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 (0.01 seconds)

1. Two types of nylon-6 supports (small cylinders and a sponge-like derivative) were prepared for immobilizing enzymes. Nylon-6 beads were solubilized by immersion in 80% formic acid and then reprecipitated using two different types of non-solvent solutions (distilled water or a 1:1 acetone:water solution) giving rise to a sponge-like derivative and to a colloidal suspension, respectively. The latter was molded into a thin thread which was cut into small cylinders. 2. Trypsin (EC 3.4.21.4) was covalently bound to glutaraldehyde-activated nylon-6 cylinders as well as to the sponge-like derivative. The maximum (100%) apparent initial enzymatic activity was found for the trypsin bound to small cylinders, while the initial activity of trypsin bound to the sponge-like material was 61% in comparison with that of trypsin-small cylinders, under the same conditions of enzyme immobilization reaction (1 g of nylon support and 5 ml of 1.3 mg/ml trypsin in 0.1 M sodium phosphate buffer, pH 8.5, at 10 degrees C for 18 h) and of enzymatic reaction (1 g of trypsin-nylon in a batch reactor, 2 ml of 0.7% w/v azocasein solution in 50 mM borate buffer, pH 8.5, at 37 degrees C, with shaking, for 1 h). However, the decrease of activity after enzyme immobilization was more conspicuous for the trypsin-small cylinders than for the trypsin-sponge. The former retained approximately 25% of its initial activity, while the latter retained approximately 67% of its initial activity, after seven cycles of utilization for 1 h, pH 8.5, at 37 degrees C and 8 days of storage, pH 8.5, at 4 degrees C in the presence of azocasein. 3. Scanning electron microscopy was performed to visualize the surface of the support after each step of the immobilization process. The electron micrographs show that the two types of nylon supports had a rough surface, which became rougher and full of craters after treatment with 5 N HCl. On the other hand, the partially hydrolyzed nylon surface acquired the appearance of Swiss cheese after treatment with 2.5% glutaraldehyde. After reaction with the enzyme molecules the surface became rougher again.
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PMID:Nylon-6 cylinders and a sponge-like derivative as supports for immobilizing trypsin. 787 18

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.
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PMID:Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue. 798 25

Multidrug resistance protein (MRP) is a Mr 190,000 integral membrane phosphoglycoprotein which has been shown by transfection studies to confer multidrug resistance. We have previously raised and characterized a panel of MRP-specific monoclonal antibodies (MAbs) which detect distinct epitopes in the MRP molecule (D. R. Hipfner et A, Cancer Res., 54. 5788-5792, 1994), and, in the present study, we have identified the epitope of one of these, MAb QCRL-1. Immunoblot analysis of MRP fragments generated by digestion with formic acid or trypsin suggested that the MAb QCRL-1 epitope was located in the region connecting the two halves of MRP. Subsequent analyses of a series of truncated bacterial glutathione S-transferase fusion proteins containing segments of human MRP further localized the MAb QCRL-1 epitope to a region encompassing amino acids 903-956. Similar experiments with an analogous segment of murine MRP demonstrated that MAb QCRL-1 was highly specific for the human protein. The reactivity of MAb QCRL-1 with a series of overlapping hexapeptides and heptapeptides within this region identified the human MRP-specific heptapeptide SSYSGDI (corresponding to amino acids 918-924) as the epitope, and this peptide was shown to specifically inhibit MAb QCRL-1 binding to MRP. The results of these studies confirm that this epitope has a cytoplasmic location consistent with the topology of MRP predicted from hydrophobicity analyses. These experiments also revealed the presence of a number of protease-sensitive sites on either side of the MAb QCRL-1 epitope in the cytoplasmic domain connecting the two halves of MRP. Future epitope-mapping studies with other MRP-specific MAbs win provide additional insights into the topology of MRP, and may help to identify functionally important regions of this protein. Moreover, definition of the epitope recognized by MAb QCRL-1 as well as the other MAbs will facilitate the use of these reagents for immunohistological studies of MRP expression in drug-resistant tumors.
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PMID:Location of a protease-hypersensitive region in the multidrug resistance protein (MRP) by mapping of the epitope of MRP-specific monoclonal antibody QCRL-1. 876 26

In the venom of Vipera palaestinae an unusual, two-component toxin was found. The two components of the toxin are an acidic phospholipase A2 (VpaPLA2) and a basic protein, both with an apparent molecular mass of about 15 kDa. Each component alone is not toxic; however, their mixture is lethal. We have determined the amino acid and cDNA sequences of VpaPLA2. The protein primary structure was solved by sequencing the peptides generated by chemical cleavage of the molecule using CNBr, formic acid and hydroxylamine-hydrochloride and by enzymatic fragmentation with trypsin and chymotrypsin. VpaPLA2 consists of 122 amino acid residues and has all the structural characteristics of subgroup IIA PLA2s. It shows the highest amino acid similarity to a non-toxic phospholipase A2 from Eristocophis macmahoni (82%), whereas the most similar toxic phospholipases A2 share about 70% of residues with VpaPLA2. The substitution of His20 for a hydrophobic residue (Leu) in VpaPLA2 might be one of the reasons that its complex with the basic protein could not be observed.
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PMID:Protein and cDNA structures of an acidic phospholipase A2, the enzymatic part of an unusual, two-component toxin from Vipera palaestinae. 887 23

