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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new methodology utilizing an enzyme inactivator covalently attached to poly(ethylene glycol) (PEG) is described in which the PEG affords facile and mild quantification, isolation, and identification of the site of enzyme inactivation. As proof of concept, the known affinity labeling agent for alpha-chymotrypsin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), was linked to PEG. The synthesis of the PEG-bound inactivator PEG-TPCK was carried out in good yields using standard solution-phase chemistry. Inactivation of alpha-chymotrypsin with PEG-TPCK was monitored via UV-vis spectroscopy in aqueous conditions, which resulted in less than 3% remaining activity, indicating that 97% of the alpha-chymotrypsin was covalently modified with PEG-TPCK. The MALDI-TOF mass spectrum showed only one new peak that was distinct in shape and corresponded to the mass of PEG-TPCK-alpha-chymotrypsin. Following proteolytic digestion, the PEG-TPCK-peptide was easily discernible from the rest of the digest in a HPLC trace because of its characteristic prolonged retention time and broad polymer shape. MALDI-TOF MS was used to determine the mass of the PEGylated peptide. Without prior removal of the PEG, the amino acid site to which PEG-TPCK covalently bound was determined via Edman sequencing. In comparison to other methods, the PEG-supported inactivator system is significantly cheaper and safer than the synthesis of radiolabeled compounds; furthermore, isolation of the PEGylated peptide is milder and more selective than standard affinity binding columns. Edman sequencing provides an exact determination of the site of inactivator covalent attachment without extensive, tedious LC-MS analysis of a complex peptide mixture. The method described here could be applied to a variety of enzymes as an alternative to current techniques.
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PMID:Poly(ethylene glycol)-supported enzyme inactivators. Efficient identification of the site of covalent attachment to alpha-chymotrypsin by PEG-TPCK. 1526 52

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. The enzyme is often isolated with varying amounts of tightly bound PEP substrate. To better understand the role of tightly bound PEP in E. coli KDO8P synthase catalysis, a combination of transient kinetic methodologies including rapid chemical quench and mass spectrometry techniques such as time-resolved electrospray ionization mass spectrometry (ESI-TOF MS) were used to study the enzyme purified both in the PEP-bound state and in the unbound state. Pre-steady state burst and single-turnover experiments using radiolabeled [1-(14)C] and [(32)P]A5P revealed significant kinetic differences between these enzyme preparations. The active sites concentrations for the bound and unbound states of the enzyme were almost the same (approximately 100%) and the product release for both states of the enzyme was rate limiting. However, the rate constant of product formation for the PEP-bound enzyme (125 s(-1)) was higher than that of the unbound enzyme (46 s(-1)). This was further confirmed by single-turnover experiments using radiolabeled [(32)P]A5P. Interestingly, when PEP was removed from the PEP-bound enzyme and external PEP was added before the kinetic experiments, both the pre-steady state burst and the single-turnover kinetic parameters were similar to those of the enzyme purified in the unbound state. The rate constants of product formation were determined as 44 s(-1) (burst experiment) and 48 s(-1) (single-turnover experiment). The reaction kinetics of the E. coli KDO8P synthase was also followed by time-resolved ESI mass spectrometry. To validate the suitability of this technique for conducting enzyme kinetics, the standard reaction of p-nitrophenyl acetate hydrolysis by chymotrypsin was analyzed by stopped-flow and time-resolved ESI-TOF MS. The rate constant of p-nitrophenol formation followed by stopped-flow spectrophotometry matched perfectly the rate constant of acetyl-chymotrypsin intermediate formation followed by time-resolved ESI-TOF MS (0.1 s(-1)). The catalytic properties of the PEP-bound and unbound states of the E. coli KDO8P synthase were then studied on a millisecond time scale. The changes in the intensity of E*PEP, E*KDO8P, and E*intermediate complexes as a function of time were quantified and the reaction kinetics were modeled using KinTekSim simulation software. An analysis of the reaction kinetics established the kinetic competence of the intermediate based upon the rate constants for substrate decay and product formation. The ability of time-resolved ESI-TOF MS to detect and monitor the kinetics for the reaction intermediate constitutes a significant advantage over the traditional rapid chemical quench technique. For all three states of the enzyme (PEP-bound, unbound, and PEP removed from the PEP-bound state) the rate constants obtained by time-resolved ESI-TOF MS matched the pre-steady state rates determined by rapid chemical quench. A comparison of reaction time courses for each state of the enzyme revealed that, in the case of PEP-bound enzyme, the enzymatic reaction reached completion faster than that for the unbound state. In summary, these studies led to the conclusion that bound PEP has an important role in catalysis, maintaining the enzyme in a conformational state optimal for catalytic activity, and established the kinetic competence of the reaction intermediate. This technique has broad applicability for the kinetic analysis of any enzyme system where the substrates, products, or intermediates are eluding the common detection techniques or as a method alternative to the widely used radioactivity assays.
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PMID:Probing the role of tightly bound phosphoenolpyruvate in Escherichia coli 3-deoxy-d-manno-octulosonate 8-phosphate synthase catalysis using quantitative time-resolved electrospray ionization mass spectrometry in the millisecond time range. 1597 47

