Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Activation of acetylated chymotrypsinogen with trypsin leads to catalytically active acetylated delta-chymotrypsin containing NH2-terminal isoleucine. The importance of the cationic terminus to the control of the active conformation of acetylated delta-chymotrypsin has been demonstrated (Oppenheimer, H. L., Labouesse, B., and Hess, G. P. (1966) J. Biol. Chem. 241, 2720). Later studies appeared to suggest that the modification of isoleucine-16 of delta-chymotrypsin is not accompanied by the loss of catalytic activity as measured by the hydrolysis of N-acetyl-L-tyrosine ethyl ester (Agarwal, S. P., Martin, C. J., Blair, T. T., and Marini, M.A. (1971)Biochem. Biophys. Res. Commun. 43, 510; Blair, T. T., Marini, M. A., Agarwal, S. P., and Martin, C. J. (1971) FEBS Lett. 1486) or by the loss of active site content (Ghelis, C., Garel, J. R., and Labouesse, J. (1970) Biochemistry 9, 3902). In the present studies, controlled acetylation of the terminal alpha-aminogroup of acetylated delta-chymotrypsin with acetic anhydride led to a progressive loss of active sites of the enzyme. Determination of the catalytic and kinetic properties of the modified enzyme with the specific ester substrate N-acetyl-L-tyrosine ethyl ester or the nonspecific substrates p-nitrophenyl acetate and cinnamyol imidazole gave nearly identical results. With N-acetyl-L-tyrosine ethyl ester as substrate, the Km (app) values for acetylated delta-chymotrypsin (1.0 plus or minus 0.1 mM) and the modified enzyme (0.67 plus or minus 0.05 mM) are nearly identical and the kcat value is reduced to about 25% in the latter enzyme species. This value correlates well with about 20% of the active sites in this enzyme as measured by the rapid initial liberation of p-nitrophenol. With p-nitrophenyl acetate as substrate, the acylation rate constants (0.13 plus or minus 0.04 s(-1) at pH 6.0, 25 degrees, in 3.3% acetonitrile) and the deacylation rate constants (0.01 s(-1) at pH 8.5, 25 degrees, in 3.3% acetonitrile) are identical for the acetyl isoleucine-16 and the isoleucine-16 enzymes. Furthermore, the residual enzyme activity could be correlated well with the residual NH2-terminal isoleucine content and with the moles of [1--14C]acetyl groups incorporated per mol of the enzyme. The activity associated with the modified enzyme can be attributed to the enzyme species in which isoleucine-16 of acetylated delta-chymotrypsin is not acetylated. These data are in general agreement with the studies of Ghelis et al. (1970) but are in disagreement with the results of Blair et al. (1971) and of Agarwal et al. (1971) and confirm the hypothesis that the final conformation of acetylated delta-chymotrypsin containing an acetylated NH2 terminus is catalytically inactive and resembles acetylated zymogen in many of its physical properties.
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PMID:Modification of isoleucine-16 acetylated delta-chymotrypsin. 114 Dec 36

Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.
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PMID:Separation and characterization of the precursor and activated forms of porcine and human pancreatic colipase by reversed-phase liquid chromatography. 147 89

Two tripeptide amides with stuctures similar to thyrotropin releasing hormone were isolated from human seminal fluid and their amino acid sequences determined. The peptides were purified by gel exclusion from Sephadex G50 and were detected by radioimmunoassay with thyrotropin releasing hormone antibody; in addition, N-terminally extended forms were demonstrated by radioimmunoassay after trypsin digestion. Further purification of the tripeptides was by chromatography on SP-Sephadex C25 and by high performance liquid chromatography on C18 Microbondapak using an HCl/acetonitrile gradient. After exclusion from mini-columns of SP-Sephadex C25 and DEAE-Sephadex A25, two neutral peptides were obtained in homogeneous form by high performance liquid chromatography with an HCl/methanol gradient. Amino acid analysis gave the following compositions: Glu, 0.74, Phe, 1.0, Pro, 1.0; and Glu, 1.72, Pro, 1.0. Both peptides possessed a blocked N terminus, but after opening the pyroglutamyl ring the sequences Glu-Phe-Pro and Glu-Glx-Pro were demonstrated. The chromatographic properties of the endogenous peptides were identical to the properties of the corresponding synthetic peptides. The structure of pGlu-Phe-Pro (where p-indicates pyro-) amide was confirmed by fast atom bombardment mass spectrometry. The presence in human semen of three structurally related peptides, pGlu-Phe-Pro amide, pGlu-Gln-Pro amide, and the previously reported pGlu-Glu-Pro amide (Cockle, S. M., Aitken, A., Beg, F., and Smyth, D. G. (1989) J. Biol. Chem. 264, 7788-7791), suggests that this series of peptides may have evolved to fulfil complementary biological roles.
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PMID:Isolation and identification of two neutral thyrotropin releasing hormone-like peptides, pyroglutamylphenylalanineproline amide and pyroglutamylglutamineproline amide, from human seminal fluid. 155 84

