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)

The effects of various amino acids and phosphorylated forms of glucose on the release of digestive enzymes from particulate cellular pools, particularly zymogen granules, were evaluated in rat pancreas. Whole tissue homogenates, as well as zymogen granules isolated either by differential centrifugation in 0.3 M sucrose or by preparation in buffered sucrose and subsequent centrifugation in a Percoll gradient, were studied. The basic amino acids L-arginine and L-lysine, sites of tryptic cleavage, caused the release of trypsinogen, but not chymotrypsinogen, whereas the aromatic amino acids L-phenylalanine and L-tryptophan, sites of chymotryptic cleavage, caused release of both trypsinogen and chymotrypsinogen. Neither led to the release of the starch-splitting enzyme amylase. All effects occurred within the range of normal plasma concentrations for these amino acids in the rat. Two amino acids, L-threonine and hydroxy-L-proline, that are not sites of cleavage by trypsin or chymotrypsin, and a nonmammalian amino acid, aminoadipic acid, did not lead to release of trypsinogen, chymotrypsinogen, or amylase. Two phosphorylated forms of glucose, glucose 1-phosphate and glucose 1,6-diphosphate, caused the release of amylase, but of neither trypsinogen nor chymotrypsinogen. Contrary to previous results, D-glucose was without effect, as was glucose 6-phosphate. We propose that certain digestive end products, by direct action on zymogen granules, cause the selective release of the enzymes involved in their evolution from polymeric substrates during digestion.
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PMID:Digestive end products release pancreatic enzymes from particulate cellular pools, particularly zymogen granules. 242 Mar 68

With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.
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PMID:Photoaffinity labeling of T4 bacteriophage 32 protein. 243 10

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].
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PMID:The conformational changes of alpha 2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes. 244 Apr 24

The primary structure of a 9-kDa basic protein from rice seeds was determined by gas-phase sequencing of the intact protein and peptides derived from it by digestion with trypsin, chymotrypsin, and endopeptidase Lys-K. The protein consists of a single polypeptide chain of 91 amino acid residues with a calculated molecular mass of 8909 Da. It is rich in alanine, serine, glycine, and cysteine. The eight cysteines form four disulfide bonds. There is no methionine, histidine, phenylalanine, or tryptophan. The sequence is highly homologous with an alpha-amylase inhibitor, I-2, from seeds of Indian finger millet [F. A. P. Campos and M. Richardson (1984) FEBS Lett. 167, 221-225] and a 10-kDa barley seed protein, also called a probable amylase/protease inhibitor [B. Svensson et al. (1986) Carlsberg Res. Commun. 51, 493-500; J. Mundy and J. C. Rogers (1986) Planta 169, 51-63]. In analogy with the barley protein, the purified protein is tentatively called a rice probable amylase/protease inhibitor (PAPI). The rice PAPI does not show inhibitory activities against proteases and amylases tested. The amino acid sequence is as follows: Ile-Thr-Cys-Gly-Gln-Val-Asn-Ser-Ala-Val(10)-Gly-Pro-Cys-Leu-Thr-Tyr- Ala-Arg-Gly-Gly(20)-Ala-Gly-Pro-Ser-Ala-Ala-Cys-Cys-Ser-Gly(30)-Val-Arg- Ser-Leu-Lys-Ala-Ala-Ala-Ser-Thr(40)-Thr-Ala-Asp-Arg-Arg-Thr-Ala-Cys- Asn-Cys(50)-Leu-Lys-Asn-Ala-Ala-Arg-Gly-Ile-Lys-Gly(60)-Leu-Asn-Ala-Gly- Asn-Ala-Ala-Ser-Ile-Pro(70)-Ser-Lys-Cys-Gly-Val-Ser-Val-Pro-Tyr-Thr(80)- Ile-Ser-Ala-Ser-Ile-Asp-Cys-Ser-Arg-Val-Ser(91).
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PMID:Amino acid sequence of a probable amylase/protease inhibitor from rice seeds. 245 99

Photoreactivating enzyme activity has been quantitated in human fetal skin, kidney, lung, liver, brain and intestine, and in neonatal human foreskin. In all the tissues examined there were at least two activities: one nominally greater than 10,000 Da, and one nominally less than 10,000 Da. Both can photolyze pyrimidine dimers in DNA using only light of wavelengths greater than 320 nm, thus excluding tryptophan-mediated dimer splitting as an important mechanism for these activities. The activities are inactivated by digestion with trypsin or pronase, and decreased partially or totally by heating to 65 degrees C. The activities from all six tissues, as well as that from neonatal foreskin, act catalytically in dimer photolysis. The properties of macromolecular size, heat lability, protease sensitivity and catalytic pyrimidine dimer photolysis by a non-tryptophan-mediated mechanism correspond to those of a true photoreactivating enzyme.
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PMID:DNA photoreactivating enzyme from human tissues. 211 1

Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.
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PMID:Organization of mitochondrial UDP-glucose:dolichylmonophosphate glucosyltransferase in the outer membrane. 253 36

A fluorescent peptide substrate to explore the protease specificity for the amino acid regions C- and N-terminal to the cleavage site has been designed. Intramolecular quenching of indole fluorescence by an N-terminal dansyl group separated by six amino acid residues forms the basis of this assay. For a particular enzyme, specificity can be designed into the peptide sequence by means of the number of residues that separate the two chromophores. In the present instance, the heptapeptide Dns-Gly-Lys-Tyr-Ala-Pro-Trp-Val is used to assay angiotensin converting enzyme (ACE), Astacus protease, carboxypeptidase A, alpha-chymotrypsin, and trypsin, all of which cleave the peptide in accord with their known specificity: Trypsin and Astacus protease hydrolyze only the Lys-Tyr and Tyr-Ala bonds, respectively. alpha-Chymotrypsin primarily cleaves the Tyr-Ala bond while ACE makes three successive dipeptidyl cleavages from the C-terminus. Carboxypeptidase rapidly hydrolyzes first the Trp-Val and then the Pro-Trp bond. For all of the enzymes, catalytic activity (kcat/Km) is in the range from 10(5) to 10(6) M-1 s-1. Hydrolysis causes a fluorescence increase in the 310 to 410 nm region of 8.6- to 13.6-fold depending on the enzyme that is assayed. Assays can be designed based on the increase in tryptophan fluorescence or by individual product analyses using thin-layer or high-performance liquid chromatography. The specificity and sensitivity of such internally quenched fluorescent oligopeptides would seem to be ideal for the assay of specific endoproteases.
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PMID:A fluorescent oligopeptide energy transfer assay with broad applications for neutral proteases. 255 28

The effects of citrate ion concentration and pH on the optical spectra and fluorescence decay have been measured for several tyrosine model compounds and lima bean trypsin/chymotrypsin inhibitor, a protein containing one tyrosine at position 69 and seven disulfides but no tryptophan, in order to determine the location and environment of Tyr 69. Tyrosine in the protein is protected from citrate collisional quenching, as indicated by the dynamic quenching constant 9 to 15 times smaller than those for the model peptides. Static quenching remains, with a Stern-Volmer constant of about 1.0 M-1, somewhat smaller than those of L-tyrosine, tyrosine-glutamate, and leucine-tyrosine-leucine. The elevated pKa of Tyr 69, greater than or equal to 11.6, also indicates protein protection from solvent ions. Though Coulomb repulsion of the Glu 70/citrate pair may play a role in the shielding of Tyr 69 from citrate, our measurements indicate that steric effects of the protein structure are more important. Tyrosinate emission in the protein at neutral pH is minimal.
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PMID:Spectroscopy and fluorescence quenching of tyrosine in lima bean trypsin/chymotrypsin inhibitor and model peptides. 262 88

Several protease inhibitors were found in the seeds of a Cucurbitacea, Ecballium elaterium, and were separated from one another by affinity and molecular sieve chromatography. Three main trypsin isoinhibitors were purified by ion-exchange chromatography and the sequence of the major one, EETI II, was elucidated and compared with other inhibitors of the squash family. It is a peptide of M.W. 3020 of strong inhibitory activity (Ka = 8 x 10(11) M-1) against trypsin, showing high Gly content, six half-cystine residues, but devoid of histidine, threonine, tryptophan, and tyrosine residues.
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PMID:Protease inhibitors from Ecballium elaterium seeds. 265 42

The interaction of tryptophan, lysozyme and tyrosine with ninhydrin in strong acid media has been investigated at 20, 25, 30, and 35 degrees C by spectrophotometry. Second-order rate constants and molar absorptivity values have been evaluated from an analytical point of view. Optimum conditions for the selective estimation of tryptophan, tryptophan residues in intact proteins, and indoles--without the disturbing effect of tyrosine--have been given. Under optimum conditions, in the concentration range from 2.5 X 10(-8) to 3.0 X 10(-7)M, molar absorptivity values and reproducibility data for various reactants have been reported. Molar absorptivity values (Am X 10(-3)/M X cm) of tryptophan (21.35), lysozyme (19.33), bovine serum albumin (21.05), human serum albumin (21.00), casein (17.85), alpha-chymotrypsin (18.28), trypsin (14.43), indole (5.03), and indole-3-acetic acid (13.75) have been measured with a standard error of 2.3% or less for any particular reactant.
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PMID:Spectrophotometric determination of tryptophan in intact proteins by the acid ninhydrin method. 274 46


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