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)

Cell walls were prepared from freeze-dried samples of 7 strains of Methanobacterium by mechanical disintegration of the cells followed by incubation with trypsin. Electron microscopy revealed the presence of sacculi exhibiting the shape of the original cells, on which no surface structure could be detected. Ultrathin sections of the isolated sacculi showed a homogenously electron dense layer of about 10--15 nm in width. The ash content varied between 8 and 18% of dry weight. The sacculi of all the strains contained Lys: Ala:Glu:GlcNAc or GalNAc in a molar ratio of about 1:1.2:2:1. In one strain (M. ruminantium M1) alanine is replaced by threonine, however, Neutral sugars and--in some strains--additional amounts of the amino sugars were present in variable amounts, and could be removed by formamide extraction or HF treatment without destroying the sacculi. No muramic acid or D-amino acids typical of peptidoglycan were found. Therefore, the sacculi of the methanobacteria consist of a different polymer containing a set of three L-amino acids and one N-acetylated amino sugar. From cells of Methanospirillum hungatii no sacculi, but tube-like sheaths could be isolated, which tend to fracture perpendicularly to the long axis of the sheath along the fibrills seen on the surface. The sheaths consist of protein containing 18 amino acids and small amounts of neutral sugars. They are resistent to the proteinases tested and are not disintegrated by boiling in 2% sodium dodecylsulfate for 30 min. The three Gram-negative strains Black Sea isolate JR-1, Cariaco isolate JR-1 and Methanobacterium mobile do not contain a rigid sacculus, but merely a SDS-sensitive surface layer composed of regularly arranged protein subunits. This evidence indicates that, within the methanogens, different cell wall polymers characteristic of particular groups of organisms may have evolved during evolution, and supports the hypothesis that the evolution of the methanogens was separated from that of the peptidoglycan-containing procaryotic organisms at a very early stage.
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PMID:Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria. 69 4

We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical.
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PMID:Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2. 72 6

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

Synthetic procedures have been developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2C1 and Pro-Phe-ArgCH2C1, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. For example, Pro-Phe-ArgCH2C1 inactivates plasma kallikrein 50% in 24 min at a concentration of 2 x 10(-8)M, while other trypsin-like proteases are less susceptible in inactivation than kallikrein, differing by a factor of 48 for plasmin and factors of 10(2)-10(5) for factor Xa, thrombin, and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2C1 (Ki = 0.078 micron) is about 60 times that for Ala-Phe-LysCH2C1(Ki = 4.9 micron), whereas human plasmin exhibits about the same affinity for the former affinity label (Ki = 1.3 micron) as for the latter (Ki = 0.83 micron). The rate constants for the irreversible step of the affinity labeling reaction, k2, are similar for affinity labels tested with the individual proteases: 0.35 min-1 for plasma kallikrein and 0.18 min-1 for plasmin.
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PMID:Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein. 72 86

Lysyl-tRNA synthetase was purified to 70-90% of homogeneity from Escherichia coli K-12. The enzyme was purified from wild-type cells grown in minimal medium, or minimal medium containing either 20 mM L-alanine or 3 mM glycly-L-leucine. The synthetase was similarly purified from a mutant strain grown in minimal medium plus 20 mM L-alanine. Results based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and trypsin inactivation studies indicate (A) that the presence of L-alanine of glycyl-L-leucine in the culture medium alters the properties of the wild-type enzyme; (B) that the alteration of the synthetase by l-alanine and glycyl-L-leucine is different; and (c) that the molecular weight of lysyl-tRNA synthetase is at least 135000--140000. The results suggest that most likely the metabolites modify the structure of lysyl-tRNA synthetase, but the possibility that the metabolites induce the synthesis of a new lysyl-tRNA synthetase cannot be completely eliminated.
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PMID:An in vivo effect of the metabolites L-alanine and glycyl-L-leucine on the properties of lysyl-tRNA synthetase from Escherichia coli K-12. I. Influence on subunit composition and molecular weight distribution. 77 46

