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)

Prolyl endopeptidase (EC 3.4.21.26) was purified from human brain by a series of column-chromatographic steps using DEAE-cellulose DE-52, hydroxyapatite, phenyl-Sepharose, Sephacryl S-200 and f.p.l.c. (Mono Q). The enzyme was purified by a factor of 943 and was homogeneous in a SDS/polyacrylamide gel as judged by Coomassie Blue staining. The Mr estimated by SDS/PAGE is 79,600, and under native conditions on Sephacryl S-200 it is 85,600. Therefore the enzyme exists as a monomer. With benzyloxycarbonylglycylproline p-nitroanilide as substrate, the optimum pH of the enzyme is 6.8, and with the substrate concentration between 0.059 mM and 0.37 mM the Km is 9.0 x 10(-4) M. The pI of the enzyme is 4.75. The enzyme is classified as a serine proteinase, as it is strongly inhibited by di-isopropyl fluorophosphate. However, other serine proteinase inhibitors do not inhibit the enzyme significantly, suggesting that the active site of prolyl endopeptidase differs from that of classical serine proteinases such as trypsin. Polyclonal antibodies were raised against purified human brain prolyl endopeptidase in rabbits. Western-blot analysis, enzyme-inhibition assays, antibody binding and immunoprecipitation experiments indicated that the polyclonal antibodies are both specific and inhibitory to the enzyme activity.
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PMID:Purification and characterization of human brain prolyl endopeptidase. 164 30

Macrophages express a receptor on the cell surface that functions to clear glycoproteins from the extracellular milieu. The activity of this receptor is sensitive to treatment with trypsin. In inflammatory situations, macrophages are activated and exposed to increased levels of extracellular proteases. Under these conditions, mannose receptor activity on the macrophages is diminished. We therefore decided to study the effects of trypsin treatment on the structure and activity of cell-associated and purified receptor that might contribute to the activation-associated receptor down-regulation. Trypsin treatment (1 microgram/ml for 3 h) resulted in the production of a 140 kDa, trypsin-resistant fragment from both intact cells and isolated receptor. This fragment was no longer able to bind ligand. The remaining 35 kDa fragment apparently is further degraded into smaller fragments, since no evidence of this domain was found on Coomassie Blue-stained gels. The 140 kDa fragment retained immunoreactivity and contained at least a portion of the iodinated tyrosine residues following surface labelling with Na125I. Neither calcium nor ligand protected the receptor from proteolysis. In addition, prior treatment with oxidants did not increase the susceptibility of the receptor to trypsin digestion. We conclude from these results that the macrophage mannose receptor is clipped by the serine protease trypsin at the cell surface, resulting in the release and further degradation of the binding domain, and the production of a membrane-associated 140 kDa fragment. This trypsin-mediated down-regulation of receptor activity might be important in controlling glycoprotein clearance during inflammation.
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PMID:Modulation of mannose receptor activity by proteolysis. 224 9

With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.
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PMID:Photoaffinity labeling of T4 bacteriophage 32 protein. 243 10

An expression plasmid containing a hybrid gene encoding a protein having the primary amino acid sequence of the adenylate cyclase from Escherichia coli was constructed. When the gene was induced, the adenylate cyclase could be expressed at high levels in a cya- strain of E. coli. The majority of the enzymatic activity and protein (having a molecular weight of 95,000) induced was insoluble. However, treatment of the insoluble fraction of cell lysates with trypsin resulted in both an increase in and solubilization of the total amount of adenylate cyclase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the soluble protein produced by treatment with trypsin revealed a polypeptide having a molecular weight of 30,000. This soluble, catalytically active fragment of adenylate cyclase was purified and subjected to amino-terminal sequence analyses; two amino-terminal sequences were identified beginning at residue 82 and at residue 342 of the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified fragment followed by either silver or Coomassie Blue staining revealed the presence of only a single polypeptide having a molecular weight of 30,000; a short oligopeptide associated with the amino terminus at residue 342 could not be detected. Site-directed mutagenesis was used to place a stop codon at residue 341; the truncated enzyme was catalytically active, so the short oligopeptide is not necessary for catalysis. The Km for ATP, the Ka for Mg2+, and the Vmax determined for the product containing the 30,000-dalton fragment were similar to the values reported for the intact enzyme from E. coli.
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PMID:Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin. 304 83

The contents of a purified somatotroph and mammotroph secretory granule fraction isolated from rat anterior pituitaries were solubilized in 4 M urea and analyzed by PAGE. In gels electrophoresed under a variety of conditions and stained with Coomassie Blue only two major bands, identified as GH and PRL, were present. In gels stained with Stains-All (which stains anionic substances), several additional bands were detected. When quarter pituitaries were labeled with a [3H]amino acid mixture, GH and PRL accounted for greater than 95% of the radioactivity incorporated into the granules. After labeling with [35S]sulfate, two classes of radiolabeled sulfated components were detected in the granules: a class of trypsin-sensitive macromolecular components which were coincident with two of the bands seen after Stains-All, and a class of low molecular weight components. In order to examine the distribution of the two classes of sulfated components within somatotroph and mammotroph granules, granules were suspended in 0.4% (w/v) Lubrol PX at pH 4.0, a treatment which has been shown to selectively solubilize somatotroph granule contents leaving mammotroph granule cores intact. This treatment was found to solubilize greater than 95% of the GH and greater than 99% of the radiolabeled, low molecular weight sulfated components; in contrast, there was virtually no solubilization of either PRL or macromolecular sulfated components. The findings indicate (a) that [35S]sulfate is incorporated into both somatotroph and mammotroph granules, and (b) that the low molecular weight sulfated components are associated with somatotroph granules whereas the macromolecular sulfated components are associated with mammotroph granules.
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PMID:Characterization of rat somatotroph and mammotroph secretory granules. Presence of sulfated molecules. 615 2

