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)

Polymyxin acylase isolated from Pseudomonas sp. M-6-3 was used as an N-myristoyl cleaving enzyme in order to determine a part of the N-terminal amino acid sequence of N-myristoyl proteins. The enzyme hydrolyzed a number of N-myristoyl oligopeptides at various hydrolysis rates but not N-myristoyl proteins. The oncogenic protein (N-myristoyl-pp60c-src) was isolated from human colon adenocarcinoma cell line COLO 320DM by two-dimensional polyacrylamide gel electrophoresis. The protein was digested with trypsin and the resultant tryptic N-myristoyl tetrapeptide (N-myristoyl-Gly-Ser-Asn-Lys) was purified by HPLC and the structure was determined both by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MASS) and by a gas-phase protein sequencer before or after treatment with the polymyxin acylase. The results suggest that the N-myristoyl peptide sequence derived from N-myristoyl proteins was clearly determined by the combined use of MALDI TOF MASS and the N-myristoyl cleaving enzyme.
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PMID:Determination of N-myristoyl peptide sequence both by MALDI TOF MASS and with an N-myristoyl cleaving enzyme (polymyxin acylase). 750 45

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.
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PMID:Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. 753 May 43

Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10-3H]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size-exclusion chromatography and purified by narrowbore reverse-phase HPLC. Analysis of the purified product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI-TOF-MS or fractionated by microbore reverse-phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C-terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.
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PMID:Localization of the palmitoylation site in the transmembrane protein p12E of Friend murine leukaemia virus. 755 84

Incubation of the C225S mutant of the R1 subunit of ribonucleotide reductase from Escherichia coli with the R2 subunit and nucleoside diphosphates leads to fragmentation of the polypeptide backbone of R1 [Mao, S. S., Holler, T. P., Bollinger, J.M., Jr., Yu, G. X., Johnston, M.I., & Stubbe, J. (1992) Biochemistry 31, 9744--9751]. The 26 and 60 kDa cleavage fragments were purified to homogeneity. The 26 kDa polypeptide was digested with Lys-C, and the peptides were partially purified by RP-HPLC. Mass spectrometric analysis (MALDI-TOF) of the HPLC fractions allowed the identification of the C-terminal peptide. The molecular mass of this peptide (2176) revealed that serine-224 constitutes its C-terminus, and further analysis of the distribution of its monoisotopic masses by FAB-MS indicated that Ser224 possesses a carboxamide rather than a carboxylate group. Treatment of the 60 kDa cleavage fragment with cyanogen bromide and subsequent MALDI-TOF analysis of the partially RP-HPLC purified peptides yielded a fraction containing its N-terminal peptide. This peptide was digested with trypsin, and the digestion mixture was purified by HPLC. Analysis of the fractions by MALDI-TOF identified the N-terminal peptide and determined a mass of 2222. This mass suggested valine 226 was the N-terminal residue (modified by an adduct of 28 mass units). Larger amounts of the C-terminal tetrapeptide of the 60 kDa fragment (V226LIE229) were obtained by complete digestion of the crude reaction mixture with endoproteinase Glu-C. The peptide mixture was then purified on an immunoadsorbent column containing immobilized antibodies raised against a synthetic peptide with the sequence KVLIE. After elution of the affinity-bound peptide, it was analyzed by CID-MS verifying that an adduct of 28 mass units was attached to valine 226. These results indicated that the amino group of Val226 is formylated. The localization of the residues at the cleavage site of C225SR1 provides a biochemical identification of the active site region of the R1 subunit of RDPR from E.coli. The details of the mechanism of cleavage remain to be elucidated.
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PMID:Identification of an active site residue of the R1 subunit of ribonucleotide reductase from Escherichia coli: characterization of substrate-induced polypeptide cleavage by C225SR1. 875 68

The amino acid p-benzoyl-L-phenylalanine, (p-Bz)Phe, has been incorporated into substance P (SP), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, to localize the agonist-binding domains of the human neurokinin-1 (NK-1) receptor overexpressed in a transfected mammalian cell line. The NK-1-specific agonist [Pro9]SP was modified at position 8 by (p-Bz)Phe and acylated at the N-terminus by a biotinyl sulfone reporter via a 5-aminopentanoyl spacer. After photolysis, the biotinyl sulfone moiety allowed easy and efficient removal of biotinylated fragments from the complex incubation mixture with streptavidin-coated beads. Direct elution from the beads with the matrix used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS), which was facilitated by saturation of streptavidin sites with biotin, and subsequent MALDI-TOF mass spectrometry analysis allowed identification of the NK-1 fragments obtained after photolysis and proteolytic digestion. Trypsin digestion and combined trypsin/Staphylococcus aureus V8 protease enzymatic cleavage established that the site of covalent attachment of the photolabelled SP resides in the second extracellular loop Thr173-Arg177. Cyanogen bromide cleavage shows that the probe is covalently attached to the methyl group of a methionine residue from human NK-1. These experiments identified Met174 as the modified residue.
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PMID:The use of photolabelled peptides to localize the substance-P-binding site in the human neurokinin-1 tachykinin receptor. 879 56