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.
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PMID:Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis. 1610

Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and cone snails, produce a variety of highly stable cystine-constrained peptide scaffolds as part of their neurochemical strategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designated framework 14). Three peptides of this family (flf14a-c) were isolated from the venom of Conus floridanus floridensis, and one (vil14a) was isolated from the venom of Conus villepinii, two worm-hunting Western Atlantic cone snail species. The primary structure for these peptides was determined using Edman degradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combination of CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of these conotoxins directly isolated from the cone snails revealed a highly helical secondary structure for the four conotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs alpha/alpha scorpion toxins kappa-hefutoxin, kappa-KTx1.3, and Om-toxins, which adopt a stable three-dimensional fold where the two alpha-helices are linked by the two disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately 6A, which is a conserved structural feature in K(+) channel blockers. The presence of this framework in scorpions and in cone snails indicates a common molecular imprint in the venom of apparently unrelated predatory animals and suggests a common ancestral genetic origin.
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PMID:A novel conotoxin framework with a helix-loop-helix (Cs alpha/alpha) fold. 1633 58

A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.
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PMID:A five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities. 1651 41

Stomatin-like protein 2 (SLP-2) (syn.: EPB72-like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP-2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two-dimensional gel electrophoresis and SLP-2 was identified by MALDI-TOF/TOF. SLP-2 was digested with trypsin, chymotrypsin, Lys-C, and de novo sequencing studies as well as Nano-HPLC-ESI-MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N-terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI-TOF/TOF, Nano-HPLC-ESI-MS/MS and de novo sequencing completed unambiguous analysis of SLP-2 primary structure of approximately = 90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP-2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance.
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PMID:Mass spectrometrical verification of stomatin-like protein 2 (SLP-2) primary structure. 1667 Oct 55

A novel elastase inhibitor from Aspergillus flavus (AFLEI) was isolated, and biochemical properties of AFLEI were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and Sephadex G-75 was used to purify the inhibitor. The final preparation was found to be homogeneous as indicated by a single band after disc polyacrylamide gel (PAGE) and isoelectric focusing electrophoreses. AFLEI had a molecular weight of 7,525.8 as determined by TOF-MS (time of flight mass spectrometry). The elastolytic activity of elastases from A. flavus, A. fumigatus and human leukocytes were inhibited by AFLEI. However, this activity from porcine pancreas elastase, trypsin, chymotrypsin, thrombin, and Ac1-Proteinase from snake venom was not affected by AFLEI. The fibrinogenase activity of the elastase from A. flavus was inhibited by AFLEI. AFLEI was inhibited by alpha2-macroglobulin. However, ethylenediaminetetraacetic acid (EDTA-2Na), benzamidine, chymostatin, tosyl phenylalanine chloromethyl ketone (TPCK) and dithiothreitol (DTT) did not show any inhibitory effect on the elastase inhibitory activity of AFLEI.
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PMID:Isolation and characterization of a novel elastase inhibitor, AFLEI from Aspergillus flavus. 1694 Sep 57

