must be extracted from pancreatic glands. 

Phenylmethanesulfonyl fluoride (PMSF) [329-98-6] M 174.2, m 90-91 , 92-93 . Purified by recrystn from ""CgHe, pet ether or CHCl3-pet ether. [Davies and Dick J Chem Soc 483 1932 cf Tullock and Coffman J Org Chem 23 2016 I 960.] It is a general protease inhibitor (specific for trypsin and chymotrypsin) and is a good substitute for diisopropylphosphoro floridate [Fahrney and Gould 7 Am Chem Soc 85 997 1963]. 

Figure 2.14 shows examples of both cases, an isolated ribbon and a p sheet. The isolated ribbon is illustrated by the structure of bovine trypsin inhibitor (Figure 2.14a), a small, very stable polypeptide of 58 amino acids that inhibits the activity of the digestive protease trypsin. The structure has been determined to 1.0 A resolution in the laboratory of Robert Huber in Munich, Germany, and the folding pathway of this protein is discussed in Chapter 6. Hairpin motifs as parts of a p sheet are exemplified by the structure of a snake venom, erabutoxin (Figure 2.14b), which binds to and inhibits... 

Sprang, S., et al. The three-dimensional structure of Asn ° mutant of trypsin role of Asp ° in serine protease catalysis. Science 237 905-909, 1987. 

FIGURE 5.20 Trypsin is a proteolytic enzyme, or protease, that specifically cleaves only those peptide bonds in which arginine or lysine contributes the carbonyl function. The products of the reaction are a mixture of peptide fragments with C-terminal Arg or Lys residues and a single peptide derived from the polypeptide s C-terminal end. 

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

There are indications that the crystal protein is subject to proteolytic enzymes when separated from the sporangium. The crystal protein has also been shown to be degradable by fairly nonspecific proteases such as pepsin and trypsin. 

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