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)

Boar spermatozoa were radioactively labeled by either lactoperoxidase-catalysed iodination or galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. Plasma membrane glycoproteins were solubilized with the non-ionic detergent Nonidet P40 and separated by affinity chromatography on concanavalin A-Sepharose. A major water-soluble concanavalin A receptor of molecular weight greater than 160 000 was isolated by gel filtration and ion-exchange chromatography. Its amino acid and carbohydrate composition were determined. This glycoprotein is susceptible to digestion by trypsin or chymotrypsin.
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PMID:The isolation and characterization of a concanavalin A receptor from boar spermatozoa surface. 723 91

Viable merozoites of Plasmodium knowlesi were isolated and the proteins that were labeled on intact merozoites by lactoperoxidase-catalyzed radioiodination were identified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of Triton soluble extracts of labeled merozoites demonstrated eight major bands ranging in apparent molecular weight from 150,000 D to 22,000 D. Exposure of intact merozoites to trypsin (10 microgram/ml) for 10 min resulted in the loss of the two highest molecular weight proteins (150,000 D and 105,000 D) and the appearance of two new bands at 70,000 D and 62,000 D. Trypsin treatment under these conditions also removed the receptor(s) for merozoite attachment to erythrocytes. Therefore, these high molecular weight proteins are candidates for the merozoite component that attaches to erythrocytes. There was no evidence that the labeled membrane components were serum or erythrocyte membrane components, two potential contaminants in the preparation. Anti-rhesus erythrocyte antibody did not precipitate labeled merozoite proteins. Furthermore, the immunoprecipitation of labeled merozoite proteins by rhesus anti-merozoite serum was not inhibited by erythrocyte ghosts.
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PMID:Identification of surface proteins on viable Plasmodium knowlesi merozoites. 727 54

T200 glycoprotein, a major cell surface component of murine hematopoietic cells, is a phosphorylated transmembrane glycoprotein. Two distinct regions of the molecule can be defined by radiolabeling with a variety of metabolic precursors or by lactoperoxidase-catalyzed iodination, in combination with protease treatments, immunoprecipitation techniques, and peptide "mapping" analysis. A relative protease-resistant domain, which is exposed on the cell surface and contains the antigenic site recognized by a monoclonal anti-T200 antibody known to react with the exterior cell surface, contains most if not all of the mannose-containing oligosaccharide units of the glycoprotein and all of the amino acid residues labeled by lactoperoxidase-catalyzed iodination of intact viable cells. This protease-resistant fragment migrates with an apparent molecular weight of approximately 100,000 in sodium dodecyl sulfate-polyacrylamide gels. The remaining portion of the molecule contains a region, extensively digested by trypsin, which is exposed on the cytoplasmic side of the plasma membrane and contains phosphoserine residues which can be labeled with 32PO4 in vivo. A 125I-labeled tryptic peptide derived from this region of the molecule was obtained if membrane preparations from cells disrupted by nitrogen cavitation were labeled by lactoperoxidase-catalyzed iodination.
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PMID:Disposition of T200 glycoprotein in the plasma membrane of a murine lymphoma cell line. 735 47

We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 degrees C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37 degrees C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 degrees C followed by washes and iodination (addition of 125I and glucose) at 4 degrees C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO ad GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, > 75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypeptide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-J774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components.
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PMID:Selective iodination and polypeptide composition of pinocytic vesicles. 741 Apr 75

Macrophages secrete a large number of proteases, implying in vivo exposure of the cell surface to proteolytic conditions. Mild trypsin treatment of 125I-labeled guinea pig peritoneal macrophages preferentially cleaves one surface component of apparent 160,000 mol wt. Similar trypsin treatment of macrophages with 3H-labeled carbohydrate surface moieties also cleaves a single 3H-labeled 160,000 mol wt glycoprotein, referred to as gp160. Nonreducing sodium dodecyl sulfate (SDS)-electrophoresis established that gp160 of trypsinized cells remains assembled in the membrane as a multichain disulfide-bonded molecule. gp160 was purified by detergent extraction, L. culinaris lectin affinity chromatography and DEAE-cellulose chromatography. The corresponding molecule from trypsinized cells was purified by the same procedure. Reducing SDS-electrophoresis of purified trypsinized 125I-labeled gp160 revealed two proteolytic fragments with apparent molecular weights of 85,000 and 71,000. Thus, mild trypsin treatment of macrophages preferentially cleaves a single surface protein, possibly at a single site. Because the two fragments of gp160 are accessible to lactoperoxidase and trypsin, both must be exposed on the membrane surface. The reactive carbohydrate site was found on the 85,000 mol wt fragment, which alone contains the 3H-label introduced into intact cells by neuraminidase, galactose, oxidase, and [3H]KBH4.
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PMID:Macrophage component gp160, a major trypsin-sensitive surface glycoprotein. 745 50

