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 inactivation of tetrameric isocitrate lyase from Escherichia coli by 3-bromopyruvate, exhibiting saturation kinetics, is accompanied by the loss of one sulfhydryl per subunit. The substrates glyoxylate and isocitrate protect against inactivation whereas the substrate succinate does not. The modification by 3-bromopyruvate (equimolar to subunits) imparts striking resistance to digestion of isocitrate lyase by trypsin, chymotrypsin, and V8 protease as well as a major decrease in the intensity of tryptophan fluorescence. After alkylation, the sequence Gly-His-Met-Gly-Gly-Lys is found following the modified Cys residue in the tryptic peptide representing positions 196-201. Thus Cys195 is alkylated by 3-bromopyruvate.
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PMID:Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate. 218 22

The protein glia maturation factor beta, isolated from bovine brain, has been sequenced by automated Edman degradation and tandem mass spectrometry of overlapped peptide fragments generated by cyanogen bromide cleavage and enzymatic digestion with trypsin, chymotrypsin, and endoproteinases Asp-N and Lys-C. The protein has 141 amino acid residues and possesses no potential N-glycosylation sites. It contains three cysteines (at positions 7, 86, and 95), three methionines (at positions 33, 101, and 102), and one tryptophan (at position 132). The blocked amino terminus as determined by tandem mass spectrometry is an N-acetylated serine. The carboxyl terminus is a histidine. To our knowledge, the sequence shows no significant homology with other sequenced proteins. The molecular weight calculated from the sequence information is 16,582.
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PMID:Complete amino acid sequence of bovine glia maturation factor beta. 219 64

Equilibrium binding studies on the interaction between the anthracycline daunomycin and plasma membrane fractions from daunomycin-sensitive and -resistant murine leukemia P-388 cells are presented. Drug binding constants (KS) are 15,000 and 9800 M-1 for plasma membranes from drug-sensitive and drug-resistant cells, respectively. Drug binding to the membranes is not affected by either (i) thermal denaturation of membrane proteins or (ii) proteolytic treatment with trypsin, thus suggesting that the protein components of the membranes do not have a major role in determining the observed drug binding. Also, fluorescence resonance energy transfer between tryptophan and daunomycin in the membranes indicates that interaction of protein components with the drug should not be responsible for the observed differences in drug binding exhibited by plasma membranes from drug-sensitive and -resistant cells. Plasma membranes from drug-sensitive cells contain more phosphatidylserine and slightly less cholesterol than membranes from drug-resistant cells. Differences in the content of the acidic phospholipid between the two plasma membranes seem to produce a different ionic environment at membrane surface domains, as indicated by titration of a membrane-incorporated, pH-sensitive fluorescence probe. The possible role of membrane lipids in modulating drug binding to the membranes was tested in equilibrium binding studies using model lipid vesicles made from phosphatidylcholine, phosphatidylserine, and cholesterol in different proportions. The presence of phosphatidylserine greatly increases both the affinity and the stoichiometry of daunomycin binding to model lipid vesicles. The similarity between the effects of phosphatidylserine and other negatively charged compounds such as dicetyl phosphate, cardiolipin, or phosphatidic acid suggests that electrostatic interactions are important in the observed binding of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of membrane lipids in the interaction of daunomycin with plasma membranes from tumor cells: implications in drug-resistance phenomena. 220 6

Data on alpha-chymotrypsin interactions with hydrophobic low-molecular compounds have been generalized. Existence of two sites of noncovalent interaction with hydrophobic nuclei of a ligand molecule is shown. When the substance to be bound contains only one hydrophobic nucleus, the interaction is mediated by a "hydrophobic pocket" of the enzyme--a binding site of amino acid residues which are, in the P1-position relative to the cleaved bond. Under these conditions substances with an asymmetric hydrophobic nucleus (of the tryptophan type) are better ligands for binding. In case of compounds containing several hydrophobic groups scattered in the space, interaction with the enzyme proceeds in two binding sites. New data are presented on the ligand specificity for binding sites of chymotrypsin in lower vertebrates. Relative position of hydrophobic groups of the ligand is shown as that of great importance for interaction with the enzyme. It is concluded that the binding sites of trypsin- and chymotrypsin-like proteinases of the lower vertebrates differ but less from each other as compared to binding sites of trypsin and chymotrypsin in mammals.
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PMID:[Hydrophobic interaction of alpha-chymotrypsin with low molecular weight compounds]. 227 Jun 21

