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)

The hybrid glycophorin in Dantu-positive human erythrocytes of the N.E. variety was not cleaved by treatment of intact cells with various proteases, in contrast to normal glycophorins. Therefore, it could be purified by phenol/saline extraction of membranes from trypsin-treated and chymotrypsin-treated red cells and subsequent gel filtration in the presence of Ammonyx-LO. The complete structure of the hybrid molecule, comprising 99 amino acid residues, was elucidated by sequence analyses of peptides prepared by chymotrypsin, trypsin, cyanogen bromide or V8 proteinase treatment. The N-terminal 39 residues and the glycosylation of the molecule were found to be indistinguishable from those of blood-group-s-specific glycophorin B. Conversely, the residues 39-99 were shown to be identical with the residues 71-131 of the major blood-group M-active or N-active sialoglycoprotein (glycophorin A). Hemagglutination inhibition assays revealed that the Dantu antigen represents a labile structure. The receptor might be located within the residues approximately 28-40 of the hybrid glycophorin, as judged from the effects of modifications of membranes. Our data provide an explanation for the previous findings that Dantu-positive cells (N.E. type) exhibit a protease-resistant N antigen and a qualitatively altered s antigen.
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PMID:Hybrid glycophorins from human erythrocyte membranes. I. Isolation and complete structural analysis of the hybrid sialoglycoprotein from Dantu-positive red cells of the N.E. variety. 359 15

Destabilization of liposomes composed of phosphatidylethanolamine (PE) and purified glycophorin of human erythrocytes was studied with the release of an entrapped fluorescent dye, calcein. Proteolytic cleavage of liposomes by trypsin induced a rapid increase of turbidity and the leakage of calcein from the liposomes. Kinetic experiments indicated that the destabilization was a second order reaction, i.e. it required liposome collision. Using N-(7-nitro-2,1,3-benzoxadiazol-4-yl) PE as a fluorescent probe for the formation of hexagonal phase of PE, tryptic digestion of the liposomes resulted in a higher tendency of the PE bilayer to transform into the hexagonal phase. We propose that hexagonal (or inverted micellar) structures are involved in the trypsin induced liposome destabilization.
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PMID:Trypsin induced destabilization of liposomes composed of dioleoylphosphatidylethanolamine and glycophorin. 381 29

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.
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PMID:Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles. 384 Dec 89

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5-7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol.53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.
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PMID:Anionic sites of human erythrocyte membranes. I. Effects of trypsin, phospholipase C, and pH on the topography of bound positively charged colloidal particles. 412 Dec 89

Both native and recombined membrane systems from the human erythrocyte membrane and the rabbit sarcoplasmic reticulum have been studied with 31P Nuclear Magnetic Resonance (NMR). We compare intensities of the anisotropic 31P resonance exhibited by these membranes with the intensity expected from the known phospholipid content of the membranous sample. In a recombinant with human erythrocyte glycophorin, a component of the phospholipid is "missing" from the 31P NMR resonance, apparently due to a severe broadening of the resonance of that component. Approximately 29 phospholipid molecules were found immobilized per glycophorin molecule in the membrane, regardless of the phospholipid:protein ratio. Cholesterol may inhibit the immobilization of phospholipids by glycophorin. Recombinants with band three from the human erythrocyte membrane contain an immobilized phospholipid component, analogous to the results with glycophorin. 31P NMR data from the native sarcoplasmic reticulum membrane also revealed an immobilized phospholipid component whose magnitude is independent of temperature between 30 degrees C and 45 degrees C. Extensive papain proteolysis of the membrane completely digests the Ca++ Mg++ ATPase and removes the immobilization of phospholipids noted in the intact membrane. Limited trypsin cleavage, however, does not completely remove the immobilized component; salt reduces the immobilized component.
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PMID:31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes. 612 13

Human red cells deficient in glycophorin B are partly resistant to invasion by Plasmodium falciparum and become completely resistant when glycophorin A is removed from their surface by trypsin treatment. Similar treatment of cells which have a hybrid glycophorin molecule renders them glycophorin-deficient and resistant to invasion. Tn and Wrb -ve cells with defined alterations in glycophorin A or B are also resistant to invasion. These findings suggest that both glycophorins A and B are involved in parasite invasion, indicate which parts of these molecules may be involved in this process, and provide the basis for a tentative model of parasite/red-cell interactions.
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PMID:Glycophorin as a possible receptor for Plasmodium falciparum. 612 59

