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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)

Homotropic cooperative binding was observed at vapor sorption of organic solvents (acetonitrile, propionitrile, ethanol, 1-propanol, 2-propanol, nitroethane) by dried solid trypsin from porcine pancreas (0.05 g H2O/g protein). The vapor sorption isotherms were obtained by the static method of gas chromatographic headspace analysis at 298 K for 'vapor solvent+solid trypsin' systems in the absence of the liquid phase. All isotherms have a sigmoidal shape with significant sorbate uptake only above the threshold of sorbate thermodynamic activity. On the sorption isotherms of non-hydroxylic sorbates the saturation of trypsin by organic solvent was observed above the sorbate threshold activity. The formation of inclusion compounds with phase transition between solvent-free and solvent-saturated trypsin is supposed. Approximation of obtained isotherms by the Hill equation gives the inclusion stoichiometry S, inclusion free energy, and the Hill constant N of clathrates. The inclusion stoichiometry S depends significantly on the size and shape of sorbate molecules and changes from S=31 mol of sorbate per mol of trypsin for ethanol to S=6 for nitroethane. The inclusion free energies determined for the standard states of pure liquid sorbate and infinitely dilute solution in toluene are in the range from -0.5 to -1.2 kJ/mol and from -3.1 to -8.1 kJ/mol, respectively, per 1 mol of sorbate. The Hill constants are relatively high: from N=5.6 for 1-propanol to N approximately equal to 10(3) for nitroethane. The implication of the obtained results for the interpretation of solvent effects on the enzyme activity and stability in low-water medium is discussed.
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PMID:Homotropic cooperative binding of organic solvent vapors by solid trypsin. 1134 57

1. Several esters of the alpha-N-toluene-p-sulphonyl and N-benzoyl derivatives of l-lysine and S-(beta-aminoethyl)-l-cysteine have been synthesized. 2. The kinetics of hydrolysis of the esters by bovine trypsin have been compared. Values of k(0) are similar for corresponding derivatives of the isosteric amino acids and deacylation of an acyl-enzyme appears to be rate-determining in each case. There are, however, some quantitative kinetic differences between the various series of substrates.
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PMID:Kinetics and mechanism of catalysis by proteolytic enzymes: The kinetics of hydrolysis of derivatives of l-lysine and S-(beta-aminoethyl)-l-cysteine(thialysine)by bovine trypsin. 1674 86

An organic-solvent-tolerant bacterium strain YP1 producing organic-solvent-stable protease was isolated from crude oil contaminant soil. Strain YP1 was strictly aerobic, motile, gram positive, spore-forming, and rod shaped. The YP1 strain was identified as Bacillus licheniformis using culture system BIOLOG analysis (SIM = 0.62, 16-24h). The 16S rDNA sequence analysis (GenBank accession number EF105377) suggested that strain YP1 was clustered together with B. licheniformis in phylogenetic tree. Based on all the taxonomy,strain YP1 was identified as B. licheniformis. YP1 strain could tolerant organic solvents at different levels, especially it can grow well in the presence of water-miscible solvents dimethylformamide (DMF, logP = -1.0) and dimethylsulphoxide (DMSO, logP = -1.35) at a concentration of 10% [V/V]. Strain YP1 can also tolerant middle concentrations of NaCl and extra alkaline conditions (pHl2). More than 80% of the biomass remained at pH range 10.5-12. However strain YP1 was sensitive to antibiotics such as ampicillin, tetracycline, kanamycin and chloromycetin. The protease production could be enhanced by acetone and repressed by alkanols such as dodecylalcohol and octanol during the fermentation. Compared to trypsin, the YP1 protease had a wider tolerance for organic solvents. YP1 protease tolerated up to at least 11 organic solvents with logP ranging from -1.35 to 5.6 including benzene, toluene, DMSO and DMF etc at 50% (V/V) concentration. Moreover, when solvents such as decane and dodecyl alcohol with log P values above 4.0 were added to the crude protease, the enzyme activity levels were 1.08 and 1.21 times higher than the control respectively. Its high tolerance for water-miscible solvents DMF and DMSO makes it an ideal catalyst for kinetic- and equilibrium-controlled synthesis. This organic solvent stable protease could be used as a biocatalyst for enzymatic synthesis in the presence of organic solvents.
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PMID:[Screening and identification of an organic solvent-stable protease producer]. 1827 Dec 59

