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)

Red algae of the species Porphyridium cruentum were grown in a minimum sulfate medium containing 35SO42-. 35S-labeled phycoerythrin was extracted. B Phycoerythrin, b phycoerythrin and R phycocyanin could be separated from other proteins by using a carrier-free electrophoresis on columns. The final ratio A545/A280 of B phycoerythrin thus obtained was greater than or equal to 5. 35S-labeled B phycoerythrin was digested proteolytically with trypsin and pepsin. The resulting 35S-containing bilipeptides were separated by isoelectric focusing. Zones of enhanced chromophore concentration always showed an enhanced radioactivity. Peptide fractions with a low molar ratio sulfur/chromophore (1.1-1.8) were purified to remove sucrose and the carrier ampholyte. A modified, optimized Edman degradation followed. A butylacetate-soluble, red Edman product was obtained that contained most of the chromophore and the bulk of the radioactivity. This product was purified by two-dimensional thin-layer chromatography. The main spot of the chromatogram was subjected to acidic hydrolysis. The major part of the radioactivity in the hydrolysate cochromatographed with cysteine. That proves cysteine to be the binding amino acid in all cases investigated.
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PMID:The protein-chromophore bond in B phycoerythrin from Porphyridium cruentum. Radiosulfur labeling experiments. 52 Mar 25

Aspartate transaminase from chicken heart cytosol was immobilized covalently on activated thiol-Sepharose and digested with trypsin. After washing, the thiol-containing peptides were eluted with 2-mercaptoethanol and further purified by gel-filtration and paper chromatography. Three pure cysteinyl peptides were isolated. One of them may be represented as Ile-(Asp, Met, Cys, Gly, Leu, Thr2)-Lys; this peptide is identical to the fragment comprizing residues 387--395 in the peptide chain of aspartate transaminase from pig heart cytosol. It thus contains a cysteine residue homologous to Cys-390 of the pig heart enzyme. The second cysteinyl peptide had the following composition and partial sequence: Tyr-Phe-Val-Ser-Glu-Gly-Phe-Glu-Leu-Phe (Cys, Ala, Glu, Ser2, Phe)Lys, which corresponds to the sequence 242--258 of the pig enzyme and thus contains a cysteine residue homologous to Cys-252. The third cysteinyl peptide was similar to the tryptic peptide of the pig enzyme containing Cys-191.
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PMID:[Thiol peptides from the aspartate transaminase of chicken heart cytosol]. 59 23

Ehrlich ascites tumour cells contain a neutral protease, capable of solubilising fluorescein-labelled telopeptides from fluorescein-labelled polymeric collagen fibrils. The cells also contain an inhibitor for this enzyme and for trypsin. The enzymically inactive enzyme-inhibitor complex can be dissociated with the mercurial thiol agent, mersalyl, with the consequent regain of enzymic activity. The reactivated neutral protease and also trypsin can be inhibited by addition of thiols such as cysteine, mercaptoethanol and dithiothreitol. Trypsin can be protected from inactivation by the tumor inhibitor by addition of cystine or L-1-tosylamido-2-phenylethyl chloromethyl ketone(TosPheCH2Cl)-inactivated chymotrypsin. The evidence suggests that the inhibitor contains a reactive thiol group which exchanges with one or more significant disulphide bridges in trypsin and the neutral protease, resulting in enzyme-inhibitor complex formation and loss of activity. Similarly, thiols interact with these enzymes resulting in a corresponding loss of enzymic activity. The evidence obtained with Tos-PheCH2Cl-inactivated chymotrypsin, which reactivated previously inhibited trypsin and neutral protease, demonstrates that the active site of the enzyme is not involved in the interaction with the thiol of the inhibitor but that the significant disulphide bond in the enzyme is required for the maintenance of the active site conformation. This disulphide exchange mechanism is therefore a form of reversible allosteric control of proteolytic activity and has been shown to be distinct from the mechanism by which soya bean trypsin inhibitor interacts with trypsin.
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PMID:Evidence for the inhibition of trypsin by thiols. The mechanism of enzyme-inhibitor complex formation. 62 6