The interaction with calmodulin of the 17-residue C-terminal fragment M5 of myosin light chain kinase has been studied by several physical techniques. Circular dichroism measurements suggest that M5 exists within the complex primarily as an alpha-helix. Fluorescence intensity measurements of the single tryptophan of M5 (Trp-4) indicate that it is in a relatively nonpolar environment and is shielded from solvent. Dynamic measurements of fluorescence anisotropy decay indicate that Trp-4 changes from a freely rotating fluorophore to one which is largely immobilized upon complex formation. Static fluorescence measurements show that 2,6-TNS is displaced from its binding site on calmodulin by M5. The binding of M5 also partially inhibits the proteolytic scission by trypsin of the bond between residues 77 and 78.
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PMID:The interaction of calmodulin with the C-terminal M5 peptide of myosin light chain kinase. 229 18

We have investigated the effect of limited trypsin digestion of chymotryptic myosin Subfragment-1 (S-1) on its kinetic properties. We find that Vmax (i.e., the extrapolated maximal ATPase activity at infinite actin) remains approximately constant, independent of the period of digestion. We also find that the apparent actin activation constant, KATPase, and the apparent dissociation constant, Kbinding, are both significantly weakened by trypsin digestion of S-1, and that these kinetic parameters change in concert. In addition, we investigated the effect of limited trypsin digestion on the initial phosphate burst. We find that trypsin digestion has no effect on the rate of the tryptophan fluorescence enhancement that occurs after ATP binds to digested S-1, but that the magnitude of the fluorescence enhancement falls approximately 40% with digestion. Digested S-1 also showed anomalous behavior in that the fluorescence magnitude increased and the fluorescence rate dropped in the presence of actin. Trypsin digestion also decreased the magnitude of the chemically measured Pi burst approximately 35%, but this magnitude was essentially unaffected by actin. A possible explanation for this behavior is discussed.
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PMID:Effect of limited trypsin digestion on the biochemical kinetics of skeletal myosin subfragment-1. 234 Mar 42

1. Lugworm protease C further purified by benzamidine-affinity chromatography, exhibited peptidase specificity for arginyl and lysyl bonds. 2. Protease C consisted of a single polypeptide with a mol. wt of ca 23,000 as determined by SDS polyacrylamide gel electrophoresis, exhibited a u.v. absorption maximum at 280 with an (mg/ml) extinction coefficient of 0.93 and fluorescence spectra typical of a protein containing tryptophan, and had an amino acid composition similar to trypsins. 3. The Kms of the cleavages of the arginyl bond of oxidized insulin B chain and of the lysyl bond of the gly23-ala30 fragment were determined to be 0.72 and 0.96 mM; the corresponding kcats were 38 sec-1 and 1.5 sec-1. The Km and kcat for TAME were 0.042 mM, and 110 sec-1. 4. Lugworm protease C was confirmed to be a trypsin.
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PMID:Characterization and peptidase specificity of lugworm (Arenicola cristata) protease C. 234 31

A number of different influenza C virus strains were tested for their fusion properties using a resonance energy assay which allows direct monitoring of fusion between virus membranes and artificial lipid vesicles. The fusion pH of various strains was found to range between 5.6 and 6.1. Haemolytic activity of the different strains with chicken erythrocytes was observed at slightly lower pH values and varied between 5.1 and 5.7. Studies of the kinetics of influenza C virus fusion showed distinct characteristics in fusion activity. A lag before onset of fusion was found with influenza C virus which was not observed for influenza A or B viruses. In addition, studies on the rate of conformational change of the influenza C virus glycoprotein, as determined by morphological changes and endogenous tryptophan fluorescence, suggest that the conformational change is rate-limiting in the fusion process, whereas for influenza A viruses the glycoprotein conformational change is fast and a later step in the fusion process is rate-limiting. Monitoring the conformational change of influenza C virus glycoprotein by the onset of trypsin susceptibility showed, however, that membrane fusion occurred in some cases without onset of trypsin susceptibility, indicating that the trypsin-susceptible conformation is a post-fusogenic conformation.
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PMID:Fusion characteristics of influenza C viruses. 234 68

