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)

A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.
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PMID:Glycophorin-enriched vesicles obtained by a selective extraction of human erythrocyte membranes with a non-ionic detergent. 44 5

Glycophorin from porcine erythrocyte membranes was digested with trypsin and chymotrypsin. Some of the peptides were isolated by conventional techniques. The amino acid sequence was determined for two isolated peptides: a chymotryptic glycopeptide of 19 residues and a tryptic peptide of 36 residues which represented the carboxy terminal of the glycophorin.
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PMID:Partial amino acid sequence of glycophorin from porcine erythrocyte membranes. 54 38

Human red blood cell membranes were labeled from within the lipid bilayer by the apolar photosensitive reagent, 5-[125I]iodonaphthyl-1-azide. Glycophorin, the major sialoglycoprotein of the red cell membrane, was purified by two different methods; it contained approximately half of the total label incorporated into membrane proteins. The label was confined to the trypsin-insoluble peptide of glycophorin that includes a sequence of 20, mainly apolar, amino acids. These findings provide direct evidence that the labeled segment resides within the membrane in direct contact with the lipid bilayer, and support the suggestion that glycophorin spans the bilayer through its hydrophobic domain.
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PMID:Red cell membrane glycophorin labeling from within the lipid bilayer. 66 61

Glycophorin was purified from human erythrocyte ghosts by the lithium diiodosalicylate -phenol procedure utilizing 125I-labeled lithium diiodosalicylate. The glycophorin preparation was found to contain 8.9 +/- 2.1 mol lithium diiodosalicylate per mol glycophorin. This bound lithium diiodosalicylate cannot be removed by extensive washings with a variety of polar organic solvents nor by treatment with the detergent, sodium deoxycholate. Further, the hydrophobic peptide produced from glycophorin by trypsin digestion contained 3.4 mol lithium diiodosalicylate per mol peptide.
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PMID:Binding of lithium diiodosalicylate to glycophorin. 67 46

The topography of the external surface of the human red cell membrane has been studied using an impermeant radioactive probe, [125I]diazodiiodosulfanilic acid, which binds covalently to protein groups of the membrane following reaction with intact cells. The pattern of labeling was assessed by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis followed by sequential analysis of single gels for carbohydrates (by staining with the periodic acid-Schiff (PAS) reagent), for proteins (by staining with Coomassie blue), and for radioactivity (by counting gels sliced in 2-mm segments). The radioactive probe bound to membrane polypeptides with apparent molecular weights of 94,200, 58,100, and 46,500 (Peaks A, B, and C, respectively). Peak A co-migrated with a small periodic acid-Schiff-positive band and protein Band 3 (nomenclature of Steck) (Steck, T.L. (1974)J. Cell Biol. 62: 1-19). Peak B migrated with protein Band(s) 4.5 slightly ahead of the major membrane glycoprotein (PAS-1). Peak C migrated like glycoprotein PAS-2 and protein Band 5, the actin-like, water-soluble membrane protein. In contrast to lactoperoxidase iodination and a number of other probes, [125I]diazodiiodosulfanilic acid reacted minimally with the major membrane glycoprotein, glycophorin. When it was reacted with isolated ghosts, all molecular weight classes of polypeptides were labeled. Treatment of labeled cells with neuraminidase or trypsin altered the glycoprotein staining pattern, but not the radioactive peaks. On the other hand, Pronase eliminated the Mr=94,200 radioactive peak, diminished the other two radioactive peaks, and profoundly changed the glycoprotein and protein staining patterns. Treatment of the membranes of labeled cells in a low ionic strength alkaline medium did not alter radioactive peaks and demonstrated that Peak C differed from the actin-like membrane protein. A nonionic detergent, Triton X-100, solubilized all radioactive components. The studies have defined the binding of [125I]diazodiiodosulfanilic acid to external proteins of the human red cell membrane. Its pattern of reaction differs quantitatively and qualitatively from other commonly used reagents, and it provides a useful additional vectorial probe for the study of membrane topography. Its reactions provide further evidence of the organizational complexity of the red cell membrane and emphasize the fact that interpretation of information derived from the use of membrane probes must take into account the differences resulting from the properties of the probing reagents themselves.
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PMID:Topography of the external surface of the human red blood cell membrane studied with a nonpenetrating label, [125I]diazodiiodosulfanilic acid. 83 50