Macromomycin is a protein isolated from the culture filtrate of Streptomyces macromomyceticus. It is an antibiotic and also cytotoxic to a broad spectrum of carcinoma cells, the ID50 for P388 leukemia cells being 1 X 10(-9) M. Macromomycin binds rapidly and tightly to the P388 cell membrane and the eventual death of the cell cannot be reversed by either washing the toxin away or treating the cell with trypsin. The cytotoxicity does not appear to be specific for any phase of the P388 cell cycle. Macromomycin is a single polypeptide, pI 5.38, devoid of methionine and arginine residues and contains 4 cysteine residues joined by two intramolecular disulfide bonds. The cytotoxicity results in inhibition of DNA, RNA, and protein synthesis in P388, the latter inhibition occurring a few hours after the inhibition of nucleic acid synthesis. The antibiotic and antitumor activities are destroyed rapidly by ultraviolet light, which gives a product that differs little in amino acid composition, molecular weight, and antigenic property, but can be separated from the native macromomycin by ion exchange chromatography. It is proposed that macromomycin has an ultraviolet-sensitive prosthetic group upon which much of the biological activity is based.
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PMID:Studies on macromomycin, an antitumor protein. 64 Oct 69

Rabbit skeletal alpha-tropomyosin, separated by hydroxyapatite chromatography, was treated with trypsin (1/100 wt/wt) at 0 degrees C for 24 h. Trypsin-resistant fragments of tropomyosin were separated into the precipitate and supernatant fractions at pH 4.3 in 1 M KCl, and these were subjected to QAE-Sephadex A50 column chromatography for further purification. SDS-gel electrophoresis showed 16,000 and 14,000 dalton bands for the supernatant (s-fragment) and an 11,500 dalton band for the precipitate (p-fragment). We obtained a 13,500 dalton chain (13,500 dalton fragment) in addition to the s- and p-fragments upon treatment with more dilute trypsin (1/500 wt/wt) for 48 h at 0 degrees C. Both the p- and 13,500 dalton fragment had the same C-terminal portion as intact alpha-tropomyosin, and could form an intra-chain disulfide bond on oxidation. Therefore, these two fragments were deduced to be polypeptides from some points on the N-terminal side of Cys 190 to the intact C-terminal. The s-fragment, on the other hand, did not contain any cysteine, Phe, or His residues according to amino acid analysis, suggesting that the fragment is derived from the N-terminal side from Cys 190. Tentative assignment of the fragments was carried out by amino acid analysis, and C- and N-terminal determination. The p-, s-, and 13,500 dalton fragments appear to be in coiled-coil form in solution, having alpha-helical contents of 77,71, and 64%, respectively, and are able to interact with intact tropomyosin to reduce the viscosity of tropomyosin solution. The s-, p-, and 13,500 dalton fragments have little binding capacity individually to troponin, but the mixture, i.e., the s- and p-fragments, the 13,500 dalton fragment and the N-chain, which was obtained by cleavage at Cys 190, showed clear binding with troponin independent of Ca2+ in solution as detected by gel electrophoresis. The p-fragment showed some binding to troponin, since cross-linkage to troponin was possible by treatment with dimethyl suberimidate. From the result, it can be inferred that the troponin binding regions in tropomyosin are located on both sides of Cys 190, where trypsin attacks more easily than at other parts of the molecule, leaving two trypsin-resistant fragments.
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PMID:Tropomyosin fragments obtained by tryptic digestion. 65 5

Homogeneous fragment B, obtained through nicking of diphtheria toxin with insoluble trypsin, was cleaved with cyanogen bromide in 70% formic acid. After citraconylation, the cleavage products were separated by gel filtration on Sephadex G--75 and purified by gel filtration, ion-exchange and thin-layer or paper chromatography. Six CNBr peptides were characterized, the composition of which account for the total amino acid content of fragment B. Their apparent molecular weights are: CB 1, 12 000; CB 2, 14 000; CB 3, 8000; CB 4a, 2400; CB 4b, 2200; CB 5, 2200. CB 4a is the NH2--terminal peptide; it contains the cysteine residue of the disulfide bridge linking fragment B to fragment A. CB 3 is the COOH--terminal peptide; it bears the disulfide bridge of fragment B. Characterization of two CNBr--derived overlapping peptides provided the positioning of CB 4b and CB 2 and allowed an alignment of the CNBr peptides of fragment B to be proposed.
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PMID:Isolation and characterization of the cyanogen bromide peptides from the B fragment of diphtheria toxin. 66 18