The graft copolymer, poly(maleic anhydride/styrene)-co-polyethylene was prepared. The copolymer immobilized bovine serum albumin (BSA), but the amount coupled appeared to be effected by the amount of styrene in the graft copolymer, temperature, and pH of the coupling medium. Competition existed between hydrolysis of the grafted anhydride groups and the protein. A graft copolymer with 66% add-on immobilized 4.5 mg/glucose oxidase/g copolymer, 4.6 mg alkaline phosphates/g copolymer and 0.2 mg cell of Bacillus stearothermophilus/g copolymer. A number of copolymers containing poly(maleic anhydride/vinyl acetate)-co-polyethylene were prepared to cover a range of grafting levels. These immobilized larger quantities of BSA, alkaline phosphatase, and cells of B. stearothermophilus than did the styrene graft copolymer. The copolymer was also hydrolyzed to release the hydroxyl group from the poly(vinyl acetate) component of the grafted chains. Using p-benzoquinone as the "activating agent," the copolymer coupled to BSA and to acid phosphatase. Using p-toluene-sulfonyl chloride, the copolymer was very effective in immobilizing trypsin.
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PMID:The immobilization of enzymes and cells of Bacillus stearothermophilus onto poly(maleic anhydride/styrene)-Co-polyethylene and poly(maleic anhydride/vinyl acetate)-Co-polyethylene. 1855 12

Attempts were made to solubilize acetylcholinesterase from Wistar rat brains by extraction with dilute buffer, Triton X-100 and proteolytic digestion. About 13% of the total enzyme activity could be solubilized with 30 mM sodium phosphate buffer (pH 7) and the remainder brought into solution with 1% w/v Triton X-100. Storage of the brains in dry toluene for 3-6 months followed by extraction did not improve the yield and resulted in the loss of about half of the enzyme activity. Digestion with trypsin or collagenase was totally ineffective in solubilizing the enzyme from fresh or toluene-stored brains. The enzyme in the buffer and detergent extracts of fresh and toluene-stored brains was very stable when stored at -20 degrees C for several months although some activity was found in a 100,000 g pellet obtained by centrifugation of the thawed extracts. All enzyme preparations showed inhibition by excess substrate and an optimum substrate concentration of 2 mM acetylcholine. The Km of the crude tissue suspension was 80 microM acetylcholine while that of the buffer-soluble enzyme was 91 microM and that of the detergent-solubilized enzyme was 250 microM. Storage of the brains in toluene had little effect on these values. Starch-block electrophoresis and polyacrylamide gel electrophoresis showed up to five bands with different net charge while gradient gel electrophoresis revealed up to eleven forms with molecular weights ranging from 39,000 to 450,000. The electrophoretic pattern obtained depended on the preparation and extraction of the tissue as well as the temperature and the presence of salt, mercaptoethanol and inhibitors. Storage of the tissue in toluene does affect the yield and the properties of acetylcholinesterase obtained from rat brain thus emphasising the need to clearly define the methods and conditions of solubilization when reporting the presence of multiple molecular forms of acetylcholinesterase.
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PMID:The effect of solubilization on the properties and molecular forms of rat brain acetylcholinesterase. 1964 67