The insoluble peptide, T(is), prepared by trypsin hydrolysis of the MN-glycoprotein (glycophorin) of the human erythrocyte has been incorporated into phospholipid membranes in the form of liposomes and black lipid membranes. The permeability of liposome membranes to 42K+ and of black lipid membranes to water and ions is increased significantly by the presence of the T(is) peptide. Electrophoresis measurements indicate that these effects are not due to the T(is) peptide carrying a net charge. The results suggest that the peptide causes local disordering of the bilayer membrane structures. This is considered in the light of findings published elsewhere: that the MN-glycoprotein penetrates through the cell membrane via a non-polar segment of its polypeptide chain, which is contained intact within the T(is) peptide; that the T(is) peptide is partially helical when associated with phospholipid and forms multimeric 8.0 nm structures within the hydrophobic plane of phospholipid bilayers.
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PMID:The effects of the membrane-penetrating polypeptide segment of the human erythrocyte MN-glycoprotein on the permeability of model lipid membranes. 112 22

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.
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PMID:Interaction of the 140/130/110 kDa rhoptry protein complex of Plasmodium falciparum with the erythrocyte membrane and liposomes. 188 71

Degradation of glycophorin by trypsin in intact red cells results in an increase in hemoglobin bound to the membrane. Incubation of resealed ghosts made from these cells demonstrated that the hemoglobin was bound to the intracellular membrane surface. We found that treatment of hemoglobin with KCNO inhibited the ability of hemoglobin to bind to the membrane. Addition of KCNO to intact cells followed by trypsin treatment abolished the additional membrane-bound hemoglobin, indicating that the bound hemoglobin resulted from increased Band 3 binding. Treatment of intact cells with neuraminidase also resulted in increased membrane-bound Hb, which correlated with the amount of sialic acid released. Scatchard analysis revealed that enzyme treatment increased the affinity of hemoglobin for the high affinity Band 3 binding site, while KCNO treatment abolished this binding. Taken together, these studies demonstrate that extracellular proteolytic degradation of glycophorin by proteases similar to those released by cells of the reticuloendothelial system results in an increased ability of hemoglobin to bind to Band 3.
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PMID:Degradation of erythrocyte glycophorin results in increased membrane bound hemoglobin. 199 Sep 74

We have studied the effects of band 4.1 phosphorylation on its association with red cell inside-out vesicles stripped of all peripheral proteins. Band 4.1 bound to these vesicles in a saturable manner, and binding was characterized by a linear Scatchard plot with an apparent Kd of 1-2 x 10(-7) M. Phosphorylation of band 4.1 by purified protein kinase C reduced its ability to bind to membranes, resulting in a reduction in the apparent binding capacity of the membrane by 60-70% but little or no change in the apparent Kd of binding. By contrast, phosphorylation of band 4.1 by cAMP-dependent kinase had no effect on membrane binding. Digestion of the stripped inside-out vesicles with trypsin cleaved 100% of the cytoplasmic domain of band 3 but had little or no effect on glycophorin. Binding of band 4.1 to these digested vesicles was reduced by 70%. Phosphorylation of band 4.1 by protein kinase C had no effect on its binding to the digested vesicles, suggesting that the cytoplasmic domain of band 3 contained the phosphorylation-sensitive binding sites. This was confirmed by direct measurement of band 4.1 binding to the purified cytoplasmic domain of band 3. Phosphorylation of band 4.1 by protein kinase C reduced its binding to the purified 43-kDa domain by as much as 90%, while phosphorylation by cAMP-dependent kinase was without effect. These results show a selective effect of protein kinase C phosphorylation on the binding of band 4.1 to one of its membrane receptors, band 3, and suggest a mechanism whereby one of the key red cell-skeletal membrane associations may be modulated.
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PMID:Selective modulation of band 4.1 binding to erythrocyte membranes by protein kinase C. 230 15

1. An anti-N lectin was extracted from Vicia unijuga leaves with phosphate-buffered saline (PBS). Purification of the lectin was achieved, after pretreatment of the PBS extract by ammonium sulfate fractionation and absorption with human M erythrocytes, by using a combination of conventional chromatographic techniques with asialoglycophorin AN-Sepharose CL-4B affinity chromatography. Purification steps were followed by increase of specific activity. 2. Homogeneity of the purified lectin was demonstrated by HPLC and SDS-PAGE. The purified lectin was a glycoprotein with 11.4% carbohydrate and relatively high percentages of serine, threonine and aspartic acid residues and had a Mw of 120,000 Da. 3. This lectin agglutinated human N and MN erythrocytes, but did not agglutinate M erythrocytes. Hemagglutination of the lectin was inhibited by glycophorin AN and N-active sialoglycopeptide released from human N erythrocytes by treatment with Pronase or trypsin. However, it was not inhibited by any of mono- and di-saccharides, ABH-active glycoproteins, glycophorin AM and M-active sialoglycopeptide liberated from human M erythrocytes by treatment with Pronase or trypsin.
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PMID:Purification and characterization of anti-N lectin from Vicia unijuga leaves. 232 20


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