Molecular weight determinations of immunoglobulin D suggest the presence of an extra region of the delta chain. In an attempt to locate this region, an IgDlambda myeloma protein (Gur), was digested with trypsin for 4 min at 56 degrees and the Fc fragment isolated by ion exchange chromatography. N-terminal analysis of this fragment showed a heterogeneity in the site of splitting by trypsin. The Fc was digested with pepsin and trypsin and the cysteine containing peptides isolated by a two dimensional paper high voltage electrophoresis (diagonal map) at pH 3.5. Further purification of these peptides was carried out by HVE at pH 6.5 and 2.1 and their amino acid composition and partial sequence were determined. Only five cysteic acid peptides were obtained, one corresponding to the inter heavy-heavy bridge and the other four, to intra chain bridges. This finding would exclude the possibility of an extra "classical domain" in this region. The position of these peptides in the delta chain has been arranged based on homology with the other classes of immunoglobulins.
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PMID:Structure of immunoglobulin D: evidence for the absence of an extra disulfide bridge in the Fc fragment. 67 26

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

Native spectrin has trypsin-susceptible sites spaced at a constant molecular weight interval. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of spectrin treated with trypsin at low salt concentrations shows a ladder of fragments spaced at approximately 8,000-dalton intervals, from the intact Band 1 (240,000 daltons) and Band 2 (220,000 daltons) down to about 150,000 daltons. The five largest fragments were identified as products of Band 2 using tryptic 125I-peptide mapping of protein from gel slices. Endogenously incorporated [32P]phosphate is absent from the largest fragment, indicating that all phosphorylation sites on spectrin are within 8,000 daltons of a terminal of Band 2. Mapping of both [14C]carboxyamidomethylated cysteine-containing tryptic peptides and 125I-peptides reveals extensive sequence homology between the spectrin subunits. Further, only somewhat over half of the distinct spots expected from the cysteine content are found in both Band 1 and Band 2 peptides. These and the tryptic susceptibility results are interpretable as evidence for a repeating structure in spectrin.
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PMID:Structural studies on human spectrin. Comparison of subunits and fragmentation of native spectrin. 76 4

Native tryptophanyl-tRNA synthetase purified from Escherichia coli B has on each identical subunit a single thiol group which rapidly forms a mixed disulfide with a thionitrobenzoate moiety of 5,5'-dithiobis(2-nitrobenzoic acid). The reaction and the concomitant inactivation of the enzyme are both reversible by reductive removal of the thionitrobenzoate with dithiothreitol. Iodoacetamide and N-ethylmaleimide also react with the thiol group required for enzyme activity, but iodoacetic acid inactivates the enzyme through another mechanism. Three or 4 half-cystine residues/subunit were detected by amino acid analysis and by titration of the denatured enzyme with 5,5'-dithiobis(2-nitrobenzoic acid); no disulfide bonds were detected by borohydride reduction. Cleavage of the subunit (molecular weight 37,000) with 2-nitro-5-thiocyanobenzoic acid gave fragments of molecular weights 32,000, 27,000, and 9,500. Five carboxymethylated peptides were isolated from the trypsin products of the denatured enzyme after treatment with iodo[14C]lacetate. Three of these peptides represented unique sequences surrounding thiol groups in the enzyme. One cysteine-containing nonapeptide has a heptapeptide sequence homologous to a heptapeptide sequence in a cysteine containing decapeptide from the tryptophanyl-tRNA synthetase of human placenta. The nonapeptide appears to bear the thiol group required for enzyme activity.
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PMID:Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. Character of required thiol group and structure of thiol peptides. 77 64


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