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)

The enzymic degradation of beta-casein by immobilized trypsin is described and its use as a model system to study proteolytic reactions which occur in milk during processing is examined. Quantitative determination of proteolysis can be obtained by densitometric transmission measurement of patterns obtained by polyacrylamide-gel electrophoresis. Staining properties of some of the individual components in the electrophoretic patterns were determined by calibration with pure protein and peptide preparations. The isolation of pure beta- and gamma-caseins and the preparation of several forms of immobilized trypsin are outlined.
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PMID:Model proteolysis of beta-casein by immobilized trypsin. 46 47

We have demonstrated a procedure for the rapid (minutes), sensitive (less than pmol), and sequence-specific identification of phosphopeptides in unfractionated digests of phosphoproteins using matrix-assisted UV laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The mass-dependent identification of one specific 13-residue phosphopeptide (S105-K117), observed among the 153 possible trypsin digest fragments of human beta-casein (211 residues), was confirmed by amino acid sequence analysis of the 33P-labeled peptide after isolation by reverse-phase HPLC. MALDI-TOF was also used to monitor the rate and extent to which an 18-residue N-terminal beta-casein peptide (R1-K18) was phosphorylated in vitro. These results demonstrate that MALDI-TOF may be used (i) to facilitate the identification of sequence-specific sites of protein phosphorylation and dephosphorylation, (ii) to monitor protein and peptide phosphorylation and dephosphorylation reaction rates, even in complex unfractionated mixtures, (iii) to determine the minimum primary structure necessary for the phosphorylation of specific protein surface domains, and (iv) to evaluate the effects of intact protein phosphorylation and dephosphorylation on susceptibility to subsequent proteolytic events.
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PMID:Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 149 23

The effect of magnesium ions on the catalytic activities of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. Mg2+ markedly stimulated the breakdown of dephosphorylated beta-casein (caseinolytic activity) and the hydrolysis of Cbz-Leu-Leu-Glu-2-naphthylamide (peptidylglutamyl peptide bond hydrolyzing activity) by a 1700-fold purified preparation of MPC. Cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide (trypsin-like activity) was strongly inhibited and cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide (chymotrypsin-like activity) was weakly inhibited. Similar results were produced when enzymatic activities in the absence of Mg2+ were measured at 52 degrees C rather than at 37 degrees C. Trace protein impurities were removed by phenyl-Sepharose chromatography. This additional chromatographic step, while not changing the specific activities of hydrolysis of the three synthetic chromogenic substrates, led to a marked activation of the breakdown of dephosphorylated beta-casein. Mg2+ was not able to further stimulate the caseinolytic activities of either the phenyl-Sepharose-treated preparation or the preparation measured at 52 degrees C. Mg2+ therefore converts a "repressed" form of MPC to an "activated" form, possibly by promoting dissociation of a protein inhibitor, and may serve as a physiological regulator of this enzyme complex.
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PMID:Enzymatic changes of the bovine pituitary multicatalytic proteinase complex, induced by magnesium ions. 155 Mar 35

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.
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PMID:3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex. 156 24

A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular energy transfer from DACM to FITC. The caseinolytic activity is then monitored by the fluorescence increase. The activities of both proteases obey Michaelis-Menten kinetics with Km = 1.6 +/- 0.2 microM for trypsin and Km = 13.2 +/- 1.9 microM for transin. Protease concentrations as low as 10 ng/mL can be utilized. The pH dependence of the caseinolytic activity has been determined for both enzymes.
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PMID:A continuous fluorescent assay for measuring protease activity using natural protein substrate. 178 89

Casein phosphopeptide (CPP), a highly phosphorylated peptide fragment which inhibits the formation of hydroxyapatite crystal was isolated from pooled ileal contents of rats fed a semi-synthetic diet containing bovine beta-casein. The estimated amino acid sequence of the CPP was shorter than that of the trypsin-digested beta-casein but the core region consisting of consecutive bindings of phosphoserine was fully conserved. A moderate and exchangeable binding to Ca2+ of the CPP molecule well substantiates the high absorbability of calcium from milk and dairy products.
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PMID:Characterization of phosphopeptide derived from bovine beta-casein: an inhibitor to intra-intestinal precipitation of calcium phosphate. 202 41

