Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The reactivities of phenylglyoxal (PGO), glyoxal (GO), and/or methylglyoxal (MGO) with several proteins, including ribonuclease A [EC 3.1.4.22] and its derivatives, alpha-chymotrypsin [EC 3.4.21.1], trypsin [EC 3.4.21.4], lysozyme [EC 3.2.1.17], pepsin [EC 3.4.23.1], rennin [EC 3.4.23.4], thermolysin, and insulin and its B chain, have been examined. From analyses of the reaction products, PGO was shown to be the most specific for arginine residues. GO and MGO also reacted rapidly with arginine residues, but they also reacted with lysine residues to a significant extent. A side reaction with N-terminal alpha-amino groups was observed with each of these reagents. 2. Two arginine residues out of four in ribonuclease A, two out of three in alpha-chymotrypsin, one out of two in trypsin, one out of two in pepsin, and one out of five in rennin appeared to react with PGO fairly rapidly, indicating a difference in the relative accessibility of these residues by the reagent. Extensive modification of the arginine residues by PGO occurred with RCM-derivatives of ribonuclease A and insulin B chain. The N-terminal isoleucine residues of alpha-chymotrypsin and trypsin appeared to be unreactive with PGO because of salt bridge formation with an aspartyl residue. The activity of alpha-chymotrypsin toward N-benzoyl-L-tyrosine ethyl ester and the lytic activity of lysozyme were lost rapidly on treatment with PGO, as in the case of ribonuclease A. Pepsin and rennin were only partially inactivated by reaction with PGO.
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PMID:Further studies on the reactions of phenylglyoxal and related reagents with proteins. 32 41

beta1-Bungarotoxin modified with p-bromophenacyl bromide (BPB) was reduced and carboxymethylated, and the resulting two constituent RCM-polypeptide chains (the RCM-A and B chains) were separated. The RCM-A chain was found to be modified by BPB by measuring its UV absorption spectrum and was shown to have lost one histidine residue by analyzing its amino acid composition. To determine the location of the modified histidine residue in the A chain of the toxin, the RCM-A chain was digested with TPCK-trypsin, and the resulting peptides were fractionated by gel filtration followed by DEAE-cellulose chromatography. The modified residue was finally identified as histidine-48 in the A chain by Edman degradation and from the amino acid composition of the BPB-modified peptide. The amino acid sequence around the modified histidine residue in the A chain is highly homologous with those of porcine pancreas phospholipase A2 and presynaptic toxin, notexin. We conclude that histidine-48 in the A chain participates in the phospholipase A activity of beta1-bungarotoxin.
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PMID:Characterization of phospholipase A activity of beta1-bungarotoxin from Bungarus multicinctus venom. II. Identification of the histidine residue of beta1-bungarotoxin modified by p-bromophenacyl bromide. 73 Jul 54

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards conformational epitopes and antibodies to disulphide bond reduced carboxymethylated RCP (RCM-RCP) are towards sequential epitopes. The major cyanogen bromide (CNBr) fragments and tryptic fragments of RCM-RCP interact with both antiserum to RCM-RCP and RCP. Passive immunization of pregnant mice with antibodies to RCM-RCP results in bioneutralization, leading to termination of pregnancy. Recently, a major tryptic fragment of RCM-RCP (24 +/- 2 kd) which could assume conformation at the antibody combining site of native RCP, obtained following mild trypsinization has been identified [Natraj et al. J. Biosci, 15 (1990) 341]. Rabbit antibodies to RCM-RCP treated with trypsin generated antibodies of low titer which interacted with RCM-RCP as well as RCP. The interaction of this antibody with RCP was of high affinity and could be displaced with RCP. The bioneutralizing ability of the antibody was demonstrated by its ability to cause termination of pregnancy in mice.
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PMID:Termination of pregnancy in mice following antiserum administration to modified chicken riboflavin carrier protein. 128 56

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.
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PMID:New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro. 257 Apr 82

A human rectal adenocarcinoma cell line, RCM-1, secreted some neutral proteinases: metallo-, serine and cysteine proteinases; and trypsin inhibitors into the serum-free conditioned medium (SFCM) in vitro. They were separated by anion-exchange and gel filtration high-performance liquid chromatography. SFCM from the cells of early passage numbers (20-24) contained native activities of serine proteinase and collagenolytic metalloproteinase. However, after several passages the native activities of them were diminished and latent form of metalloproteinase increased. In contrast to the native proteolytic activities, trypsin inhibitory activity was elevated in SFCM from the cells of passages 38-42. It inhibited the serine proteinase secreted by the same cells of early passage numbers. Furthermore, the serine proteinase was able to activate the latent form of metalloproteinase. Cooperative roles of these tumor-secreted proteinases and inhibitors may be important in tumor invasion.
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PMID:Neutral proteinases and inhibitors secreted by human rectal adenocarcinoma cell line (RCM-1). 265 68