A procedure for the generation and isolation of internal peptide fragments for less than 10 micrograms of protein bound to either polyvinylidene difluoride (PVDF) or nitrocellulose membranes after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) is presented. This technique has produced internal sequence data for 120 peptides, with an average initial yield of 20 pmol. Membrane-bound proteins were enzymatically digested with either trypsin or endoproteinase Lys-C in the presence of 1% hydrogenated Triton X-100/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 degrees C. The eluted peptides were then directly isolated by microbore HPLC for subsequent sequence analysis. One percent hydrogenated Triton X-100 did not inhibit enzymatic activity, distort HPLC resolution of peptides, or contain uv-absorbing contaminants that could interfere with peptide identification. Reproducible peptide maps and consistent recoveries are presented for standard proteins (3.5-8.0 micrograms) bound to either membrane, with higher recoveries for PVDF-bound proteins. Ninety percent of the proteins analyzed by this technique have produced results; representative peptide maps and sequence data are presented. This technique has a wide range of applications, particularly for proteins with blocked amino termini or those that can only be purified by SDS-PAGE or 2D isoelectric focusing SDS-PAGE.
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PMID:Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes. 163 12

The 180,000 molecular weight protein from [32P]phosphorylated wheat germ agglutinin-purified rat liver plasma membranes was digested with trypsin. NIH 3T3 HIR 3.5 cells were [32P]phosphate-labelled in the presence of 10(-7) M insulin, and the 185,000 molecular weight cytoplasmic protein was digested with trypsin. Digests were applied to a C18-mu Bondapak column, eluted with acetonitrile gradients, and radioactivity in the eluate was monitored. The chromatogram for the cytoplasmic protein was similar but not identical to chromatograms of trypsin digests of insulin receptor substrates from other cultured cells. Thirteen and seven phosphopeptides were obtained from the plasma membrane and cytoplasmic substrate, respectively. One phosphopeptide from the two digests eluted at the same acetonitrile concentration; however, dissimilarity in elution profiles and dissimilarity in relative yields of individual phosphopeptides, suggest that the primary structures of tyrosine phosphorylation sites in the two insulin receptor substrates are different.
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PMID:Reverse phase chromatography of trypsin digests of a plasma membrane and a cytoplasmic insulin receptor substrate. 164 43

The reactions were studied of N-acyl-L-amino acid esters with various D-amino acid amides catalyzed by free alpha-chymotrypsin, trypsin and proteinase K in acetonitrile containing 80 or 5 vol. % of water. In the medium with low water content the incorporation of D-amino acid amides into peptides proceeded with satisfactory yield sometimes approaching that of analogous L-L dipeptides. In the media with high water content negligible or low yields of L-D dipeptides were achieved. Synthesis of Boc-L-Trp-D-Phe-NH2 catalyzed by alpha-chymotrypsin was performed at molar ratio L: D = 3 : 2 in acetonitrile with 5 vol.% of water and the dipeptide was isolated in larger quantity. However, synthesis of the peptide bond did not occur at all when diastereomeric dipeptides having D-residue in the N-terminal P1' position were used even in the media with low water content.
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PMID:Serine proteinase-catalyzed incorporation of D-amino into model peptides in acetonitrile with low water content. 182 59

