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 structural and functional roles of the single asparagine (N)-linked oligosaccharide chain of Band 3 (AE1), the anion transport protein of the human erythrocyte membrane, were examined. Purified Band 3 (M(r) = 95,000) in 0.1% octaethylene glycol mono n-dodecyl ether (C12E8) detergent solution was deglycosylated using N-glycosidase F. This treatment sharpened the protein band on sodium dodecyl sulfate gel electrophoresis and decreased its apparent molecular weight by 5,000. The purified membrane domain could be deglycosylated under similar conditions, causing a shift from a broad band centered at 55 kDa to a sharp 46-kDa band. Band 3 was shown to bind tomato lectin, and loss of lectin binding on blots provided a sensitive assay for deglycosylation. Carbohydrate analysis revealed that greater than 80% of the oligosaccharide could be removed from Band 3 by N-glycosidase F digestion. The deglycosylated protein maintained its dimeric structure and level of detergent binding but had a smaller Stokes radius (RS = 72 A) than native Band 3 (RS = 75 A). The Stokes radius of the membrane domain (RS = 60 A) also decreased upon deglycosylation (RS = 58 A). Circular dichroism studies showed that deglycosylation did not change the secondary structure of Band 3 or the membrane domain. The sensitivity of Band 3 or the membrane domain to proteolytic digestion by trypsin or proteinase K was also unaffected by deglycosylation. The deglycosylated protein aggregated more rapidly and was much more readily precipitable by ammonium sulfate. The deglycosylated protein bound the anion transport inhibitor 4-benzamido-4'-amino-stilbene-2,2'-disulfonate with the same affinity (Kd = 1 microM) as the native protein. Transport studies using reconstituted Band 3 and resealed ghosts showed that deglycosylated Band 3 retained its ability to transport anions. We conclude that removal of the oligosaccharide chain from Band 3 and any resultant structural changes had no effect on the transport function of this protein.
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PMID:Enzymatic deglycosylation of human Band 3, the anion transport protein of the erythrocyte membrane. Effect on protein structure and transport properties. 160 63

We describe the successful isolation and maintenance of primary cultures of dog gallbladder epithelial cells. The surgically removed gallbladder was treated with trypsin/EDTA for 45 minutes and epithelial cells were collected and resuspended in Eagle's minimum essential medium with 10% fetal calf serum, and plated on Vitrogen-coated culture dishes. Each gallbladder yielded approximately 12 to 15 x 10(6) columnar epithelial cells, greater than 95% of which were viable by trypan blue exclusion. In culture, cells maintained their polarity. They were arranged and grew in small and tight clusters that coalesced at confluency. When examined using transmission electron microscopy, prominent and numerous microville were identified on the apical portion of the plasma membrane. Cells were connected by well-formed desmosomes. Scanning electron microscopy revealed clusters of polyhedral cells with numerous papillary projections. Immunohistochemical studies demonstrated uniform staining of cells to keratin 35BH11 and AE1. Histochemical studies were positive for gamma-glutamyl transpeptidase and negative for glucose-6-phosphatase and albumin. Cells incorporated [3H]uridine into intracellular proteins and [14C]glucosamine into tissue and secreted mucous glycoproteins linearly over 2 to 24 hours. Flow cytometry studies demonstrated a consistent and reproducible number of cells (10 to 12%) at S-phase. However, the number of cells at S-phase was dramatically reduced to almost negligible as cells reached confluency. This method of culturing primary dog gallbladder epithelial cells is highly reproducible and reliable. These cells preserve their state of differentiation, polarity, histochemical and immunohistochemical profile, morphologic, and metabolic integrity with repeated passaging or after being frozen. [3H]Thymidine uptake is well maintained, although doubling time shows a trend of decreased cell duplication with time. This technique offers the opportunity to study the electrophysiologic, metabolic, and immunologic properties of epithelial cells.
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PMID:Long-term culture and partial characterization of dog gallbladder epithelial cells. 170 26