A receptor specific for lipoglycans from Acholeplasma axanthum and Acholeplasma granularum was isolated from sheep erythrocyte stroma by extraction with n-pentanol and permeation chromatography. The purified receptor appeared as one band on sodium dodecyl sulfate-polyacrylamide gels and stained with Coomassie blue, periodate-Schiff reagent, and Sudan black. It was distinct from the erythrocyte receptor for gram-negative lipopolysaccharides and the glycophorin receptor for certain species of Mycoplasma. Periodate oxidation and trypsin did not affect the receptor activity in intact erythrocytes, but the purified receptor was susceptible to proteolytic digestion. Specific receptors, sensitive to trypsin digestion, could be isolated from rabbit kidney and cultured rabbit epidermal cell membranes. These could be distinguished from the receptor from erythrocytes by their solubility in n-pentanol. The segment of the lipoglycan molecule which binds to these receptors was not lipoidal in nature and was distinct from the specific antigenic determinants of the lipoglycans.
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PMID:Isolation and characterization of the sheep erythrocyte receptor for acholeplasmal lipoglycans. 618 20

Four mouse monoclonal antibodies directed against the red cell membrane protein glycophorin A have been isolated and characterized. They are produced by hybridomas derived from SP2/0 myeloma cells and spleen cells from Biozzi mice immunized with a mixture of human erythrocytes from homozygous blood group M and N individuals. These antibodies recognize and bind to purified glycophorin A and to glycophorin on the red cell surface. All are of the IgGl, kappa light chain subclass and bind to determinants presented on the 39 amino acid, trypsin-sensitive, N-terminal peptide of glycophorin A. Three display differential specificities for the two allelic forms of glycophorin A; two are exquisitely specific for the M-form and one preferentially binds the N-form. Treatment of red cells with neuraminidase, which removes N-acetylneuraminic acid from glycophorin A, abolishes the binding of these three antibodies. The binding of the N-specific antibody is also sensitive to modification of the amino-terminal residue of the antigen. The fourth antibody binds equally well to both the M- and N-forms as well as to neuraminidase-treated red cells; thus it recognizes a public, N-acetylneuraminic acid independent glycophorin A determinant.
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PMID:Monoclonal antibodies specific for the M- and N-forms of human glycophorin A. 619 36

To investigate the greater enzymatic activity of the alternative pathway convertase (and the subsequent greater fixation of C3b) on paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, we have examined the topography of binding of C3b to PNH and normal erythrocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the alpha-chain of C3b was found to bind via predominantly ester bonds to free hydroxyl groups on glycophorin-alpha, the major erythrocyte sialoglycoprotein. The pattern of binding of nascent C3b was the same for normal and PNH erythrocytes. Thus, although C3b binding to a different membrane constituent did not appear to account for the greater enzymatic activity of the alternative pathway convertase when affixed to PNH erythrocytes, it seemed possible that the glycoproteins to which C3b bound might be qualitatively abnormal on the PNH cells, and that structural differences in these molecules might impose modifications in the enzyme-substrate interactions of the alternative pathway convertase. Using methods for radiolabeling both protein and carbohydrate residues, we therefore compared the electrophoretic pattern of the cell-surface glycoproteins on PNH and normal erythrocytes. The glycophorin-alpha dimer was found to be qualitatively abnormal on the PNH cells as evidenced by its greater susceptibility to trypsin-mediated proteolysis. In addition, the abnormal erythrocytes from patients with PNH had fewer periodate oxidizable constituents than did normal erythrocytes, indicating a relative deficiency of cell-surface sialic acid. These investigations suggest that abnormalities in membrane glycoproteins may underlie the aberrant interactions of complement with the hematopoietic elements of PNH.
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PMID:Abnormality of glycophorin-alpha on paroxysmal nocturnal hemoglobinuria erythrocytes. 623 12

Glycophorin A, a major glycoprotein of the red blood cell, is reconstituted in small lipid vesicles (250-300 A in diameter) by using cholate detergent solubilization followed by rapid removal of cholate on a molecular sieve column. The extent of glycophorin incorporation is found to be critically dependent on the amount of cholate used, with higher amounts yielding vesicles with higher percentages of glycophorin. Vesicles with as much as 1 molecule of protein per 20 molecules of lipid can be prepared. Data on the vesicles obtained by using hydrolytic enzymes such as neuraminidase and trypsin, combined with amino acid analysis, suggest that glycophorin is incorporated in a transbilayer fashion with a high fraction of the molecules oriented with the carbohydrate-containing amino terminus to the vesicle exterior. Interaction of the protein with the hydrophobic portion of the bilayer is apparent in proton nuclear magnetic resonance spectra, and lipid line-width increases have been used to characterize the strength and stoichiometry of interaction. Glycophorin is found to affect directly as many as 40 lipid molecules per molecule of protein; however, the magnitude of the effects is not large.
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PMID:Small unilamellar vesicles containing glycophorin A. Chemical characterization and proton nuclear magnetic resonance studies. 626 87


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