Proteolyses of colicin E3 by both trypsin and subtilisin yield fragments of various molecular weights. On the basis of sodium dodecyl sulfate gel electrophoresis, tryptic cleavage yields peptides of molecular weight about 42 000 and 18 000, while the comparable pieces in a subtilisin digest have apparent weights of about 36 000 and 24 000. The digests lose almost all of their in vivo cell killing activity but the in vitro activity leading to ribosomal inactivation is augmented. Trypsin-treated colicin E3 shows a 20-30-fold increase in its ability to release the 52 nucleotide fragment from the 16S ribosomal ribonucleic acid (rRNA) and this activity is associated with the smaller fragment. Subtilisin-treated colicin E3 is only about two to three fold more active than the native protein in vitro, and the peptides obtained upon cleavage cannot be separated by gel filtration or polyacrylamide gel electrophoresis without sodium dodecyl sulfate. However, in the presence of 0.1% sodium dodecyl sulfate, subtilisin-treated E3 shows a 20-30-fold augmentation in in vitro activity which is again associated with the smaller fragment extracted from the sodium dodecyl sulfate gel. Amino terminal end-group studies showed that the two larger fragments and intact E3 have the same N-terminal residue, valine. These fragments presumably originate from the amino end of the native protein. The smaller tryptic fragment has an N-terminal alanine, while the smaller subtilisin piece has an N-terminal leucine. In addition, modification of a single carbosyl group in intact colicin E3 abolishes more than 90% of the in vivo activity with a simultaneous increase in in vitro activity. This carboxyl group is located in the larger fragments obtained in both trypsin and subtilisin cleavage. Binding of E3 to sensitive cells is drastically reduced or eliniated by this chemical modification and by both of the limited proteolytic cleavages.
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PMID:Proteolytic and chemical modification of colicin E3 activity. 78 24

C3a anaphylatoxin is a protein fragment generated enzymatically in serum during activation of the third component of complement (C3). A four-step procedure is described for the purification of human and porcine C3a anaphylatoxins from their respective sera after activation with inulin. Because serum carboxypeptidase rapidly inactivates C3a, the inhibitor epsilon-aminocaproic acid (EACA) was added during C3 activation, thus permitting isolation of fully active C3a anaphylatoxins directly from serum. A 2000-fold purification of C3a was achieved with an average 30% recovery assuming total conversion of C3 during treatment of serum with inulin. Human C3a anaphylatoxin obtained through the action of the C3 activating enzyme of the "alternate" pathway appeared nearly identical with the C3a obtained from isolated C3 after treatment with the C4,2 enzyme of the "classical" pathway or with trypsin. Comparisons were made between various properties of human and porcine C3a anaphylatoxins. The molecular weights differed only slightly. Electrophoresis on a cellulose acetate strip at pH 8.6 indicated a difference of approximately one net charge between human and porcine C3a, with the human anaphylatoxin exhibiting the more basic behavior. Although the amino acid compositions are similar, significant differences exist. The most marked difference was the total absence of threonine residues in porcine C3a. The NH2-terminal sequences of 20 amino acid residues were examined; homology existed for 16 of the 20 positions. Although partial analysis of the primary structure of human and porcine C3a indicates approximately 80% homology, no immunological cross-reactivity between the anaphylatoxins could be detected with antisera produced to either human or porcine C3a. In spite of the structural differences, the biological activities of porcine and human C3a were essentially identical. Smooth muscle contraction, increase in vascular permeability, and release of histamine from mast cells were similarly induced by equal amounts of anaphylatoxin from either human or porcine origin. Porcine C3a, like human C3a, was shown to contain a COOH-terminal arginyl residue essential for smooth muscle contraction and for induction of histamine release from mast cells. The sequence adjacent to the COOH-terminal arginine was Leu-Ala-Arg-COOH for both humans and porcine C3a. Current evidence suggests common mechanisms exist for the generation of C3a in various animal species and that the two known C3 activating enzymes in serum exhibit trypsin-like specificity.
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PMID:Purification and partial characterization of human and porcine C3a anaphylatoxin. 80 5