The effect of preliminary trypsinization on the immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissues of a variety of tumors (squamous cell carcinomas, adenocarcinomas, mesotheliomas, and transitional cell carcinomas) was evaluated. Three types of trypsin (Type II and Type IX porcine trypsin and Type III bovine trypsin) and varying concentrations of trypsin were assessed. Immunoreactivity of keratin proteins was determined using rabbit anti-keratin antibodies and monoclonal antibodies (combination of AE1 and AE3) and immunoperoxidase techniques. Preliminary trypsinization was mandatory for optimal immunoreactivity of keratin proteins using either polyclonal or monoclonal antibodies. Excellent results were obtained using Type II porcine trypsin at concentrations of 25 mg/dl for 30-45 min or 50 mg/dl for 20 min, at 37 degrees C. Trypsin treatment with excessive concentrations of enzyme and/or extended incubation times promoted tissue digestion and in some cases, yielded decreased immunoreactivity and altered staining patterns.
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PMID:Optimal immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumours using polyclonal and monoclonal antibodies. 258 Aug 83

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55,000 and 64,000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44,000 and 57,000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18,000-23,000 and 43,000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.
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PMID:Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes. 314 74

The "gold standard" inflow cytometric DNA analysis of breast cancer uses fresh tumor cells simultaneously labeled for cytokeratin (CK) and DNA. We developed a 2-parameter CK-DNA flow assay suitable for archival, paraffin-embedded tissue (PT). Six anti-CK monoclonal antibodies were tested by immunocytochemistry and our assay for staining of nuclei extracted from PT breast cancers by combination pepsin-trypsin digestion. Clone CAM 5.2 was inadequate for PT nuclear suspensions, but a cocktail of 2 anti-CK clones (AE1/AE3 and KL-1) distinguished epithelial from nonepithelial nuclei in 2-parameter flow dot plots. We studied 82 routine PT breast tumors by our assay and used a univariate flow DNA histogram based on fresh biopsy tissue for comparison. Three histogram data quality indicators were improved. A trend toward higher S-phase fractions was found for DNA diploid PT tumors, although when inflammation was evident histologically, the increment in S-phase fraction with gating was often marked. CK gating identified PT tumors containing concurrent CK-positive DNA diploid and nondiploid populations (27 of 56 DNA nondiploid histograms). By excluding nonepithelial nuclei, 2-color CK-DNA flow methods may increase the accuracy of ploidy and S-phase fraction measurements. Our method appears superior to previous techniques using clone CAM 5.2 for labeling of archival breast cancers.
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PMID:Flow cytometric DNA analysis with cytokeratin gating of formalin-fixed deparaffinized breast cancer nuclei. 970 23

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.
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PMID:Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses. 1046 Jan 74

The topology of the band 3 (AE1) polypeptide of the erythrocyte membrane is not fully established despite extensive study. Residues near lysine 743 (K743) have been reported to be extracellular in some studies and cytoplasmic in others. In the work presented here, we have attempted to establish the sidedness of K743 using in situ proteolysis. Trypsin, papain, and proteinase K do not cleave band 3 at or near K743 in intact red cells, even under conditions that cause cleavage on the C-terminal side of the glycosylation site (N642) in extracellular loop 4. In contrast, trypsin sealed inside red cell ghosts cleaves at K743, as does trypsin treatment of inside-out vesicles (IOVs). The transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H(2)DIDS), acting from the extracellular side, blocks trypsin cleavage at K743 in unsealed membranes by inducing a protease-resistant conformation. H(2)DIDS added to IOVs does not prevent cleavage at K743; therefore, trypsin cleavage at K743 in IOVs is not a consequence of cleavage of right-side-out or leaky vesicles. Finally, microsomes were prepared from HEK293 cells expressing the membrane domain of AE1 lacking the normal glycosylation site. This polypeptide does not traffic to the surface membrane; trypsin treatment of microsomes containing this polypeptide produces the 20 kDa fragment, providing further evidence that K743 is exposed at the cytoplasmic surface. Therefore, the actions of trypsin on intact cells, resealed ghosts, unsealed ghosts, inside-out vesicles, and microsomes from HEK293 cells all indicate that K743 is cytoplasmic and not extracellular.
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PMID:Topology of the anion exchange protein AE1: the controversial sidedness of lysine 743. 1187 46