A protein antigen with an apparent molecular weight (Mr) of 31,000 was isolated from 0.2 M glycine hydrochloride (pH 2.2) extracts of a typical human fecal isolate, Campylobacter jejuni VC74. The protein was purified to homogeneity on a preparative scale by immunoaffinity chromatography followed by molecular sieving with a Superose 12 column. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.3, and amino acid composition analysis showed that the protein was unusually rich in lysine, containing 14.9 mol% of this basic amino acid. Cysteine and tryptophan were absent. The protein also contained approximately 35% hydrophobic amino acid residues, and N-terminal amino acid analysis showed that 17 of the first 38 residues were hydrophobic. This amino-terminal sequence to residue 22 was virtually identical to that of an antigenically cross-reactive 31,000-Mr protein isolated from another C. jejuni strain belonging to a different heat-labile serogroup. Western blotting (immunoblotting) of glycine extracts of other C. jejuni, Campylobacter coli, and Campylobacter laridis strains belonging to different thermolabile and thermostable serotypes, as well as Campylobacter fetus, with a rabbit polyclonal antiserum raised against the purified C. jejuni VC74 protein showed that all C. jejuni, C. coli, and C. laridis strains tested contained a 31,000-Mr protein with epitopes which were antigenically cross-reactive with the C. jejuni VC74 protein. The antigenically cross-reactive epitopes of this protein were also readily detected by immunodot blot assay of glycine extracts of C. jejuni, C. coli, and C. laridis with monospecific polyclonal antisera to the 31,000-Mr protein, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of these important pathogens. Slide agglutination reactions, immunofluorescence assay, and immunogold electron microscopy with antisera to purified 31,000-Mr protein and trypsin treatment of whole cells indicated that the cross-reactive epitopes of the 31,000-Mr protein were not exposed on the cell surface. Cell fractionation analysis and immunogold electron microscopy located the protein on the outer surface of the cytoplasmic membrane. This finding suggests that the 31,000-Mr protein is not a good candidate for inclusion in a monovalent subunit Campylobacter vaccine.
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PMID:Purification, characterization, and localization of a protein antigen shared by thermophilic campylobacters. 238 Mar 60

Trp repressor of Escherichia coli K-12 is a dimeric protein (monomer size, 108 amino acids) that acquires high affinity for certain operator targets in double-stranded DNA upon interaction with L-tryptophan. High titer antiserum directed against E. coli Trp repressor protein, elicited in rabbits, was monospecific toward native or denatured Trp repressor. Using an enzyme-linked immunosorbent assay to measure antigen-antibody reaction, we found that the binding of L-tryptophan to Trp repressor was associated with a marked decrease in antibody reactivity that presumably accompanied a conformational change in this protein to a state with strong affinity for trp operator-bearing DNA. We analyzed the pattern of cleavage of Trp repressor by chymotrypsin and trypsin and the effect of L-tryptophan on such hydrolytic cleavages. Chymotrypsin cleaved Trp repressor mainly between residues 71 and 72. In the presence of L-tryptophan this cleavage was slowed. The first-order rate constants for chymotryptic digestion of Trp repressor were 7.6 X 10(-2) and 4.6 X 10(-2) min-1 in the absence and presence of L-tryptophan, respectively. Tryptic digestion was more complex. Initial cleavage of Trp repressor occurred with approximately equal facility between residues 69-70 or 84-85. Subsequent tryptic hydrolyses led eventually to a major core fragment containing the first 54 amino acids of Trp repressor plus four other fragments from the carboxyl-terminal half of the protein. In the presence of L-tryptophan, cleavage by trypsin between residues 54-55 and 84-85 was retarded, even when a previous hydrolytic event elsewhere in the protein had occurred. Tryptophan had essentially no effect on the tryptic hydrolysis of peptide bond 97-98, but accelerated cleavage at peptide bond 69-70. The first-order rate constants for the first tryptic cleavage of Trp receptor were 1.55 X 10(-1) and 1.33 X 10(-1) min-1 in the absence and presence of ligand, respectively. Our results are compatible with a structural model wherein certain amino acid side chains and peptide bonds of Trp repressor (specifically, those of residues 69-85) lie on or near the surface of the protein. This region of Trp repressor has been predicted to contain the operator recognition site. The susceptibility to proteolytic attack of at least four peptide bonds in this area changes when the protein interacts with L-tryptophan.
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PMID:Ligand-mediated conformational changes in Trp repressor protein of Escherichia coli probed through limited proteolysis and the use of specific antibodies. 241 31


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