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 dimethyl ester of bovine pancreatic ribonuclease-A (dimethyl RNAase-A), the initial product of esterification of RNAase-A in anhydrous methanolic HCl, was isolated in a homogeneous form. The two carboxy functions esterified in this derivative are those of glutamic acid-49 and aspartic acid-53. There were no changes in the u.v.-absorption spectral characteristics, the accessibility of the methionine residues, the resistance of the protein to proteolysis by trypsin and the antigenic behaviour of RNAase-A as a result of the esterification of these two carboxy groups. Dimethyl RNAase-A exhibited only 65% of the specific activity of RNAase-A, but still had the same K(m) value for both RNA and 2':3'-cyclic CMP. However, the V(max.) was decreased by about 35%. On careful hydrolysis of the methyl ester groups at pH9.5, dimethyl RNAase-A was converted back into RNAase-A. Limited proteolysis of dimethyl RNAase-A by subtilisin resulted in the formation of an active RNAase-S-type derivative, namely dimethyl RNAase-S, which was chromatographically distinct from dimethyl RNAase-A and had very nearly the same enzymic activity as dimethyl RNAase-A. Fractionation of dimethyl RNAase-S by trichloroacetic acid yielded dimethyl RNAase-S-protein and dimethyl RNAase-S-peptide, both of which were inactive by themselves but regenerated dimethyl RNAase-S when mixed together. Dimethyl RNAase-A-peptide was identical with RNAase-S-peptide. RNAase-S-protein could be generated from dimethyl RNAase-S-protein by careful hydrolysis of the methyl ester groups at pH9.5. The interaction of dimethyl RNAase-S-protein with RNAase-S-peptide appears to be about 4-fold weaker than that between the RNAase-S-protein and RNAase-S-peptide. Conceivably, the binding of the S-peptide ;tail' of dimethyl RNAase-A with the remainder of the molecule is similarly weaker than that in RNAase-A, and this brings about subtle changes in the geometrical orientation of the active-site amino acid residues of these modified methyl ester derivatives. It is suggested that these changes could be responsible for the generation of the catalytically less-efficient RNAase-A and RNAase-S molecules (dimethyl RNAase-A and dimethyl RNAase-S respectively).
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PMID:Structure and enzymic activity of ribonuclease-A esterified at glutamic acid-49 and aspartic acid-53. 70 73

When purified rabbit sciatic nerve myelin, whether lyophilized or not, is treated with low amounts of trypsin (25 microgram/ml) for 0.5, 3, or 24 h the resulting protein patterns viewed on sodium dodecyl sulfate (SDS) gel electrophoresis are similar. The most striking feature of the trypsinized myelin is the accumulation of a heavy band at the basic protein position, molecular weight 19 000, which is accounted for as a degradation product of the PO protein, referred to as the TPO protein. The PO protein, the major glycoprotein of sciatic nerve myelin, as well as the 23K and P2 proteins and albumin, an absorbed component, are all partially degraded; most high molecular weight bands are lost. The TPO protein, isolated by gel filtration in 2% SDS on an agarose column, like the PO protein, is highly insoluble in aqueous solvents. It is a glycoprotein (8% carbohydrate), staining with periodic acid-Schiff reagent; containing 3 mannose, 1 galactose, 3 N-acetylglucosamine, 1 sialic acid, and 1 fucose residues and is identical to the nonasaccharide of the parent PO protein. The amino acid composition of the TPO protein, is similar to the PO protein, but has a much higher content of hydrophobic residues and begins with NH2-methionine. This suggests that the PO protein is an amphipathic membrane protein in which its more polar character is confined to the first third of its NH2-terminus. This polar domain is probably positioned above the lipid leaflet where it is accessible to trypsin which cleaves a sensitive lysinyl (or argininyl)-methionine linkage. The more hydrophobic domain (the TPO protein) is buried in the myelin bilayer where it is protected from further tryptic attack. Thus trypsin can serve as a useful probe of myelin structure.
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PMID:Isolation of a product from the trypsin-digested glycoprotein of sciatic nerve myelin. 70 55

Human trypsins 1 and 2 both converted Met-Lys-bradykinin to bradykinin and released bradykinin from kininogen in human plasma as measured by bioassay with the isolated guinea pig ileum. Porcine kallikrein did not act on Met-Lys-bradykinin and released kallidin from human kininogen. Since human trypsin 1 is only partially and trypsin 2 completely inhibited by soybean trypsin inhibitor, these data show that the criterion of susceptibility to soybean trypsin inhibitor cannot be used to discriminate between trypsin and kallikrein of different species.
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PMID:Comparison of the kininogenase activity of human pancreatic trypsins and porcine Kallikrein on Met-Lys-bradykinin and human plasma kininogen. 71 Nov 61