1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcus protease and with chymotrypsin, separately. 2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined. 3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analysis of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase Ms was compared with those of RNase T1 (Asp. oryzae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these for RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu57 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His 91 (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr67, Phe100, and Cys103 in RNase Ms. Since among these peptides His39, Glu57, His91, and Arg76 in RNase Ms corresponded to His40, Glu58, His92, and Arg77 in RNase T1 which are known to be involved in the active site of RNase T1, the possible role of these amino acids in the active site of RNase Ms is discussed. 5. The sequence similarity of RNase Ms to that of RNase T1 was about 60% and to those of RNase U1 and RNase U2 was about 30%. 6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.
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PMID:Primary structure of a minor ribonuclease from Aspergillus saitoi. 709 2

beta 2-Bungarotoxin (beta 2-toxin) was isolated from the venom of Bungarus multicinctus by means of CM-Sephadex C-25 column chromatography, Sephadex G-75 gel filtration and CM-Sephadex C-25 column rechromatography. beta 2-Toxin consisted of two dissimilar polypeptides, a (120 amino acid residues) and B (60 amino acid residues) chains, crosslinked by an interchain disulfide bond. The neurotoxicity (LD50) and phospholipase activity of beta 2-toxin were 0.029 micrograms/g of mouse and 48.9 units/mg of toxin, respectively, and both the activities were slightly weaker than those (0.019 micrograms/g and 60.9 units/mg) of beta 1-bungarotoxin (beta 1-toxin). beta 2-Toxin was reduced and carboxymethylated and then its RCM-A and -B chains were separated. Each RCM-chain was maleylated and then digested with TPCK-trypsin. The tryptic peptides were sequences by manual Edman degradation or the dansyl-Edman method, and the total alignment of the tryptic peptides from each RCM-chain was deduced based on the amino acid sequences of the A and B chains of beta 1-toxin. The amino acid sequence of the B chain of beta 2-toxin differed from that of the B chain of beta 1-toxin by 22 amino acid substitutions, while those of their A chains were identical. We concluded that the variation in the amino acid sequence of the B chains did not significantly affect the neurotoxicity of the beta-toxins. The amino acid sequences of the B chains of the two beta-toxins were homologous to those of proteinase inhibitors from snake venoms and mammalian pancreas, but no inhibitory activity of the two beta-toxins on proteinases was observed.
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PMID:Amino acid sequence of beta 2-bungarotoxin from Bungarus multicinctus venom. The amino acid substitutions in the B chains. 709 4

The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.
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PMID:Amino acid sequences of three beta-bungarotoxins (beta 3-, beta 4-, and beta 5- bungarotoxins) from Bungarus multicinctus venom. Amino acid substitutions in the A chains. 709 5

Bungarus multicinctus phospholipase A was reduced and carboxymethylated. The RCM-enzyme was digested with TPCK-trypsin or cleaved with cyanogen bromide followed by chymotrypsin digestion. The resulting peptide mixtures were fractionated by gel filtration on Sephadex G-50 and G-25 columns or by DEAE-cellulose (DE-32) column chromatography. Further purification of the peptide mixtures was performed by paper electrophoresis at pH 3.5 or 6.5 or by paper chromatography. The sequences of isolated peptides were determined by the manual Edman or dansyl-Edman method. From the sequences of these peptides the whole enzyme sequence (total 118 residues) was deduced. The complete sequence of the enzyme is similar to those of phospholipases A2 from other snake venoms and mammalian pancreas. Further, a 58% sequence homology was found between the present phospholipase A and the A chain of beta 1-bungarotoxin, a presynaptic neurotoxin having weak phospholipase A activity, contained in the same venom.
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PMID:Amino acid sequence of phospholipase A from Bungarus multicinctus venom. 721 37

A431 cells grown as three-dimensional spheroids show growth stimulation in response to nanomolar concentrations of EGF in contrast to monolayer cultures that show inhibition. In investigating the alterations in EGF signal transduction that underlie this modification of the proliferative response, we have compared the expression of EGF receptors on A431 cells under these conditions and related our findings to tyrosine phosphorylation and the growth response. EGF receptors were measured by 125I-EGF binding to trypsin-dispersed cells. Unexpectedly, dispersion of the monolayers caused an 80% decrease in surface EGF receptor, although, after dispersion, EGF receptor was digested by trypsin with a half-life of 69 +/- 32 min. No evidence for a comparable loss of cellular EGF receptor was seen on trypsin dispersion of spheroids. After allowing for this effect, we found that the receptor density on nondispersed monolayers (5 x 10(6) per cell) was twentyfold greater than that on spheroids (0.25 x 10(6) per cell). EGF-induced tyrosine phosphorylation was confined to the outermost cells of the spheroid, although the presence of surface-expressed EGF binding sites could be demonstrated throughout the structure and the number of EGF receptors/cell on dispersed spheroid cells showed a single distribution peak by flow cytometry, with no evidence for more than one population. Using RCM-lysozyme as a substrate, tyrosine phosphatase activity in spheroids lay within the range observed in monolayer cultures. Autophosphorylation of the EGF receptor following EGF stimulation in monolayer cultures of A431 cells rose rapidly in the first 10 seconds and then slowly increased for at least 3 h. In spheroids, it reached a maximum within 10 seconds and then declined over 3 h. Since the microenvironment within a tumor resembles that in a spheroid, a similar reduction in surface EGF receptor expression may be expected in tumors relative to monolayer cultures, together with corresponding growth stimulation in response to EGF.
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PMID:Adaptation of EGF receptor signal transduction to three-dimensional culture conditions: changes in surface receptor expression and protein tyrosine phosphorylation. 796 22


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