To clarify the function of the hydrophilic carboxyl-terminal tail of human erythrocyte membrane band 3 protein (HEM-B3), we purified two peptides, C1 (Ala893-Val911) and KS4 (Gly647-Arg656), from human erythrocyte band 3 protein preparations. Purified C1 peptides at concentrations from 5 to 80 microM were incubated with fresh human erythrocyte white ghosts. The C1 peptide demonstrated a novel protease activity, which cleaved glycophorin A (GPA) at Leu118-Ser119 in a dose-dependent manner. This activity was eliminated by trypsin. In a control experiment, the KS4 peptide did not cleave GPA under the same conditions. To help substantiate that the band 3 C-terminal tail peptide (C1) alone possesses the protease activity, two experiments were performed. First, the plasmids pGBKT(7)-GPA-Ct and pGADT(7)-AE1-Ct were cotransformed into the yeast strain AH109. The pGBKT(7)-GPA-Ct plasmid contains the cDNA of the 33 amino acid residue section of GPA (Tyr93-Asn125) fused with the pGBKT(7) vector. The plasmid pGADT(7)-AE1-Ct contains the cDNA of the C-terminal 33 amino acid residues of HEM-B3 fused with the GAL4 DNA-binding domain in the pGADT(7) vector. The results of the cotransformation experiment indicated that the C-terminal 33 amino acid residues of HEM-B3 interacted directly with the GPA C-terminal segment defined above. Second, we used a mammalian two-hybrid analysis to confirm the interaction relationship between the band 3 C-terminal segment and the GPA C-terminus. The C-terminus of GPA and the C-terminal 33 amino acid residues of HEM-B3 were subcloned into the DNA-binding domain and transcription activation domain vectors of the two-hybrid system, respectively. They were then cotransfected along with a chloramphenicol acetyltransferse (CAT) reporter vector into HeLa cells. The CAT activity measured in this experiment also indicated that there was interaction between the C-terminal 33 amino acid residues of HEM-B3 and the C-terminus of GPA.
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PMID:Purification and characterization of the human erythrocyte band 3 protein C-terminal domain. 1476 40

An R664X nonsense mutant AE1 is responsible for dominant hereditary spherocytosis in cattle and is degraded by the proteasomal endoplasmic reticulum-associated degradation. The present study demonstrated that R664X AE1 translated in vitro had the trypsin-sensitve site identical to that of the wild-type AE1. The P661S/R664X mutant containing a possible N-glycosylation site at Asn660 showed an increase in size by 3 kDa both in the cell-free translation system and in transfected HEK293 cells. Moreover, steady state levels of R664X and P661S/R664X in HEK293 cells were markedly increased in the presence of a proteasome inhibitior. These findings indicate that the truncated C-terminal region of R664X AE1 has lumenal localization in the endoplasmic reticulum and is not accessible to proteasomal machineries in the cytosol.
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PMID:Lumenal localization in the endoplasmic reticulum of the C-terminal tail of an AE1 mutant responsible for hereditary spherocytosis in cattle. 1740 56

To date, the most recent specific diagnostic investigations for amniotic fluid embolism have been unable to conclusively identify any mechanism of disease other than a physical block to the circulation. We selected eight fatal cases in previously healthy women with uneventful singleton term pregnancies who presented to tertiary care centers in Italy for delivery. Pathologic features were assessed immunohistochemically using anti-fibrinogen, anti-tryptase, anti-C(3a), and anti-cytokeratin antibodies. AE1/AE3 cytokeratin stains proved positive, and tryptase-positive material was documented outside pulmonary mast cells. In all studied cases, expression of complement C(3a) was twofold lower than in the control group, suggesting a possible complement activation in AFE, initiated by fetal antigen leaking into the maternal circulation.
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PMID:Complement C3a expression and tryptase degranulation as promising histopathological tests for diagnosing fatal amniotic fluid embolism. 1917 92


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