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)

Transmembraneous back-insertion of a solubilized membrane protein, glycophorin A, has been obtained in 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (Pam2GroPCho) cell-size-like liposomes by submitting the lipid/protein mixture to calibrated electric field pulses. Field conditions which are prone to trigger glycophorin insertion are similar to those which mediate lipid layer electropermeabilization. The efflux of calcein, trapped in the liposomes during their preparation, was observed only when field strength is higher than 1.3 kV/cm. Electroinsertion was detected only above the same critical field intensity. Calcein efflux as well as glycophorin insertion were increased by increasing field intensity, pulse duration and/or number of pulses. Experimental evidence of protein insertion was provided by physico-chemical as well as biochemical methods. Direct observation of the pulsed vesicles under the microscope revealed the insertion by means of immunofluorescence and fluorescence. Electroinsertion of fluorescent glycophorin A revealed that the inner bilayers were also labeled. The gel-to-liquid phase transition temperature of Pam2GroPCho decreased after insertion but its cooperativity was not affected. A narrow 31P-NMR peak was observed after electroinsertion showing that the polar headgroups of phospholipids had been altered. Analysis of trypsin-digested peptides revealed that the two trans-orientations of the protein across the external lipid layer were present after electroinsertion. Localized perturbation of the polar headgroup region of phospholipids, which supports the transient permeabilization of lipid layers, allows spontaneous transinsertion of glycophorin across the lipid bilayers.
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PMID:Insertion of glycophorin A, a transmembraneous protein, in lipid bilayers can be mediated by electropermeabilization. 760 45

A fraction of band 3 protein, the major transmembrane protein of erythrocyte membranes, is held to the cytoskeletal protein spectrin via noncovalent interactions with the protein ankyrin (band 2.1). In this study, trypsin was used under defined conditions to selectively proteolyze ankyrin and thereby destroy the band 3-ankyrin linkage on the cytoplasmic side of erythrocyte ghost membranes. Electron paramagnetic resonance (EPR) spectroscopy, in conjunction with selective spin labeling methods, was used to monitor conformational changes occurring in cytoskeletal proteins or cell-surface carbohydrates as a result of this treatment. Treatment of RBC ghosts with TPCK-trypsin for 5 s at 0 degrees C caused an approx. 56% increase in the relevant EPR parameter of a maleimide spin label bound to spectrin (P < 0.004), indicative of increased segmental motion of the spin label and decreased protein-protein interactions. Analysis of the apparent rotational correlation time parameter tau of a spin label covalently and selectively bound to terminal sialic acid residues of glycophorin showed no significant effect from trypsin treatment. However, tau of spin label covalently and specifically bound to terminal galactose residues of cell-surface glycoconjugates of band 3 and other transmembrane glycoproteins significantly decreased with tryptic uncoupling of the ankyrin linkage (P < 0.005). These results suggest a marked conformational alteration in both cytoskeletal and transmembrane proteins as a result of uncoupling from ankyrin. Spermine (N,N'-bis(3-aminopropyl)tetramethylenediamine), a naturally occurring polyamine known to strengthen cytoskeletal protein-protein interactions (Wyse and Butterfield (1988) Biochim. Biophys. Acta 941, 141-149), was used to partially reverse the trypsin-induced cytoskeletal alterations. Addition of 2 mM spermine to ghosts previously treated with trypsin increased cytoskeletal protein-protein interactions as indicated by EPR (P < 0.002). SDS-PAGE was used to confirm the integrity of spectrin, band 3, and band 4.1 in all experiments. The results are discussed with reference to transmembrane signaling mechanisms and membrane-associated pathologies.
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PMID:Alteration of the erythrocyte membrane via enzymatic degradation of ankyrin (band 2.1): subcellular surgery characterized by EPR spectroscopy. 838 64

A lectin was isolated from the hemolymph of Asian horseshoe crab Tachypleus tridentatus by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The lectin's molecular weight was approx. 533 kDa; being a simple protein comprised of two non-identical subunits with molecular weights of 30 and 32 kDa. The hemagglutinating activity of the lectin against equine erythrocytes was strongly inhibited by several sialoglycoproteins and weakly inhibited by sialic acid, although not inhibited by neutral sugars, hexosamines, N-acetylhexosamines, glucuronic acid, or several asialoglycoproteins. In addition, glycophorin HA was more effective than glycophorin HA digested with trypsin in inhibiting hemagglutination of the lectin. These results suggest that the purified lectin specifically reacts with sialic acids containing glycoprotein.
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PMID:Isolation and characterization of a sialic acid-specific binding lectin from the hemolymph of Asian horseshoe crab, Tachypleus tridentatus. 846 14