The 1H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d3/aqueous cosolvent. Resonances from 69 of 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel beta-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end of their respective region of the antiparallel beta-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors.
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PMID:1H assignments and secondary structure determination of the soybean trypsin/chymotrypsin Bowman-Birk inhibitor. 201 1

A high-performance liquid chromatographic method involving fluorescence derivatization followed by separation on a reversed-phase polymer (octadecylated polyvinylalcohol copolymer gel) column is described for the determination of opioid peptides in rat brain tissues. The peptides extracted from brain tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate. The derivatives were separated on an Asahipak ODP-50 column by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 9.5). The detection limits (S/N = 3) for the peptides were 0.33-1.21 pmol per 100 microliters injected. The method actually permit the determination of leucine enkephalin, methionine enkephalin, methionine enkephalin-Arg-Phe and methionine enkephalin-Arg-Gly-Leu in the tissues. The method is also applied to the characterization of the peptides in the tissues by means of enzymatic degradations with carboxypeptidase A and trypsin.
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PMID:Pre-column fluorescence derivatization high-performance liquid chromatography of opioid peptides in rat brain and its use for enzymatic peptide characterization. 204 96

Previous studies identified synapsin I as a potential substrate for a newly discovered growth factor-sensitive, proline-directed protein kinase originally isolated from rat pheochromocytoma. The present study describes the site-specific phosphorylation of synapsin I by highly purified preparations of proline-directed protein kinase. The incorporation of [32P]phosphate into bovine brain synapsin I was dependent upon both the amount of kinase present and the time of incubation. The maximum stoichiometry of phosphorylation approached 1 mol of phosphate/mol of synapsin I protein. When analyzed by sodium dodecyl sulfate-gel electrophoresis and autoradiography, [32P]phosphate was found to be incorporated into both synapsin Ia and Ib. Phosphoamino acid analysis demonstrated that serine residues were phosphorylated exclusively. Digestion of phosphorylated synapsin I with trypsin followed by high performance liquid chromatography (HPLC) phosphopeptide analysis indicated that the tryptic peptide containing the major phosphorylation site eluted as a single peak at approximately 17% acetonitrile. The primary structure of this phosphopeptide, determined by gas-phase sequencing, was found to be Gln-Ser-Arg-Pro-Val-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Pro-Ala-Thr-Arg-Pro-Pro- Ala-Ser-Pro-Ser-Pro-Gln-Arg. Sequential Edman degradation of this HPLC-purified tryptic phosphopeptide revealed that serine 20 of this peptide was the major phosphorylated residue. This phosphoacceptor site is immediately flanked by a carboxyl-terminal proline residue, an observation that further verifies the proline-directed nature of this protein kinase. The tryptic phosphopeptide corresponds exactly to a sequence in the collagenase-sensitive, proline-rich "tail" region of bovine synapsin I. This novel phosphorylation site is close to but distinct from phosphorylation sites 2 and 3, which are known to be phosphorylated by calcium/calmodulin-dependent protein kinase II and are considered to be of regulatory importance.
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PMID:Phosphorylation of synapsin I at a novel site by proline-directed protein kinase. 210 63

Human lung cancer cell line, T3M-30, has been shown to produce a growth factor that stimulates proliferation of peripheral blood monocytes. In the presence of this factor, human circulating monocytes were able to proliferate in vitro. Gel exclusion chromatography of the conditioned medium revealed a single peak of monocyte growth-promoting activity at an apparent molecular weight of 16,000. The growth-promoting activity was adsorbed to an anion-exchange column, Mono Q, and eluted with a salt gradient as a single peak of bioactivity at 300 mM NaCl. When the sample was applied to a Vydac C4 column, a reverse-phase high-performance liquid chromatography column, a single peak of activity was observed at a concentration of 76% acetonitrile in 0.1% trifluoroacetic acid. The monocyte growth-promoting activity was heat stable at 56 degrees C. It was partially destroyed by trypsin. The activity was lost after treatment with 2-mercaptoethanol.
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PMID:A human monocyte growth factor produced by lung cancer cells. 216 45


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