The location with respect to the plasma membrane of tyrosine 486 in the native anion exchanger of human erythrocytes has been determined by site-directed immunochemistry. Intact erythrocytes and inside-out vesicles were [125I]radioiodinated by lactoperoxidase in the same vessel. After the erythrocytes and inside-out vesicles had been separated by differential centrifugation, the modified polypeptide of the anion exchanger was isolated from each sample and digested with the proteinase from Staphylococcus aureus strain V8 and trypsin to generate the peptide YIVGR. An immunoadsorbent that was specific for the carboxy-terminal sequence -IVGR was used to purify the peptide YIVGR, which contains tyrosine 486 of the anion exchanger, from the products of the digestion. The [125I]radioiodinated peptides isolated by the immunoadsorbent were submitted to high-pressure liquid chromatography, and their respective mobilities were compared to those of synthetic peptides that had been iodinated at tyrosine. By applying this technique, the peptide containing tyrosine 486 was unambiguously identified, and the incorporation of [125I]iodine into this residue in anion exchanger could be monitored. When inside-out vesicles and intact cells were [125I]radioiodinated in the same suspension, tyrosine 486 was modified to at least a 6-fold greater specific radioactivity in the inside-out vesicles than it was in the intact cells. This amino acid, therefore, was assigned to the cytoplasmic surface of native anion exchanger. It follows that the polypeptide of anion exchanger spans the membrane three times before it reaches the extracellular region surrounding glutamine 550.
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PMID:Topological disposition of tyrosine 486 in anion exchanger from human erythrocytes. 854 83

Previous studies indicate that when low iodine thyroglobulin (Tg) is iodinated enzymatically with thyroid peroxidase (TPO), the tyrosyl residues that are used for the formation of thyroid hormone (hormonogenic sites) are selected for early iodination. The aim of the present study was to assess the relative importance of the substrate (Tg) and the enzyme (TPO) in the selection of the early tyrosyl sites that undergo iodination. For this purpose, low iodine human Tg (2.0 atoms I per 660,000 dimer) was iodinated chemically with (125)I-(3) and enzymatically with TPO + 125I- to a matched low level of iodination (approximately 8 added I atoms per molecule). After reduction and alkylation, the two Tg preparations were digested with trypsin, and the tryptic digests were separated by reverse-phase HPLC into 10 125I-containing pools. Each pool was further fractionated by HPLC to provide purified 125I-peptides suitable for sequence analysis. From the sequence information and the known amino acid sequence of Tg, it was possible to define the location of the iodinated tyrosyl residues. Surprisingly, almost identical results were obtained with chemically and enzymatically iodinated Tg. Not only were the 125I-peptide maps very similar, but all of the recovered 125I in the purified peptides from both samples was located in only three different tyrosyl sites, 5, 2553, and 2520. Tyr 5 and Tyr 2553 are well-established sites of thyroxine formation, while Tyr 2520 has previously been proposed by us to be a donor site. Our observation that the same hormonogenic tyrosyl sites are iodinated by chemical as well as enzymatic iodination indicates that preferential iodination of hormonogenic sites is dependent primarily on the native structure of Tg. TPO plays a minor role, if any, in the selection of early tyrosyl iodination sites in Tg. Consistent with this conclusion was our finding that chemical iodination, as well as enzymatic iodination, led to formation of uniformly iodinated Tg, as determined by isopycnic centrifugation in rubidium chloride. However, we observed a slightly higher diiodotyrosine (DIT) content and a correspondingly lower monoiodotyrosine content in enzymatically iodinated Tg, compared to matched chemically iodinated Tg. This was not observed with two other proteins, bovine serum albumin and trypsinogen, or with free tyrosine, as substrates for iodination. The same preferential formation of DIT in Tg was, however, observed when lactoperoxidase was substituted for TPO. Preferential formation of DIT, therefore, appears to involve interaction between Tg and the peroxidase.
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PMID:Selectivity in tyrosyl iodination sites in human thyroglobulin. 890 Apr 3

