Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-L-[methyl-3H]methionine (8-N3-Ado[methyl-3H]Met), the binding sites for S-adenosyl-L-methionine(AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC 2.1.1.23); AdoMet:histone-arginine N-methyltransferase (EC2.1.1.23); and AdoMet:cytochrome c-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.
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PMID:Comparative studies on S-adenosyl-L-methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-L-methionine as photoaffinity probe. 814 3

It is widely believed that hydroxyl radicals generated in vivo contribute to damage to macromolecules, such as proteins and DNA. We evaluated methodology based on the transformation of protein tyrosine to L-Dopa, via aromatic ring hydroxylation, as an index of hydroxyl radical attack on proteins. The catechol structure of Dopa makes it amenable to isolation with alumina, followed by HPLC analysis, typically used for the measurement of catecholamines. Because a level of controversy exists about the formation of Dopa by hydroxyl radicals, we conducted a systematic study of the formation of Dopa from tyrosine, tyrosine dipeptides, pure proteins (chymotrypsin and myelin basic protein), and endogenous proteins in tissue homogenates (rat brain), exposed to hydroxylating conditions (Fe2+/EDTA/ascorbate at neutral pH). Dopa residues in peptides and proteins were liberated by acid hydrolysis with 6 M HCl at 145 degrees C for 1 h. A marked lability of Dopa in 6 M HCl under hydrolysis conditions was prevented with added phenol; chymotrypsin and precipitated pellets of brain protein were also protective. Overall recoveries (hydrolysis plus purification procedures) averaged 83.4 +/- 1.7%. This improved analytic procedure may be useful for studying protein damage by hydroxyl radicals.
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PMID:Protein L-dopa as an index of hydroxyl radical attack on protein tyrosine. 979 36

Myelin basic protein is known to be released into the circulation following traumatic injuries or demyelination within the central nervous system, resulting in the generation of potentially immunogenic myelin basic protein material. In this investigation we have studied the binding of bovine and human myelin basic protein to human alpha2-macroglobulin, which was found to be the only major myelin basic protein-binding protein in human plasma. Myelin basic protein bound to all three conformational forms of alpha2-macroglobulin studied, i.e., native alpha2-macroglobulin, methylamine-treated alpha2-macroglobulin, and chymotrypsin-treated alpha2-macroglobulin. Zinc chloride (1 mM) or 1 mM iodoacetamide partly blocked the complex formation between myelin basic protein and alpha2-macroglobulin, while 1 mM magnesium chloride, 1 mM calcium chloride, or 1 mM EDTA had no effect on binding. Chymotrypsin and trypsin can degrade myelin basic protein to fragments which do not bind to alpha2-macroglobulin. However, when myelin basic protein was complexed with any of the conformational forms of alpha2-macroglobulin, no significant release of Na[125I]-labeled myelin basic protein occurred after proteinase treatment. The results suggest that binding of myelin basic protein to alpha2-macroglobulin may protect extracellular compartments in vivo from immunogenic myelin basic protein fragments and alpha2-macroglobulin may participate in the specific clearance of myelin basic protein from the circulation.
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PMID:Binding of soluble myelin basic protein to various conformational forms of alpha2-macroglobulin. 980 60

Affinity cosurfactants, consisting of hydrophilic ligands derivatized with hydrophobic tails, increase the efficiency of selective protein recoveries using reversed micelles by extending the operating range of pH and salt concentration over which an extraction can be performed. Three different affinity cosurfactant-protein pairs have been used to demonstrate the principles of this extractive technique: (i) concanavalin A, a lectin, was extracted with the addition of octyl glucoside; (ii) natural amphiphiles, such as lecithin, were used to extract myelin basic protein, a membrane-associated protein known to recognize and bind the phosphatidylcholine headgroup; and (iii) alkyl boronic acids were used to extract chymotrypsin. The enhancement in protein transfer correlated with the binding strength of the free ligand and protein in aqueous solution. Several control studies confirmed the biospecificity of the interactions of protein and affinity cosurfactant.
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PMID:Affinity-based reversed micellar protein extraction: I. principles and protein-ligand systems. 1860 69

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used to create spatial distribution maps from lipids, peptides, and proteins in a variety of biological tissues. MALDI-MSI often involves trade-offs between the extent of analyte extraction and desired spatial resolution, compromises that can adversely affect detectability. For example, increasing the extraction time can lead to unwanted analyte spatial redistribution. With the stretched sample method (SSM), the extraction period can be extended, resulting in reduced analyte redistribution while suppressing detection of cationic salt adducts. The SSM involves thaw-mounting a thin tissue section onto a substrate of small glass beads embedded in Parafilm M and then stretching the membrane to fragment the tissue into thousands of bead-sized pieces. Here, we applied the SSM method to MALDI-MSI using rat spinal cord as a model. We used surface-modified beads coated with trypsin or chymotrypsin in order to facilitate controlled digestion and detection of proteins. The enzymatic reactions were maintained by repeatedly condensing water on the stretched sample surface. As a result, new peptides formed by tryptic or chymotryptic protein digestion were detected and identified using a combination of MALDI-MSI and offline liquid chromatography tandem mass spectrometric analysis. Localization of these peptides indicated the distribution of their proteins of origin, including myelin basic protein, actin beta, and tubulin alpha chain. Additionally, we used uncoated beads to create distribution maps of many endogenous lipids and small peptides. The extension of the SSM using modified beads resulted in the creation of mosaic bead surfaces where adjacent beads were coated with different enzymes or other reactive chemicals, permitting investigation of the distributions of a wider range of analytes in biological samples within a single experiment.
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PMID:The modified-bead stretched sample method: development and application to MALDI-MS imaging of protein localization in the spinal cord. 2162 33