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)

A rapid and reproducible analytical tryptic mapping method was developed as an identity test for a recombinant chimeric monoclonal antibody for lot release testing. The unfolding, reduction, carboxymethylation, trypsin digestion, and reversed-phase (RP) HPLC steps were optimized to provide a reproducible method. The optimized method requires 30 min for unfolding the protein, 30 min for carboxymethylation, 4 h for digestion with TPCK-trypsin and 140 min for RPHPLC analysis. The total time required is less than 8 h compared to conventional procedures, which must be performed over several days. The optimized method was validated for its precision, recovery, specificity, and robustness. The precision of the method was determined by repeatability and intermediate precision experiments. Relative standard deviation (RSD) values were < or = 10% for the relative peak areas of marker peaks. The mean recovery of these marker peaks was 88.4%. The specificity was demonstrated by the unique tryptic mapping patterns obtained compared with several other monoclonal antibodies. Robustness was demonstrated by the relative insensitivity of the tryptic map to small deliberate changes in key method parameters. Excessive relative peak area variability observed for one peak (RSD 52%) was traced to adsorption to glass autosampler vials. This variability was substantially reduced (RSD 11%) by substituting polypropylene autosampler vials. The data demonstrate that this method may be applicable to a wide range of pharmaceutically relevant monoclonal antibodies.
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PMID:Rapid analytical tryptic mapping of a recombinant chimeric monoclonal antibody and method validation challenges. 950 59

Using hen lysozyme in which the epsilon-carbons of two methionine residues are enriched with 13C nuclei, we found that there is a subtle difference in the chemical shift of the epsilon-carbon resonances between Met 12 and Met 105 in thermally denatured lysozyme without any reduction of disulfide bonds at pD 3.8, and also in reduced S-alkylated lysozyme at pD 3.8 and 35 degrees C. The difference in the chemical shift was abolished on digestion with TPCK-trypsin and the chemical shifts of both resonances converged to that of Met 12, whose chemical shift is identical to that in the randomly coiled state. Therefore, it is suggested that the chemical shift in the epsilon-carbon resonance of Met 105 is different from that in the randomly coiled state due to an interaction involving Met 105. In order to locate the interaction involving Met 105, fragmentation of the reduced S-alkylated lysozyme into the peptides was carried out by means of chemical cleavage or specific endoprotease digestion. As a result, the local interaction of Met 105 or the residues around Met 105 with eleven residues at the C-terminus of lysozyme is suggested to occur.
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PMID:Detection of a local interaction of hen lysozyme under highly denaturing conditions using chemically 13C-enriched methionine resonance. 953 8

As reported previously, caldecrin, a serum calcium-decreasing factor, from pancreas was found to be a serine protease, but the proteolytic activity was not necessary for its serum calcium decreasing activity. The caldecrin cDNA encoded a protease zymogen of the chymotrypsin/elastase superfamily consisting of a signal peptide, an activation peptide and a mature enzyme. On a homology search, we found that the sequence of rat caldecrin is almost identical to that of rat elastase IV (nucleotides: 99.3%175B amino acids: 90.3%) with the exception of the central region. However, it is not known whether or not elastase IV is transcribed and translated in vivo, and has proteolytic activity. In the present study, we constructed a rat elastase IV cDNA by means of combinatorial PCR, and compared the recombinant elastase IV with the recombinant caldecrin synthesized in a baculovirus expression system. The recombinant caldecrin protein was expressed in the cells and secreted mainly into the medium. In contrast, in the case of elastase IV, no recombinant protein was immunologically or enzymatically detected in the medium, while an immunoreactive protein with much lower protease activity was found in the cells in an amount comparable to that of the caldecrin protein. Using the RT-PCR method to discriminate caldecrin mRNA from elastase IV mRNA, we detected caldecrin mRNA expression in rat pancreas, but no elastase IV mRNA expression in any tissues examined. PCR analysis of rat genomic DNA revealed the presence of caldecrin and the absence of elastase IV sequences. These results indicate that caldecrin is expressed in the pancreas, but that elastase IV is an artifact produced during cloning. Furthermore, we investigated the protein-chemical and enzymological properties of the rat and human caldecrins using their recombinant proteins. Both recombinant proteins were secreted into the medium as proforms and showed protease activity after trypsin treatment. Some differences were found in the activation process and stability between human and rat caldecrins; human caldecrin was more easily activated by trypsin, but was much more labile than rat caldecrin. Although both caldecrins were found to be chymotrypsin-type proteases, on the basis of their substrate and inhibitor specificities, they were not inhibited by TPCK, suggesting that caldecrin is a novel type of serine protease.
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PMID:Caldecrin is a novel-type serine protease expressed in pancreas, but its homologue, elastase IV, is an artifact during cloning derived from caldecrin gene. 953 41