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme l, is isolated through an approach that combines proteolytic hydrolysis and reverse-phase high performance liquid chromatographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotrypsin) or a peptide-free heme (with proteinase K). Both heme l and heme l-peptide species were investigated by paramagnetic 1H NMR spectroscopy, electrospray mass spectrometry, and peptide sequence analysis. Paramagnetic 1H NMR experiments on the low spin bis(cyano)-Fe(III)heme l complex conclusively define the heme l structure as a 1,5-bis(hydroxymethyl) derivative of heme b. The electrospray mass spectrum of heme l confirms the two-site hydroxyl functionalization on this heme. Paramagnetic 1H NMR spectra of the high spin bis(dimethyl sulfoxide)-Fe(III) complexes of the isolated heme species provide information regarding peptide content. Sequence analyses of peptides released from two heme l-peptide species by base hydrolysis suggest that heme-protein ester linkages in lactoperoxidase occur between the two hydroxyl groups of heme l and the carboxylic side chains of glutamate 275 and aspartate 125. These results confirm the earlier reported structural proposal (Rae, T. D., and Goff, H. M. (1996) J. Am. Chem. Soc. 118, 2103-2104).
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PMID:The heme prosthetic group of lactoperoxidase. Structural characteristics of heme l and heme l-peptides. 977 11

Human red cells of Rh blood groups -D-/-D- ('super-D'), -/- (Rhnull) and normal Rho(D)+ cells were radioactively surface-labeled using the lactoperoxidase 125I method. Polyacrylamide gel electrophoresis in the presence of SDS followed by fluorography showed a strong enrichment of a polypeptide with an apparent mol. wt. of 28,0000-33,000 in the 125I-labeled -D-/-D- membranes. This polypeptide was specifically immune precipitated with anti-Rho(D) antiserum. Treatment of intact cells with trypsin or Pronase did not digest the protein. The Rho polypeptide migrated identically on polyacrylamide gel electrophoresis under reducing and non-reducing conditions. It was not phosphorylated after in vitro incubation of red cells with 32P. When whole labeled membranes were solubilized in neutral detergent and applied to lectin-Sepharose columns the Rho(D) polypeptide adsorbed to Ricinus communis lectin but not to wheat germ lectin or Lens culinaris lectin. The purified molecule did not adsorb to R. communis lectin-Sepharose. Treatment of the Rho(D) antigen with endo-N-acetyl glucosaminidase H, endo-beta-galactosidase or mild alkali did not lower its apparent mol. wt.
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PMID:Molecular characterization of the human red cell Rho(D) antigen. 1189 30

Milk forms a rich source of biologically interesting components. In particular, its protein fraction is known to encompass many kinds of biological functions. In this review we focus on antibacterial and antiviral properties of milk proteins and milk protein derivatives. The latter include chemically modified proteins and enzymatically induced peptides. If such peptides are released by enzymes present within the digestive tract (e.g. trypsin or pepsin), it is likely that they play a role in the health defense system. This is especially the case when the active fragments can survive the intestinal conditions long enough to arrive at the right place to exert their beneficial function. In the first part of this paper attention is paid to the antibacterial proteins lactoferrin, lactoperoxidase, and lysozyme. Furthermore, antibacterial peptides originating from caseins and whey proteins are described. The second part reports on studies of antiviral effects of milk proteins and derivatives thereof. Special focus is directed to the antiviral action towards the human immunodeficiency virus (HIV) and the human cytomegalovirus (HCMV). Unmodified milk proteins are generally not active against these viruses. An exception is lactoferrin, which shows significant antiviral activity against both HIV and HCMV. Several other milk proteins tested showed strong antiviral effects only after chemical modification, i.e. by making them polyanionic (for anti-HIV activity) or polycationic (for anti-HCMV activity). In a number of cases, conclusions are drawn concerning possible relationships between antibacterial/antiviral activity and molecular structure of the components described.
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PMID:Antibacterial and antiviral effects of milk proteins and derivatives thereof. 1276 35


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