Anterior gradient protein 2 homolog is a metastasis-inducing protein in a rat model of rat breast cancer and prognostic for outcome in hormonally treated breast cancer patients. Carrying out protein profiling in several mammalian cells and tissues, we detected this protein (synonym: secreted cement gland protein XAG-2 homolog) that was originally described in toad skin, in human bronchial epithelia. Tissues obtained from biopsies were homogenised and extracted proteins were run on two-dimensional gel electrophoresis. Following in-gel digestion with proteases trypsin, AspN, LysC and chymotrypsin, mass spectrometrical analysis was carried out by MALDI-TOF/TOF. The use of MS following multi-enzyme digestion of the protein resulted into 100% sequence coverage. MS/MS analysis enabled sequencing of 87% of the protein structure. This percentage does not include the signal peptide that was not observed in our protein due to processing. No posttranslational modifications were detectable and no sequence conflicts were observed. Complete analysis, unambiguous identification and characterisation of this biologically important protein could be shown, which is relevant for the definition of a marker protein that has been described so far by immunochemical methods only. Complete analysis is of importance as it forms the basis for all future work on this protein and, moreover, may serve as an analytical tool for further studies.
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PMID:Mass spectrometrical analysis of the processed metastasis-inducing anterior gradient protein 2 homolog reveals 100% sequence coverage. 1749 4

In the present study, a peptide having antioxidant properties was isolated from bullfrog skin protein, Rana catesbeiana Shaw. Bullfrog skin protein was hydrolyzed using alcalase, neutrase, pepsin, papain, alpha-chymotrypsin and trypsin. Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. In order to purity a peptide having potent antioxidant properties, alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10 DEAE FF anion exchange column, Superdex Peptide 10/300 GL gel filtration column and highan octadecylsilane (ODS) C18 reversed phase column. Finally, a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487 Da) by Q-TOF ESI mass spectroscopy. This antioxidant peptide from bullfrog skin protein (APBSP) inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radicals: DPPH radical (IC(50)=16.1 microM), hydroxyl radical (IC(50)=12.8 microM), superoxide radical (IC(50)=34.0 microM) and peroxyl radical (IC(50)=32.6 microM). Moreover, MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5).
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PMID:Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw. 1751 26

Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding, and the M-domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear, and the residues or regions directly involved in substrate binding have not yet been identified. Here, a hydrophobic probe, bis-ANS, was used to characterize potential substrate-binding regions. Results showed that bis-ANS binds TF with a 1:1 stoichiometry and a K(d) of 16 microM, and it can be covalently incorporated into TF by UV-light irradiation. A single bis-ANS-labeled peptide was obtained by tryptic digestion and identified by MALDI-TOF mass spectrometry as Asn391-Lys392. In silico docking analysis identified a single potential binding site for bis-ANS on the TF molecule, which is adjacent to this dipeptide and lies in the pocket formed by the C-terminal arms. The bis-ANS-labeled TF completely lost the ability to assist GAPDH or lysozyme refolding and showed increased protection toward cleavage by alpha-chymotrypsin, suggesting blocking of hydrophobic residues. The C-terminal truncation mutant TF389 also showed no chaperone activity and could not bind bis-ANS. These results suggest that bis-ANS binding may mimic binding of a substrate peptide and that the C-terminal region of TF plays an important role in hydrophobic binding and chaperone function.
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PMID:Identification of a potential hydrophobic peptide binding site in the C-terminal arm of trigger factor. 1752 65


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