Alkylresorcinol homologs form stable monomolecular layers at air-water interface. Their interaction with proteins present in the subphase results in an increase of alkylresorcinol molecular packing in the extent dependent upon the protein studied. Strongest effects were observed for proteins with large hydrophobic regions, e.g. glycophorin or serum albumin. Interaction of proteins with alkylresorcinol monolayers is stronger than with phospholipids. A decrease and a shift of intrinsic protein fluorescence upon interaction with the compounds studied support their involvement in alteration of hydrophobic regions. For trypsin, 50% quenching was observed at the alkylresorcinol/trypsin ratio of 0.75. Concomitantly, an apparent inhibition of the enzymatic activity was noted. These results indicate that direct interaction of alkylresorcinols and modulation of enzymatic activities should be recognised as a significant part of the biological effect of these cereal bran components.
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PMID:Interaction of alkylresorcinols with proteins. 858 71

LAMA-84, a human leucocytic cell line, which upon establishment was described as having megakaryocytic, erythroid and granulocytic characteristics, was analysed for expression of various differentiation markers. In addition to some of the previously described phenotypic characteristics, this cell line was found to express mRNA for several proteins characteristic for basophilic leucocytes and mast cells. The authors show that LAMA-84 cells express mRNA for the mast cell tryptase, the proteoglycan core protein, carboxypeptidase A and the alpha and beta chains of the high affinity IgE receptor (Fc epsilon RI). The authors examined the potential of LAMA-84 to differentiate in serum-free medium or after DMSO or PMA treatment. Depending on the inducing factor, surface expression of the Fc epsilon RI alpha-chain was increased from 20% to 35-50% of the cells and mRNA levels for tryptase were increased in serum-free medium and after DMSO treatment. LAMA-84 was found to express CD13, CDw17, CD29, CD33, CD40, CD45 and CD117. Furthermore, mRNA for the eosinophil/basophil markers Charcot-Leyden crystal (CLC) protein and the major basic protein (MBP), as well as the erythrocyte differentiation marker alpha-globin, was detected. However, the authors observed only trace amounts of mRNA for another erythroid differentiation marker (glycophorin), trace amounts of the megakaryocytic marker GPIIIa, and no detectable level of GPIb alpha. By comparing the expression pattern of a panel of differentiation markers in LAMA-84, and a second human cell line (KU812) expressing a basophil phenotype, it is evident that these cell lines, which presently are the only two cell lines identified with basophilic characteristics, share a large number of phenotypic characteristics.
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PMID:Characterization of a human basophil-like cell line (LAMA-84). 869 92

The discovery of a natural Gerbich antigen (anti-Ge2) in the serum of a propositus prompted us to study his red blood cells (RBCs) by using monoclonal anti-bodies (mAbs) directed against glycophorin (GP) C and GPD. An mAb directed against the Ge4 antigen (mAb NaM10-7G11) agglutinated both untreated and trypsin-treated cells, demonstrating the expression of a trypsin-resistant GPC (namely, GPC of the Gerbich type: GPCGe). Surprisingly, an anti-Ge3 antibody (mAb NaM19-3C4) agglutinated untreated cells, showing that they also express the Ge3 antigen that may be carried by normal GPC and CPD or by the abnormal GPC of the Yussef (Yus) type (GPCYus). Immunoblotting analysis performed with an mAb directed against the C-terminal portion of GPC showed that the propositus' RBCs do not contain normal GPC and GPD but both GPCGe and GPCYus. Analysis of RBCs from the family demonstrated that, like the propositus, 2 of the 3 sisters had inherited both the GYPCGe and the GYPCYus alleles from the parents, who carried either the GYPCGe or the GYPCYus allele. The third sister had inherited the normal GYPC alleles from her parents, whereas the child of the propositus had inherited the GYPCGe allele. Interestingly, natural anti-Ge2 antibodies were identified in the serum of 2 of the 3 Ge-negative individuals.
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PMID:Inheritance of abnormal glycophorin C of the Gerbich and Yussef type in a French family. 880 69

Malaria male gametocytes within a newly ingested infected blood meal in the mosquito midgut emerge from erythrocytes and extrude approximately eight flagellar microgametes in a process termed exflagellation. In culture, and in blood removed from infected patients, emerging microgametes avidly adhere to neighboring uninfected and infected erythrocytes, as well as to emerged female macrogametes, creating "exflagellation centers". The mechanism of erythrocyte adherence is not known nor has it been determined for what purpose microgametes may bind to erythrocytes. The proposition of a function underlying erythrocyte adherence is supported by the observation of species-specificity in adhesion: microgametes of the human malaria Plasmodium falciparum can bind human erythrocytes but not chicken erythrocytes, whereas avian host Plasmodium gallinaceum microgametes bind chicken but not human erythrocytes. In this study we developed a binding assay in which normal, enzyme-treated, variant or null erythrocytes are identified by a cell surface fluorescent label and assayed for adherence to exflagellating microgametes. Neuraminidase, trypsin or ficin treatment of human erythrocytes eliminated their ability to adhere to Plasmodium falciparum microgametes, suggesting a role of sialic acid and one or more glycophorins in the binding to a putative gamete receptor. Using nulls lacking glycophorin A [En(a-)], glycophorin B (S-s-U-) or a combination of glycophorin A and B (Mk/Mk) we showed that erythrocytes lacking glycophorin B retain the ability to bind but a lack of glycophorin A reduced adherence by exflagellating microgametes. We propose that either the sialic acid moiety of glycophorins, predominantly glycophorin A, or a more complex interaction involving the glycophorin peptide backbone, is the erythrocyte receptor for adhesion to microgametes.
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PMID:Adherence of erythrocytes during exflagellation of Plasmodium falciparum microgametes is dependent on erythrocyte surface sialic acid and glycophorins. 958 38