There seems to be very little doubt but that luciferase is a protein or so closely associated with proteins that their removal destroys its characteristic properties. The particular group of proteins to which it belongs may be arrived at by a process of exclusion, and only the group of albumins has properties which agree completely with those of luciferase. Dubois believes Pholas luciferase to be an oxidizing enzyme similar to the oxydones of Battelli and Stern because it is readily destroyed by fat solvents such as chloroform, strong alcohol, etc. He has detected iron in a luciferase solution which has dialyzed against running water for a long time, and believes it to be made up of protein in combination with iron and to act as an "oxyzymase ferrique." Cypridina luciferase, on the other hand, is not readily destroyed by fat solvents. Toluene and chloroform are good preservatives, and I often make use of them for this purpose, keeping the luciferase solutions for many months. Professor A. H. Phillips of Princeton University has very kindly analyzed some whole dried Cypridinoe for me and finds iron, copper, and manganese but no zinc or vanadium to be present. Whether these metals are connected with the action of Cypridina luciferase is uncertain, but it is significant that all three of the metals thought to be concerned in organic oxidations are present. Although a large amount of luciferin mixed with a small amount of luciferase will use up all the latter, I agree with Dubois that luciferase has sufficient properties in common with the enzymes as a class to be considered an enzyme. The peroxidases are well known to be used up in the reactions they accelerate. All workers on enzymes agree that the more enzymes are purified the less active they become. The chemical procedures necessary to remove foreign material bring about irreversible changes in the enzyme itself, a characteristic also of many protein groups and of the colloidal state in general. This is true in the case of luciferase, for the crude luciferase solution is the most active preparation that can be obtained. I believe that Cypridina luciferase should be placed in a class of oxidizing enzymes by itself-a group having the chemical reactions of an albumin, possibly in combination with some heavy metal, and which as far as we know, acts specifically on only one substance, Cypridina luciferin. It resembles the plant peroxidases in resisting the action of chloroform, toluene, etc., but will not oxidize any of the hydroxyphenol or aminophenol compounds so readily oxidized by the peroxidases, nor will the peroxidases oxidize luciferin with light production. Dubois' researches show that Pholas luciferase differs in some properties from Cypridina luciferase, and my own work indicates that firefly luciferase is more like that of Pholas. A comparative study of other species of luminous animals is needed in order to delimit more accurately the class of luciferases as a whole. Luciferin presents many characteristics in common with the proteins, but two, which, to say the least, throw doubt on its protein nature: (1) its peculiar solubility (in alcohols, esters, and glacial acetic acid), (2) and its resistance to digestion by proteases, even by trypsin which has almost universal digestive action. These two peculiarities have been discussed above. We can only say that if a protein, luciferin must belong to a new group differing from known natural proteins in these respects. In general characteristics this new group would fall somewhere on the border-line between the proteoses and peptones. It would not be surprising to find in nature proteoses or peptones soluble in absolute alcohol. We know also that some NH-CO linkages of proteins are broken down with great difficulty by trypsin as it is difficult to obtain a tryptic digest of protein which does not give the biuret reaction, and the work of Fischer and Abderhalden has shown that certain artificial polypeptides are not digested by pure activated pancreatic juice. We have, then, three possibilities. Luciferin is (1) either a natural proteose not attacked by trypsin, or (2) if attacked by trypsin, its decomposition products (presumably amino-acids) still contain the group oxidizable with light production, or (3) it is not a protein at all. I believe that luciferin has too many protein characteristics to conform to the last possibility. I have been unable to oxidize with light production various mixtures of amino-acids (from beef and casein) by means of luciferase and consequently am led to believe that luciferin is a new natural proteose, soluble in absolute alcohol and not digested by trypsin. Dubois believes Pholas luciferin to be a natural albumin with acid properties. Cypridina luciferin could not possibly be regarded as an albumin, but it is very likely that the luciferins of different species of luminous animals differ in certain characteristics. As in the case of the luciferases, we know that the luciferins are not identical substances, and only future work can determine in what particulars they differ. A summary of the properties of Cypridina luciferin and Cypridina luciferase will be found in the tables accompanying this paper.
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PMID:STUDIES ON BIOLUMINESCENCE : IX. CHEMICAL NATURE OF CYPRIDINA LUCIFERIN AND CYPRIDINA LUCIFERASE. 1987 44


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