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)

To clarify the properties and functions of a trypsin inhibitor from Japanese barley in comparison with the inhibitor from Pirkka barley, an inhibitor was isolated from the barley Hordeum distichum L var. emend Lamark by extraction with 1% NaCl, ammonium sulfate fractionation and repeated chromatography on DEAE-cellulose and CM-cellulose. The final purified preparation of the inhibitor was found to be homogeneous by both chromatographic and electrophoretic analysis. The inhibitor was thermostable and was stable over the broad pH range from 2 to 11. No inhibition was observed by heavy metal ions and many reagents at 10(-2) M, except that p-chloromercuribenzoate caused a 69% loss of activity. The inhibitor was subjected to isoelectric focusing at pH 7.51 and its molecular weight was calculated to be 14,200+/-900 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent dissociation constant for the complex between the inhibitor and trypsin[EC 3.4.21.4] was 1.64 X 10(-7)M with casein as a substrate. One microgram of purified inhibitor inhibited 1.5 mug of pure trypsin in the hydrolysis of alpha-N-benzoyl-DL-arginine-p-nitroanilide. By chemical modification of arginyl residues in the inhibitor with 1,2-cyclohexanedione, the inhibitor was shown to be an arginine inhibitor. The inhibitor contained relatively many basic amino acids and few half cystines as compared with Pirkka barley trypsin inhibitor.
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PMID:Studies on trypsin inhibitor in barley. I. Purification and some properties. 0 Mar 80

Location of electron transport chain components in chloroplast membranes of chlamydomonas reinhardi, y-1 was investigated by use of proteolytic digestion with soluble or insolubilized trypsin. Digestion of intact membrane vesicles with soluble trypsin inactivates the water-splitting system, the 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition site of Photosystem II, the electron transport between the two photosystems as well as the ferredoxin NADP reductase. Reduction of NADP with artificial electron donors for Photosystem I could be restored, however, by addition of purified reductase to trypsin-digested membranes. Electron transfer activities of Photosystems I and II reaction centers were resistant to trypsin digestion either from outside or from within the thylakoids when active trypsin was trapped inside the membrane vesicles by sonication and digestion carried out in the presence of trypsin inhibitor added from outside. In the latter case, the water-splitting system was also found to be resistant to digestion. Polyacrylamide-bound insolubilized trypsin inactivated only the ferredoxin NADP reductase. Photosynthetically active membranes obtained at different stages of development showed a basically similar behavior toward trypsin.
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PMID:Trypsin-sensitive photosynthetic activities in chloroplast membranes from Chlamydomonas reinhardi, y-1. 0 Apr

A technique utilizing Pregnant Mare's Serum Gonadotropin and Human Chorionic Gonadotropin treatment of hens (Gallus domesticus), followed by manual ovulation of the excised follicles, was developed to obtain a large number of mature ova. The intact ova were used to test whether acrosin, partially purified from the spermatozoa of the cock (Gallus domesticus), partially purified rabbit testicular acrosin and commercial preparations of several hydrolytic enzymes could dissolve the inner vitelline membrane. Enzymes were applied to pieces of filter paper placed on the ovum. Cock acrosin and endopeptidases such as trypsin, chymotrypsin, collagenase and elastase hydrolyzed the membrane whereas exopeptidases such as leucine aminopeptidase and carboxypeptidase A did not. Phospholipase A, sulfatase, hyaluronidase, beta-glucuronidase and rabbit testicular acrosin also failed to hydrolyze the membrane. Cock acrosin hydrolysis of the ovum surface was inhibited by soybean trypsin inhibitor. The surface of the ovum over the germinal disc region was hydrolyzed more quickly by cock acrosin than the surface over other regions of the ovum. Acrosin from cock sperm caused the release of trichloroacetic acid soluble material absorbing at 280 nm from sonicated preparations of inner vitelline membranes. Hydrolysis was greatest at pH 8.0 and was inhibited by soybean trypsin inhibitor.
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PMID:Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes. 0 Apr 54