The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si.
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PMID:Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius. 645 98

The 130- and 125-kDa heavy chains of Acanthamoeba myosins IA and IB were radioactively labeled at either the regulatory phosphorylation site or the catalytic site and then subjected to controlled proteolysis by either trypsin or chymotrypsin. The labeled and unlabeled peptides generated during the course of proteolysis were identified by autoradiography and Coomassie Blue staining after separation by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The relative positions of the phosphorylation and active sites could be deduced. The catalytic site of myosin IA is most probably within 38 kDa of one end of the 130-kDa heavy chain, and the phosphorylation site, which can be no more than 40 kDa away from the catalytic site, would then be between 38 and 78 kDa of that same end of the heavy chain. Possibly, the phosphorylation site is further restricted to the region between 38 and 64 kDa from the end of the heavy chain. The catalytic and phosphorylation sites of myosin IB are both contained within a segment of 62 kDa at one end of the 125-kDa heavy chain and are within 40 kDa of each other. The phosphorylation site may be restricted to a small segment between 60 and 62 kDa from one end of the heavy chain which would limit the possible position of the catalytic site to the region between 20 and 60 kDa of that end.
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PMID:Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB. 650 Dec 93

Mature asexual stages of the malaria parasite Plasmodium Knowlesi synthesize proteins of Mr 180 000-225 000 that are expressed on the outer membrane of infected erythrocytes and which vary antigenically such that different parasite clones are specifically agglutinated with homologous antibody. Other non-agglutinable clones have been prepared which fail to express variant antigen on infected cells. Two agglutinable clones of different variant antigen phenotypes and a non-agglutinable cone were examined to determine the proportion of total malarial proteins represented by variant antigens. Malarial proteins were labelled with various radioactive amino acids and the sodium dodecyl sulphate--polyacrylamide gel patterns for the three clones compared by fluorography. The patterns were indistinguishable, the variant antigens being undetectable in analyses of total malarial proteins. Furthermore, these antigens were not detected by Coomassie Blue-staining of total cellular proteins after electrophoresis. Sodium dodecyl sulphate and Triton X-100 extracts of labelled cells were immunoprecipitated using a panel of sera of defined agglutination specificity. The variant antigens could not be detected in the fluorographic patterns of total malarial antigens immunoprecipitated by these sera. In contrast, after lactoperoxidase catalysed radio-iodination of intact schizont-infected cells, the 125I-variant antigens on the cell surface were identified by demonstrating their accessibility both to antibody and to trypsin with intact cells. Thus, the variant antigens are quantitatively very minor malarial proteins that can only be detected by methods which selectively analyse the subset of proteins on the erythrocyte surface.
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PMID:Protein antigens of Plasmodium knowlesi clones of different variant antigen phenotype. 671 54

The mitochondrial porin or VDAC (Voltage-Dependent Anion Channel), the pore-forming structure responsible for the high permeability of the outer mitochondrial membrane, was found to be one of only three mitochondrial proteins bound by [14C]dicyclohexylcarbodiimide (DCCD) at low dosages (1.5 nmol/mg of mitochondrial porin) (De Pinto, V., Tommasino, M., Benz, R., and Palmieri, F. (1985) Biochim. Biophys. Acta 813, 230-242). Treatment of intact mitochondria with DCCD results in the inhibition of their ability to binding hexokinase (Nakashima, R. A., Mangan, P. S., Colombini, M., and Pedersen, P. L. (1986) Biochemistry 25, 1015-1021). In the present study, mitochondrial porin was purified from [14C]DCCD-labeled mitochondria. The purified labeled porin was treated with the cleavage reagent CNBr and with the endoproteases trypsin and V8 from Staphylococcus aureus and blotted to polyvinylidene difluoride membrane. The transferred peptides were detected with Coomassie Blue dye, excised, and sequenced. The sequences of several labeled and unlabeled peptides were obtained and then overlapped. The region containing the [14C]DCCD radioactivity was limited to 50 amino acid residues and completely sequenced. Covalently incorporated [14C]DCCD was exclusively released at the position corresponding to glutamate 72. The DCCD-reactive residue is located in the 4th of 16 predicted transmembrane amphipathic beta-strands. When the sequence surrounding the DCCD site was compared to those surrounding the DCCD-reactive residue of other membrane proteins, no homology was apparent.
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PMID:Location of the dicyclohexylcarbodiimide-reactive glutamate residue in the bovine heart mitochondrial porin. 768 55

The alpha-I fragment of human spectrin that carries the binding site on the alpha-chain of spectrin for the beta-chain has been purified from limited trypsin digests of spectrin by means of FPLC. The self-association of spectrin and the binding of the alpha-I fragment to spectrin heterodimers and to tetramers have been quantified through the use of gel electrophoresis, staining with Coomassie Blue, and quantification of the bound dye following elution with pyridine. The parameters of self-association were found to be consistent with those estimated from sedimentation equilibrium analysis. The data were consistent with a model in which both self-association and the binding of the alpha-I fragment are considered to occur through an intermediate in which the alpha-beta interface is initially dissociated. The alpha-beta interface in the heterodimer was found to be less stable than that of higher oligomers by approx. 3 kJ/mol.
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PMID:Quantitative assessment of the association of the alpha-I fragment of spectrin with oligomers of intact spectrin. 808 17


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