The translation of ovomucoid mRNA in a reticulocyte lysate protein-synthesizing system yields a precursor form which contains an NH2-terminal extension of 23 amino acid residues. Edman degradation of radioactive translation products (pre-ovomucoid) identified the following sequence: formula : (see text), where the initiator methionine (in parentheses) is the only residue cleaved from the NH2 terminus during cell-free synthesis and the vertical line indicates the site at which pre-ovomucoid is cleaved in vivo to yield ovomucoid. The precursor sequence differs from those of two other proteins (pre-lysozyme and pre-conalbumin) secreted by the same cell, but resembles these and other secretory protein "signal peptides" in both length and hydrophobicity. Pre-ovomucoid does not interact with trypsin in the same manner as mature ovomucoid.
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PMID:Precursor of egg white ovomucoid. Amino acid sequence of an NH2-terminal extension. 72 26

Casein was modified by use of a series of active N-hydroxy-succinimide esters of amino acids in order to study the effects of new covalently linked hydrophobic or hydrophilic groups on its physical and nutritional properties. Tryptophan was used to determine the best conditions for the chemical reaction and to study the stability of the newly formed amide linkage (isopeptide bond). Casein was also modified with glycine, alanine, methionine, N-acetyl-methionine and aspartic acid. In vitro hydrolysis studies using bovine chymotrypsin, pancreatine and rat bile-pancreatic juice indicated that digestibility of the modified casein derivatives was lower than that of the untreated protein. Since solubility was not significantly changed (except for tryptophyl-casein), the decreased in vitro digestibility is probably due to other factors such as steric hindrance as well as decrease in lysine residues available to trypsin in pancreatin and rat pancreatic juice. Plasma amino acid patterns for rats fed a 10% protein diet of highly modified glycyl-casein or methionyl-casein suggest that the epsilon-aminolysyl derivatives are readily hydrolyzed in vivo. This was confirmed by the growth response of rats fed the following isonitrogenous diets (protein source listed only): casein, casein + free methionine, methionyl-casein, casein + free N-acetyl-methionine, N-acety-methionyl-casein. Covalently attached methionine appeared to be as readily available as the free amino acid; bound N-acetyl-methionine was also available but to a slightly lower extent. Although this study is preliminary, the covalent attachment of amino acids to proteins appears to be a promising method for improving the biological value of food proteins.
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PMID:A method for improving the nutritional value of food proteins: covalent attachment of amino acids. 72 27

The conditions of digestion of colicin E3 with trypsin were examined to obtain an active fragment (T2A) of colicin E3, and a method suitable for large-scale preparation of T2A was developed. The T2A preparation thus obtained was homogeneous on SDS-polyacrylamide gel electrophoresis and the molecular weight was estimated to be about 11,000. T2A was composed of 97 amino acid residues and was rich in basic amino acids; methionine, valine, cysteine, and cystine were absent. The N-terminal residue was lysine and the structure near the C-terminus was -Lys-Lys-Tyr-Leu. Since T2A had no lysine or arginine residue at the C-terminus and since the C-terminal structure was identical to that of protein A, it was concluded that T2A was derived from the C-terminal region of protein A. No clear differences were detected among T2A preparations obtained from 3 different fractions of colicin E3, suggesting that the apparent homogeneity of colicin E3 does not involve the T2A region.
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PMID:Preparation and characterization of an active fragment of colicin E3. 73 Jul 46

Protein S21 was digested with trypsin before and after maleylation, with chymotrypsin, thermolysin and a glutamyl-specific protease. The resulting peptides were isolated and their amino acid composition determined. The amino acid sequences of selected peptides were determined either by the manual subtractive Edman method or by the dansyl-Edman procedure. Additional information was obtained from the automatic Edman degradation of the whole protein in a modified Sequenator. All these results combined yielded the sequence shown in Fig. 1. Protein S21 consists of 70 amino acids (Asp1,Asn2,Thr3,Ser2,Glu8,Pro3,Gly1,Ala9,Val6,Cys1,Ile1,Leu4,Tyr2,Phe3,His1,Lys9 and Arg14). It contains neither Met nor Trp. The molecular weight amounts to 8359. Clustering of basic amino acids is observed in five regions. We also include a prediction for regions with alpha-helices and with beta-sheets.
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PMID:Determination of the complete amino acid sequence of protein S21 from Escherichia coli ribosomes. 76 57

A protein proteinase inhibitor was isolated and purified from eggplant exocarp by heat treatment, ammomium sulfate fractionation, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-25 and G-50. The final purified preparation of the inhibitor was found homogeneous by electrophoretic analysis. The inhibitor showed strong and stoichiometric inhibition on trypsin whereas it showed weak inhibition on alpha-chymotrypsin. It displayed no inhibiting characteristics on pepsin. The molecular weight of the inhibitor was estimated to be approximately 6000. This finding, with the trypsin inhibition data, suggested that the inhibitor combined trypsin in the molar ratio of 1:1. The amino acid analysis indicated that the inhibitor is rich in half-cystine, glycine and aspartic acid, and contains no tryptophan, histidine, methionine or valine.
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PMID:Purification and partial characterization of a protein proteinanse inhibitor isolated from eggplant exocarp. 78 32