Bombyx mori was found to be more sensitive to the protoxins of HD-1 than Heliothis armigera. SDS-PAGE analysis showed that a large amount of activated toxin was yielded from protoxin by B. mori gut juice while little was yielded by H. armigera. Further degradation of activated toxin was observed in H. armigera midgut juice detected by SDS-PAGE. pH influenced the proteolytic activity of the midgut juice significantly, but there was no obvious effect of pH on the degradation of activated toxin. Specific inhibitor study revealed the presence of trypsin, chymotrypsin, and elastase in the midgut juice. TLCK, TPCK, elastatinal and some general serine protease inhibitors successfully prevented the excessive degradation of protoxin in H. armigera midgut juice. Chymotrypsin inhibitors showed strong inhibitory effects against the further degradation of activated toxin, indicating that chymotrypsin played a major role in the process. It was presumed that the excessive degradation of protoxin in H. armigera midgut juice was responsible for the low sensitivity of the insect to Bt. Further study demonstrated that the excessive degradation in vitro was triggered by SDS treatment. However, all of the tested serine protease inhibitors expressed synergism with protoxin against H. armigera larvae, suggesting that the excessive degradation of protoxin may occur in vivo to some extent and may be triggered by receptor binding of activated toxin.
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PMID:Processing of delta-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 in Heliothis armigera midgut juice and the effects of protease inhibitors. 964 4

Horse cytochrome c was reacted with the spin label (succinimidyl-2,2, 5,5-tetra-methyl-3-pyrroline-1-oxyl-carboxylate) using optimized conditions and the reaction products were separated by a combination of cation-exchange chromatography and HPLC. The purified cytochrome c derivatives were digested with TPCK treated trypsin and the resulting peptides were separated by reverse phase HPLC. The modified Lys residues were subsequently characterized by Edman degradation and mass spectrometry. These analyses showed that five distinct cytochrome c derivatives had been produced which were modified at the specific Lys residues including Lys8, Lys25, Lys72, Lys86 or Lys87, respectively. The electron paramagnetic resonance (EPR) spectra for each cytochrome c derivative revealed that for the spin label attached to Lys8 and Lys87 only one component contributes to the spectrum whereas for Lys25, Lys72 and Lys86 the spectrum consists of two components. The highest mobility with the rotational correlation time, tauB, of 0.38 ns was observed for Lys87. The longest tauB of 1.84 ns was obtained for Lys72. An attempt to correlate the spin label mobility with the local protein structure is presented. These mono derivatized cytochrome c molecules provide a unique tool for EPR studying the interaction between cytochrome c and the lipid bilayer, as well as cytochrome c oxidase and reductase.
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PMID:Preparation and electron paramagnetic resonance characterization of spin labeled monoderivatives of horse cytochrome c. 967 42

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.
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PMID:Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects. 1038 Jun 52

Not only does mouse complement (C) have low hemolytic activity, but mouse serum has an inhibiting effect on hemolysis by human C. To purify and identify the putative mouse serum factor inhibiting human C activity, a sequential procedure of fractionated precipitation by PEG, followed by chromatographies with a heparin-Sepharose column, a phenyl-Sepharose column, a Protein G column, and a gel-filtration column was performed. The amino acid sequence analyses of two polypeptides obtained by digestion of the purified serum factor with TPCK-trypsin revealed that it was mouse fibronectin (FN). Highly purified mouse FN, but not human FN, has an inhibiting effect on human C-dependent hemolysis. Moreover, the hemolysis of sensitized rabbit erythrocytes by mouse C was also inhibited by the addition of mouse FN in a dose-dependent fashion, but not by the addition of human FN. These results suggest that FN is the putative internal C inhibitor in the mouse system.
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PMID:Inhibition of human and mouse complement-dependent hemolytic activity by mouse fibronectin. 1040 81