Escherichia coli alpha-hemolysin (HlyA) can lyse both red blood cells (RBC) and liposomes. However, the cells are lysed at HlyA concentrations 1-2 orders of magnitude lower than liposomes (large unilamellar vesicles). Treatment of RBC with trypsin, but not with chymotrypsin, reduces the sensitivity of RBC toward HlyA to the level of the liposomes. Since glycophorin, one of the main proteins in the RBC surface, can be hydrolyzed by trypsin much more readily than by chymotrypsin, the possibility was tested of a specific binding of HlyA to glycophorin. With this purpose, a number of experiments were performed. (a) HlyA was preincubated with purified glycophorin, after which it was found to be inactive against both RBC and liposomes. (b) Treatment of RBC with an anti-glycophorin antibody protected the cells against HlyA lysis. (c) Immobilized HlyA was able to bind glycophorin present in a detergent lysate of RBC ghosts. (d) Incorporation of glycophorin into pure phosphatidylcholine liposomes increased notoriously the sensitivity of the vesicles toward HlyA. (e) Treatment of the glycophorin-containing liposomes with trypsin reverted the vesicles to their original low sensitivity. The above results are interpreted in terms of glycophorin acting as a receptor for HlyA in RBC. The binding constant of HlyA for glycophorin was estimated, in RBC at sublytic HlyA concentrations, to be 1.5 x 10(-9) m.
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PMID:Glycophorin as a receptor for Escherichia coli alpha-hemolysin in erythrocytes. 1113 7

Erythrocytes (RBCs) opsonized by IgG and complement are prevalently recognized and phagocytosed by complement receptor CR1. This mechanism, effective in senescent and damaged RBCs seems to be operative in ring-parasitized RBCs, since infection by Plasmodium falciparum induces stage-dependent binding of auto-antibodies and activated C3 to the RBC membrane. Later, parasite forms are also recognized by non-opsonic receptors, such as scavenger receptor CD36. Malaria parasites induce the oxidative formation of hemichromes which are the trigger for the auto-antigen development. Band 3 protein is the most plausible candidate of the RBC auto-antigen, induced by hemichromes. Auto-antigens isolated from trophozoites were found only in a high-molecular-weight protein aggregates not present in the normal RBC. The immunocomplex was purified by protein-A affinity chromatography, purified proteins digested by trypsin and analyzed by MALDI-TOF. Peptide mapping showed that the main antigen consisted of band 3 protein aggregates that also contained hemichromes, IgGs, complement factor 3 (C3), and traces of spectrin and glycophorin but no parasite proteins. Two cysteines located in the band 3 cytoplasmic domain were found to be particularly reactive to oxidants and mediated band 3 covalent dimerization via disulfide bonds. Thus, parasites promote oxidative alterations in the membrane of the host which lead to exposure of antigenic sites recognized by anti-band 3 auto-antibodies. Formation of band 3 clusters appears to be mediated by cytoplasmic binding of hemichromes and also by direct band 3 oxidation, whereby clustered, oxidized and antigenic band 3 was underglycosylated.
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PMID:Mechanisms of band 3 oxidation and clustering in the phagocytosis of Plasmodium falciparum-infected erythrocytes. 1496 70

The low-prevalence MNS blood group antigenTSEN is located at the junction of glycophorin A (GPA) to glycophorin B (GPB) in several hybrid glycophorin molecules. Extremely rare people have RBCs with a double dose of the TSEN antigen and have made an antibody to a high-prevalence MNS antigen. We report the first patient who is heterozygous for GYP.JL and Mk. During prenatal tests,an alloantibody to a high-prevalence antigen was detected in the serum of a 21-year-old Hispanic woman. The antibody detected an antigen resistant to treatment by papain, trypsin, alpha-chymotrypsin, or DTT. The antibody was strongly reactive by the IAT with all RBCs tested except those having the MkMk, GP.Hil/GP.Hil, or GP.JL/GP.JL phenotypes. The patient's RBCs typed M+N-S+/-s-U+, En(a+/-), Hut-, Mi(a-), Mur-, Vw-, Wr(a-b-), and were TSEN+, MINY+. Reactivity with Glycine soja suggested that her RBCs had a decreased level of sialic acid. Immunoblotting showed the presence of monomer and dimer forms of a GP(A-B) hybrid and an absence of GPA and GPB. Sequencing of DNA and PCR-RFLP using the restriction enzyme RsaI confirmed the presence of a hybrid GYP(AB). The patient's antibody was determined to be anti-EnaFR. She is the first person reported with the GP.JL phenotype associated with a deletion of GYPA and GYPB in trans to GYP.JL.
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PMID:An alloantibody to a high-prevalence MNS antigen in a person with a GP.JL/Mk phenotype. 1828 4


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