Proteinase inhibitor II, an inhibitor of chymotrypsin and trypsin, is a heat-stable protein with a dimeric molecular weight of 21 000 that is a component of Russet Burbank potato tubers. Four monomeric isoinhibitor species of molecular weight 10 500 comprise inhibitor II and were isolated by chromatography on phosphocellulose in 8 M urea. Upon removal of the urea, each monomeric species dimerized to yield homogeneous dimers. The three major protomer species, called B, C, and D, and their homogeneous dimers were further characterized. They have similar molecular weights and amino acid compositions, and each has an N-terminal alanine residue. Dimers of purified protomers B, C, and D exhibited full cross-reactivities with each other in immunological double-diffusion assays. Reconstituted dimers possess two binding sites for bovine alpha-chymotrypsin, indicating that each monomer possesses one binding site for this enzyme. Significant differences were noted among the reconstituted dimers in their isoelectric points, immunoelectrophoretic mobilities, ion-exchange properties, and their inhibitory reactivities against trypsin. The properties of the inhibitor II dimeric species are similar but not identical to inhibitors IIa and IIb reported from Japanese potatoes (variety "Danshaku-Imo"), indicating the existence of intervarietal, as well as intravarietal, differences among potato tuber inhibitor II isoinhibitors.
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PMID:Proteinase inhibitor II from potatoes: isolation and characterization of its protomer components. 82 19

During studies on the amino acid sequence of bovine nasal cartilage collagen, the cyanogen bromide peptide alpha1(II)-CB11 was degraded to smaller peptides with trypsin. One of the tryptic peptides, T5, which contained 39 residues was shown by amino acid and sequence analyses to occur in a predominant form that contained glutamine at position 5 and in a second form with leucine at this site. In addition to the heterogeneity at this position, amino acid analyses of five different preparations revealed that the peptide with leucine contained a seryl residue not found in the major form. Sequence heterogeneity at a third position of alpha1(II) was demonstrated by the isolation of a hexapeptide (T2) from the trypsin digest of alpha1(II)-CB11 which contained 0.21 residue of alanine and 0.77 of leucine. Both the leucine and alanine of T2 were removed after the second cycle of subtractive Edman degradation. These data show that at least two types of alpha1(II) chains, designated as alpha1(II)Major and alpha1(II)Minor, exist in bovine nasal cartilage. Further considerations suggest that these two chains are probably not variants derived from allelic genes but are the products of separate genes.
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PMID:The covalent structure of cartilage collagen. Evidence for sequence heterogeneity of bovine alpha1(II) chains. 83 47

Isolation of tropoelastin is complicated by the presence of a neutral protease closely associated with tropoelastin that is capable of sequentially degrading tropoelastin to small peptides. Substrate and inhibitor specificities of this neutral protease associated with purified tropoelastin were examined. The enzyme displayed proteolytic activity against casein, and esterase activity was detected when assayed against N-tosyl-L-arginine methyl ester but not against tert-butyl-oxycarbonyl-L-alanine p-nitrophenyl ester. No appreciable elastinolytic activity was detectable against either insoluble sodium dodecyl sulfate treated elastin or maleylated tropoelastin. The enzyme was not inhibited by the chymotrypsin inhibitor toluenesulfonylphenylalanine chloromethyl ketone. The enzyme was inhibited by phenylmethanesulfonyl fluoride and, to various degrees, by metal chelators. Tosyllysyl chloromethyl ketone, epsilon-aminocaproic acid, and Aprotinin (pancreatic trypsin inhibitor--Kunitz type), all inhibitors of trypsin-like enzymes, were very effective inhibitors, as were soybean trypsin inhibitor and human alpha-1-antitrypsin. The data suggest that the tropoelastin-associated enzyme is a neutral serine protease with trypsin-like specificity.
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PMID:Trypsin-like neutral protease associated with soluble elastin. 90 57


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