An analytical system is presented for rapid assessment of site-specific microheterogeneity of the two potential N-linked glycosylation sites of recombinant human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary cell culture. The target protein is first purified from culture supernatant by immunoaffinity chromatography, and the acidic eluent is neutralized via an in-line mixing tee. On-line proteolysis is rapidly performed by an immobilized trypsin cartridge, and reversed-phase chromatography isolates the two pools of glycopeptides representing the potential glycosylation sites. Following off-line analysis by matrix-assisted laser-desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry, observed mass shifts of glycopeptides relative to the known masses of their amino acid portions are correlated to site-specific oligosaccharide structures. Desialylation of glycopeptides by sialidase treatment on the MALDI sample plate allows for quantitative estimations of asialoglycan structures by MALDI/TOF. This methodology permits glycoprotein microheterogeneity to be evaluated in a time frame of approximately 2 h, utilizing as little as 0.5 microgram (25 pmol) of product. Results of monitoring a batch culture are presented as well as analysis of a culture containing deoxymannojirimycin, an inhibitor of glycoprotein processing.
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PMID:Rapid monitoring of site-specific glycosylation microheterogeneity of recombinant human interferon-gamma. 881 42

Among the milk proteins, bovine beta-casein has the peculiarity of containing in its sequence some peptides liable to interfere in mineral nutrition and some peptides with opioid (casomorphines), antihypertensive, and immunomodulatory activities. In this work we propose a novel type of multicompartment enzyme reactor, operating under an electric field, for the continuous hydrolysis of milk proteins such as beta-casein. The enzyme trypsin is trapped, with zwitterionic buffering ions and its substrate beta-casein, in solution between two isoelectric membranes having pI values encompassing the isoelectric point of the enzyme. Additionally, beta-casein is captured inside the same reaction chamber with the aid of sieving membranes, since its pI is too far away from the pI of trypsin. This setup permits the continuous operation at the pH of optimum of activity. The peptides, arising from tryptic hydrolysis of beta-casein, are removed under the influence of the electric field and collected in different chambers in which they are isoelectric and isoionic as well. The purity of the peptides collected is ascertained by capillary zone electrophoresis and their identity confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. This setup allows continuous harvesting of some biologically-active peptides in a pure form. The major advantages of such a reactor system over conventional batch reactors are the great increase in enzyme utilization efficiency and the overall reactor productivity.
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PMID:Continuous enzymatic hydrolysis of beta-casein and isoelectric collection of some of the biologically active peptides in an electric field. 919 76

A protease-inhibitor was isolated from mature ovaries of Schistocerca gregaria by a combination of trypsin-affinity chromatography and reverse-phase high performance liquid chromatography. It was characterized by aminoterminal amino acid sequencing using Edman degradation based automated microsequencing and by MALDI-TOF mass spectrometry. The N-terminal sequence (Y)XAEXDELA(A)EEY(Y)Q(Q)X(I)(L)M (X being a Cys, an irregular or modified amino acid) revealed no similarities with any other protease inhibitors isolated from invertebrate or vertebrate source. The 14 kDa inhibitor was found to be heat-stable. It shows potent inhibitory activity toward bovine trypsin and chymotrypsin, but not toward pancreatic elastase. It is likely that the characterized inhibitor will serve as an important tool for understanding its role in insect development.
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PMID:Purification of a novel, heat-stable serine protease inhibitor protein from ovaries of the desert locust, Schistocerca gregaria. 929 12

In an approach to develop a commercial-scale process for the production of casein phosphopeptides containing the cluster sequence-SerP-SerP-SerP-Glu-Glu-, we have studied the relationship between casein hydrolysis and phosphopeptide release. The degrees of hydrolysis (DH) of casein using Novo trypsin PTN 3.0 S and pancreatin 4NF independently, at enzyme to substrate (E:S) ratios of 1:50-1:1600 (by weight), were determined using the pH-stat method. Casein phosphopeptides (CPP) were selectively precipitated using Ca2+ and ethanol from the acid-clarified hydrolysates. The precipitates were analysed by high performance capillary electrophoresis to calculate individual phosphopeptide yields based on extinction coefficients of the purified peptides. Individual peptides were purified by reversed-phase HPLC and anion-exchange FPLC and characterized by MALDI-TOF mass spectrometry and amino acid sequence analysis. For both enzymes, lowering the E:S ratio resulted in reductions in the DH and the release of the CPP, and an increase in peptide chain length. The longer chain length offset the reduction in release such that the gravimetric yields of CPP preparations remained relatively constant. For Novo trypsin the highest yields of the major cluster peptides (beta-casein(CN)f(1-25), alpha s1-CNf(59-79), alpha s2-CNf(1-21), alpha s2-CNf(46-70) and related peptides) in the selective precipitates were obtained at a casein DH of 17%. At lower DH values (9-15%), there was a decrease in yield of the peptides derived from alpha s1-CN and alpha s2-CN while the yield of the beta-CN-derived cluster peptides remained relatively constant. The CPP produced using pancreatin were found to be truncated at all E:S ratios, relative to the tryptic CPP, owing to higher levels of chymotryptic and carboxypeptidase activities in pancreatin. The highest yields of the truncated forms of the major cluster peptides using pancreatin were obtained at a casein DH of 19-23%.
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PMID:Relationship between degree of casein hydrolysis and phosphopeptide release. 940 65

Rho-associated kinase (Rho-kinase), the putative target of the small GTP-binding protein Rho, phosphorylated neurofilament protein (NF-L) in vitro with approximately 1 mole phosphate per mole NF-L. Phosphorylated NF-L no longer formed the 10 nm filaments, and NF-L filaments were phosphorylated with a result of nearly complete disassembly. NF-L phosphorylated by Rho-kinase was digested with trypsin, and digested fragments were assigned by MALDI/TOF. Unique phosphorylation sites were found at Ser-26 and Ser-57 in the head domain of NF-L. These results indicate that domain- and site-specific phosphorylation by Rho-kinase may regulate the assembly-disassembly of NF-L filaments.
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PMID:Domain- and site-specific phosphorylation of bovine NF-L by Rho-associated kinase. 957 Nov 64


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