Cheviot sheep from the Neuropathogenesis Unit flock were examined for PrP in brain sections using immunocytochemistry in order to aid scrapie diagnosis. Brains were collected from sheep which had been naturally or experimentally infected with scrapie and fixed in periodate-lysine-paraformaldehyde or in formalin. Immunolabelling was achieved using a monoclonal antibody (FH11) raised to the N-terminus of recombinant PrP protein. Several pre-treatments were studied in an effort to enhance PrP immunolabelling such as trypsin, formic acid and hydrated autoclaving. Trypsin was successful in highlighting PrP staining in formalin-fixed tissue. PrP staining was regularly observed in the dorsal vagus nucleus of the medulla oblongata and in the thalamus. Differences in the distribution and intensity of PrP immunostaining were apparent between the scrapie sources ME7 and SSBP/I.
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PMID:Immunolocalisation of the prion protein (PrP) in the brains of sheep with scrapie. 895 91

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.
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PMID:Epitope and mimotope for an antibody to the Na, K-ATPase. 923 55

As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of alpha-tubulin. 3H-labeled palmitoylated alpha-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to peptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of 3H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify 3H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in alpha-tubulin. The palmitoylated residues in alpha-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.
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PMID:Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation. 924 44

An alternative method to enzymatic digestion for protein identification by mass spectrometry has been developed that is based on chemical cleavage by formic acid. This method was tested on gel-purified apomyoglobin and BSA, as well as unknown proteins that cofractionate with Tyl-virus-like particles from Saccharomyces cerevisiae. Cleavage at aspartyl residues was found to be efficient and specific, and this specificity of cleavage lent itself easily to database searches. Parallel digestions using trypsin were also performed. The formic acid cleavage method generated comparable or better results than tryptic digestion for protein identification.
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PMID:Chemical cleavage at aspartyl residues for protein identification. 1181 65

Semicarbazide-sensitive amine oxidase (SSAO) encodes a wide family of enzymes named E.C.1.4.3.6 [amine:oxygen oxidoreductase (deaminating) (copper containing)] that metabolises primary aliphatic and aromatic amines. It is present in almost all vascularised and nonvascularised mammalian tissues, and it is also present in soluble form in plasma. SSAO appears to show different functions depending on the tissue where it is expressed. Here we describe, for the first time, the activation of the SSAO from human lung by human plasma. The extent of activation was greater when the human plasma came from diabetic and heart infarcted patients. A kinetic mechanism of such effect is proposed. The activation was lost after the plasma was dialysed, indicating a low molecular weight component (MW <3800 Da) to be responsible. The activator component is heat stable and resistant to proteolysis by chymotrypsin and trypsin and also resistant to perchloric acid treatment. However, treatment with 35% formic acid, completely abolished activation, suggesting involvement of lipid material. The possibility of that lysophosphatidylcholine (LPC), an amphiphilic phospholipid derived from the phosphatidylcholine, the major component in plasma accumulated in pathological conditions, was studied. LPC was shown to behave as a "competitive activator" of human lung SSAO at concentrations below its critical micellar concentration (CMC value=50 microM). Thus LPC may be a component of the SSAO activatory material present in human plasma.
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PMID:Activation of human lung semicarbazide sensitive amine oxidase by a low molecular weight component present in human plasma. 1287 30

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides that result from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly-charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is the MS method of choice for PMF. The qualitative features of a MALDI-MS mass spectrum can be selectively tuned by varying the matrix and the solvent system used to prepare the matrix. The selective tuning of MALDI-MS mass spectra in order to optimize PMF results is addressed in this paper. Carbonic anhydrase, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin, and the digest was analyzed with MALDI-MS. 2,5-Dihydroxybenzoic acid (2,5-DHB) and alpha-cyano-4-hydroxycinnamic acid were prepared, using five different solvent systems: (A) 99% acetone; (B) 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA); (C) 75% ACN, 0.1% TFA; (D) formic acid:H(2)O: 2-propanol (1:3:2); and (E) H(2)O:MeOH (2:1). Each protein was found to have a different optimum solvent system for PMF. Generally, better PMF results were obtained with 2,5-DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest were convolved.
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PMID:A strategy to improve peptide mass fingerprinting matches through the optimization of matrix-assisted laser desorption/ionization matrix selection and formulation. 1476 Jul 19


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