The topography of bovine beta-casein at a soya oil/water interface was studied by following the kinetics of the trypsin-catalysed hydrolysis. Tryptic peptides were identified from their amino acid compositions and the kinetics were compared with those obtained from beta-casein in solution. Whereas soluble beta-casein was initially hydrolysed at a number of trypsin-sensitive bonds, the hydrolysis of the protein at the interface was a more ordered event. The crucial initiating step was the cleavage of the N-terminal peptides 1-25 and 1-28 from the molecule. Hydrolysis at other trypsin-sensitive sites could then occur. This suggests that with the exception of the large hydrophilic moiety in the N-terminal region, most of the beta-casein molecule is inaccessible to the proteinase, and lies fairly flat on the oil/water interface. After removal of the N-terminal peptide, the remaining macropeptide can reorientate and other hydrophilic regions become accessible to the proteinase.
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PMID:The topography of bovine beta-casein at an oil/water interface as determined from the kinetics of trypsin-catalysed hydrolysis. 226 66

The 700-kDa multicatalytic proteinase complex from bovine pituitaries separates in polyacrylamide gel electrophoresis under dissociating and reducing conditions into 11 components with molecular masses ranging from 21 to 32 kDa. No higher molecular mass components were detected. A rabbit polyclonal antibody raised against the complex recognizes five immunoreactive components. As reported previously, the complex exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. All three activities are rather rapidly inactivated by 3,4-dichloroisocoumarin, a general serine protease inhibitor, however, the pseudo-first-order rate constants of inactivation of the three components differ within a wide range, with the chymotrypsin-like activity being most sensitive to inhibition. The peptidylglutamyl-peptide hydrolyzing activity is greatly activated by low concentrations of sodium dodecyl sulfate and fatty acids and seems to constitute the main component responsible for degradation of protein substrates. In addition to cleaving bonds on the carboxyl side of glutamyl residues, this activity also cleaves, albeit at a slower rate, bonds on the carboxyl side of hydrophobic residues; however, the secondary specificity of this component is clearly different from the chymotrypsin-like activity. Heparin selectively activates the chymotrypsin-like activity. The complex cleaves rapidly both native and dephosphorylated beta-casein in a reaction greatly accelerated by low concentrations of sodium dodecyl sulfate. The nature of proteolytic products, and also the rate of formation of acid-soluble, ninhydrin-reactive products, is different for the phosphorylated and dephosphorylated form of beta-casein, indicating that the degree of phosphorylation influences the rate and pattern of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity. 253 72

Procedures for the reduced-scale analysis of proteins by peptide mapping have been developed, allowing peptide maps to be obtained from picomole to femtomole quantities of protein. The use of trypsin immobilized on agarose gel and placed in a small reactor column has made it possible to reproducibility digest as little as 50 ng of protein. This represents a decrease in sample size of approximately 3 orders of magnitude from conventional tryptic digestion schemes. Separations of tryptic digests were accomplished by using either microcolumn high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Separations of 100 ng (4 pmol) of tryptic digest samples of beta-casein were achieved with microcolumn HPLC, while separations of approximately 2 ng (80 fmol) of beta-casein tryptic digest (from a total sample size of 50 ng) were possible with CZE. Peptide maps from phosphorylated and dephosphorylated forms of beta-casein were readily distinguishable using both separation methods, demonstrating an ability to detect a single amino acid modification in a protein. Relative standard deviations of peak retention or migration times were less than 3% for microcolumn HPLC and less than 1% for CZE.
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PMID:High-sensitivity peptide mapping by capillary zone electrophoresis and microcolumn liquid chromatography, using immobilized trypsin for protein digestion. 259 96

Degradations by proteolytic enzymes and intestinal epithelial permeability represent two major drawbacks to the transfer of food protein antigens to blood. These steps were studied in vitro for the milk protein antigens beta-lactoglobulin (beta-Lg), alpha-Lactalbumin (alpha-La) and beta-casein (beta-cas). Pepsin-trypsin hydrolysis and permeability in isolated rabbit ileum in Ussing chamber were suited by ELISA and radiolabelled-protein measurement. Pepsin-trypsin hydrolysis showed an increasing resistance in the order beta-cas less than alpha-La less than beta-Lg. The rate of absorption of the antigenic proteins by isolated rabbit ileum was in the same order, and the rate of absorption of the whole proteins (degraded and antigenic forms) was significantly higher for beta-Lg than for alpha-La and beta-cas. These results suggest a selective intestinal permeability for milk protein antigens. This selectivity is probably important in the mechanism of food protein sensitization via the oral route.
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PMID:Permeability of milk protein antigens across the intestinal epithelium in vitro. 262 77


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