Thioredoxin from Escherichia coli (a small hydrogen transport protein containing 108 amino acid residues and having in its oxidized form a single disulfide bond) was acylated with citraconic anhydride. Citraconylation of all amino groups resulted in total loss of enzymatic activity with thioredoxin reductase and immunoprecipitin activity with antithioredoxin antibodies; both these activities were fully restored after deblocking of the citraconylated protein by acid treatment. Large enzymatically inactive peptide fragments of thioredoxin were prepared by selective cleavage at Arg-73 and Met-37, respectively, and tested for their antigenic activity with antibodies against native thioredoxin. Thioredoxin-T-(1-73) and thioredoxin-T-(74-108) were separated by Sephadex G-50 chromatography in 50% acetic acid of a deblocked trypsin digest of citraconylated thioredoxin. Thioredoxin-T-(1-73) afforded about 25% of the corresponding immunoprecipitate of native thioredoxin without significant inhibition at antigen excess. Thioredoxin-T-(74-108) gave no immunoprecipitate but was a potent inhibitor of the reaction of thioredoxin and antithioredoxin as measured by turbidity formation. A major antigenic determinant of thioredoxin was present in the COOH-terminal sequence of the molecule. An equimolar mixture of thioredoxin-T-(1-73) and thioredoxin-T-(74-108) showed full immunoprecipitation activity with antithioredoxin and significant enzymatic activity with thioredoxin reductase. Gel chromatography experiments at pH 8 with the peptide mixture showed a main symmetrical peak with elution volume and amino acid composition identical with native thioredoxin. The results strongly suggested reconstitution of these two fragments to a complex, thioredoxin-T', with a conformation similar to native thioredoxin. Reconstitution of a thioredoxin-like structure was also obtained from a mixture of the overlapping fragments thioredoxin-T-(1-73) and thioredoxin-C-(38-108), although these peptides represented more than the 108 amino acid residues of the protein. Previous results showed reconstitution of thioredoxin-C-(1-37) and thioredoxin-C-(38-108) to a complex called thioredoxin-C' (Holmgren, A. (1972) Fed. Eur. Biochem. Soc. Lett. 24, 351-354). Together with the present results, this shows that three different combinations of two larger peptide fragments obtained by cleavage at two permissible sites gives reconstitution of thioredoxin. In each case, at least one of the component peptides showed strong immunochemical activity with antibodies to native thioredoxin, Netion from a tetrameric to a dimeri
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PMID:Reconstitution of Escherichia coli thioredoxin from complementing peptide fragments obtained by cleavage at methionine-37 or arginine-73. 80 2

Information compiled by automatic Edman degradation of Streptomyces griseus trypsin coupled with previous data has permitted the assignment of the first 36 residues at the NH2 terminus of the protein. Cyanogen bromide cleavage at the three methionine residues followed or preceded by reduction and aminoethylation resulted in the production of four fragments, Cnl to Cn4, which were separated by gel filtration on Sephadex G-50 or G-75. Fragments CN4 (15 RESIDUES) AND Cn3 (5 residues) were shown to be derived from the NH2 terminus of the protein while Cn2 (47 residues and devoid of homoserine) was from the COOH terminus. The arrangement of the fragments was thus Cn4-Cn3-Cn1-Cn2. Automatic Edman degradation in the sequenator coupled with peptides derived from alpha-lytic protease and chymotryptic digestion and from the peptic and tryptic peptides previously elucidated have permitted the sequence determination of fragments Cn1 and Cn2 and therefore of the whole protein. These studies show that extensive regions of identity or similarity exist between Streptomyces griseus trypsin and bovine trypsin. These include the NH2-terminal four residues, the sequences near histidine-57 (chymotrypsinogen A numbering system), aspartic acid-102, aspartic acid-189, and serine-195, the regions of the three disulfide bridges, and the COOH-terminal end (residues 225-229) of the proteins. When aligned to maximize homology the identity of residues is 34%. This identity is increased to 54% when only those residues classified as internal by Stroud et al. (Stroud, R. M., Kay, L. M., and Dickerson, R. E. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 125) are considered. These results indicate that the folding of the polypeptide chains of the two enzymes is very similar and are in agreement with the very similar enzymic, chemical, and physical properties of the two enzymes.
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PMID:Amino acid sequence of Streptomyces griseus trypsin. Cyanogen bromide fragments and complete sequence. 80 14


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