An enzyme is designed to bind most tightly to a substrate when it is in the transition state of the reaction which the enzyme catalyses. The consequent reduction of the activation energy of the reaction constitutes the catalytic mechanism. The energetic contributions of different features of the interaction can only be crudely assessed, but they are dominated by entropically driven effects. The binding site of trypsin orients the substrate so that the reacting groups are correctly placed for reaction to occur. Apart from two side chains which take part in chemical steps of the reaction, the enzyme behaves almost as a rigid body. The full binding interactions are only developed when the substrate is in an intermediate stage of the reaction. The tightly bound complexes of trypsin with protein trypsin inhibitors have proved amenable to structural analysis. Enzyme inhibitor interactions, which account for almost 80 kJ mol-1 of interaction energy, are known fairly accurately. The similarity of the two known trypsin inhibitor structures, close to the primary binding site, indicates a high specificity, even for this simple interaction. In cases where no large conformational changes occur the specificity of an enzyme should be predictable from accurate knowledge of its tertiary structure.
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PMID:Enzyme substrate and inhibitor interactions. 0 20

The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH.
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PMID:Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins. 0 94

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.
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PMID:Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz). 0 70

The conversion of proparathyroid hormone (proparathormone) to parathyroid hormone (parathormone) by subcellular fractions of the bovine parathyroid has been investigated. The identification of the conversion product as parathormone was established by its elution postion during ion exchange chromatography and gel filtration, and by partial amino acid sequence analysis of its NH2-terminal region. Total homogenates and derived subcellular fractions (600 X g pellet, 5,000 X g pellet, 20,000 X g pellet, 190,000 X g pellet, and 190,000 X g supernatant) all catalyzed the conversion of exogenous [3H]- or [14C]prohormone. Over 60% of the converting activity was in the particulate fractions; the 190,000 X g particulate fraction contained the highest specific converting activity. The converting activity appeared to be an integral component of the membranes since it could only be partially removed by extraction with Triton X-100. The production of parathormone by the particulate converting enzyme increased with time and the concentration of enzyme protein. The optimum pH range was between 7 and 9, and the enzyme was inactive below pH 6. Conversion by the particulate enzyme was inhibited by benzamidine or chloroquine, but not by pancreatic trypsin inhibitor, indicating its dissimilarity to trypsin. When a mixture of [14C]proparathormone and [3H]parathormone was used as substrate, the particulate enzyme did not metabolize the hormone despite over 70% conversion of the prohormone to hormone and other peptides. There was a close correlation between the subcellular distribution of converting activity and that of newly formed parathormone found in the membrane fraction. These data suggest that the particulate converting activity is that concerned with the formation of parathormone in vivo.
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PMID:Conversion of proparathyroid hormone to parathyroid hormone by a particulate enzyme of the parathyroid gland. 1 Mar 4

Highly purified aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin. The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs. activity profile in the presence of added metal ions. However, S0.5 and hill coefficient for L-aspartate considerably increase upon activation. As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs. untreated aspartase appears with negative absorbance maxima at 278 and 285 nm. When the trypsin-activated enzyme is denatured in 4 M guanidine-HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.
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PMID:Studies on aspartase. III. Alteration of enzymatic properties upon trypsin-mediated activation. 1 Sep 95

Paraffin-stimulated whole mixed saliva was collected from 6 individuals and pooled. Three trypsin-like enzymes from saliva supernatant and two from the saliva sediment were isolated by use of affinity chromatography on a soybean trypsin inhibitor Sepharose 4 B column. The isoelectric points for these enzymes were pI 8.0, 6.8 and 6.4 from the supernatant and 6.4 and 6.2 from the sediment. The enzymes were characterized with respect to pH-optimum, pH-stability, temperature stability, substrate specificity and influence of metal ions and inhibitors. The Michaelis constants were determined for these enzymes on the substrates BAEE and TAME.
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PMID:Characterization of trypsin-like enzymes from human saliva isolated by use of affinity chromatography. 1 95

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.
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PMID:Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12. 2 11


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