Our previous studies have demonstrated that myoepithelial cells, which surround incipient carcinomas in situ of the breast and other organs, exert antiinvasive and antiangiogenic effects in vitro through the elaboration of a number of different suppressor molecules among which include the shed membrane CD44. The present study addresses the mechanism of this myoepithelial CD44 shedding. This CD44 shedding is enhanced by PMA pretreatment, is specific for myoepithelial CD44, and inhibited by chymotrypsin-like inhibitors (chymostatin, alpha(1)-antichymotrypsin, TPCK, and SCCA-2) but not by trypsin-like inhibitors (TLCK), nor papain-like inhibitors (SCCA-1) nor hydroxamate-based or general metalloproteinase inhibitors (BB2516 (marimastat), 1,10-phenanthroline, and TIMP-1). The effect of PMA can be mimicked by exogenous chymotrypsin but not by other proteases. The CD44 shedding activity cannot be transferred by conditioned media, cell-cell contact, peripheral membrane, or integral membrane fractions. However, cell-free purified integral plasma membrane fractions obtained from myoepithelial cells pretreated with PMA also exhibit CD44 shedding which is inhibited by chymotrypsin-like inhibitors. These findings support the presence and activation of a putative chymotrypsin-like sheddase as the mechanism of CD44 shedding in myoepithelial cells.
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PMID:Myoepithelial-specific CD44 shedding is mediated by a putative chymotrypsin-like sheddase. 1111 26

Intra-acinar cell nuclear factor-kappaB (NF-kappaB) and trypsinogen activation are early events in secretagogue-induced acute pancreatitis. We have studied the relationship between NF-kappaB and trypsinogen activation in rat pancreas. CCK analogue caerulein induces early (within 15 min) parallel activation of both NF-kappaB and trypsinogen in pancreas in vivo as well as in pancreatic acini in vitro. However, NF-kappaB activation can be induced without trypsinogen activation by lipopolysaccharide in pancreas in vivo and by phorbol ester in pancreatic acini in vitro. Stimulation of acini with caerulein after 6 h of culture results in NF-kappaB but not trypsinogen activation. Protease inhibitors (AEBSF, TLCK, and E64d) inhibit both intracellular trypsin activity and NF-kappaB activation in caerulein stimulated acini. A chymotrypsin inhibitor (TPCK) inhibits NF-kappaB activation but not trypsin activity. The proteasome inhibitor MG-132 prevents caerulein-induced NF-kappaB activation but does not prevent trypsinogen activation. These findings indicate that although caerulein-induced NF-kappaB and trypsinogen activation are temporally closely related, they are independent events in pancreatic acinar cells. NF-kappaB activation per se is not required for the development of early acinar cell injury by supramaximal secretagogue stimulation.
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PMID:Relationship between NF-kappaB and trypsinogen activation in rat pancreas after supramaximal caerulein stimulation. 1116 28

A novel type of protease (mantis egg fibrinolytic enzyme, MEF-2) was isolated from the egg cases of Tenodera sinensis. The protease was homogeneous by SDS-PAGE and its apparent molecular mass was 32,900 Da. The amino acids in the N-terminal region were Ile-Val-Gly-Gly-Glu-Glu-Ala-Val-Ala-Gly-Asp-Phe-Pro-Ile-Val-Ser-Leu-Gln-Glu. The enzyme was inhibited by PMSF, TLCK, aprotinin, benzamidine, soybean trypsin inhibitor and also slightly by elastatinal, EDTA, EGTA, cysteine and beta-mercaptoethanol, but TPCK, iodoacetate and E-64 did not affect the activity. MEF-2 was not sensitive to alpha(1)-antitrypsin but antithrombin III and alpha(2)-antiplasmin inhibited the enzyme. MEF-2 preferentially cleaved the oxidized B-chain of insulin between Arg(22) and Gly(23). Among chromogenic protease substrates, the most susceptible to MEF-2 hydrolysis was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at 30 degrees C and pH 5.0. These results indicate that MEF-2 belongs to the trypsin family. Upon incubation of crosslinked fibrin with MEF-2, a steady increase of D-dimer suggests that the enzyme has a strong fibrinolytic activity. In conclusion, MEF-2 is a new type of proteolytic enzyme and has some potential for practical application in fibrinolysis.
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PMID:Purification and characterization of a plasmin-like protease from Tenodera sinensis (Chinese mantis). 1126 96


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