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)

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.
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PMID:The human placental transferrin receptor: reconstitution into liposomes and electron microscopy. 129 37

Several high-density lipoprotein (HDL)-binding proteins, candidates for the putative HDL receptor, have recently been identified, including two membrane proteins: HB1 of 120 kDa and HB2 of 100 kDa, present in rat and human liver plasma membranes respectively. Further insights into their function however, have been hampered by poor recoveries of these hydrophobic peptides, and the present work was undertaken to improve yields and enable a more detailed investigation of their properties. A significant improvement has been achieved using two affinity chromatographic procedures, one exploiting the glycoprotein nature of the proteins and the other exploiting their ligand properties, which in combination resulted in considerable enrichment of HB1 and HB2. Thus DEAE-Sephacel fractionation (0.05-0.2 M-NaCl) of CHAPS-solubilized plasma membranes yielded active HDL-binding proteins which bound to concanavalin A-Sepharose or wheat-germ-lectin-Sepharose columns and retained their binding activity after eluting with methyl-alpha-D-mannoside or N-acetylglucosamine respectively. These glycoproteins were further purified by affinity chromatography using apo-HDL-Sepharose columns. Final purification required preparative SDS/PAGE. Investigation of the carbohydrate moieties of the proteins using glycosidases and two-dimensional gel electrophoresis revealed pI values ranging from 4.6 to 4.9 and from 4.5 to 4.7 for HB1 and HB2 respectively, which after treatment with neuraminidase shifted towards basic pH (5.4-5.7 and 5.3-5.5 respectively). The molecular masses were decreased to 115 kDa and 95 kDa respectively, demonstrating that sialic acid residues contributed significantly to the negative charge of the glycosylated peptides. Treatment with the enzyme peptide N-glycosidase F (N-glycanase) resulted in a decrease in molecular mass of HB1 and HB2 to 105 kDa and 80 kDa respectively, but endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment was not effective. Neither neuraminidase nor N-glycanase treatment destroyed activity, suggesting that sialic acids or N-linked oligosaccharides are not important determinants of HDL binding. Digestion of plasma membranes with trypsin or Pronase resulted in a loss of activity of both HB1 and HB2 that was not influenced by prior treatment with neuraminidase, suggesting that sialic acid residues play no protective role against proteolytic cleavage of HDL receptor proteins.
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PMID:Affinity purification of the hepatic high-density lipoprotein receptor identifies two acidic glycoproteins and enables further characterization of their binding properties. 131 18

This study was undertaken in order to improve photoaffinity labelling efficiency of pancreatic cholecystokinin receptor by the cleavable probe 125I-ASD-(Thr28,Ahx31)-CCK-25-33 and to further characterize the denaturated receptor and its agonist binding domain. Membrane bound pancreatic cholecystokinin receptor was specifically labelled by 125I-ASD-(Thr28,Ahx31)-CCK-25-33 as a component of Mr approximately 85,000-100,000. The efficiency of the photolabelling was 3-4%. Performing photolysis on [125I-ASD-(Thr28,Ahx31)-CCK-25-33-receptor] complexes solubilized by CHAPS did not affect specificity of the labelling reaction but enhanced its efficiency so that up to 10% of the receptor site population could be cross-linked. Several lectins were tested for their ability to recognize and purify the cholecystokinin receptor denaturated by Nonidet P-40. Wheat germ agglutinin provided the best recovery and purification rate. The receptor was fully adsorbed on immobilized wheat germ agglutinin, while only a fraction was retained on ricin II (28%) and Ulex europaeus (58%), thus suggesting that the receptor is heterogeneously glycosylated. Finally, major labelled receptor fragments were generated by enzymatic digestion. There were: endoproteinase Glu-C----Mr approximately 34,000; endoproteinase Glu-C/trypsin----Mr approximately 12,000; chymotrypsin/endoproteinase Glu-C----Mr approximately 16,000 and 12,000. The fragment of Mr approximately 34,000 was deglycosylated to a component of Mr approximately 22,000 whereas the other fragments were insensitive to deglycosylation Such results strongly suggest that cholecystokinin binding occurs in a non-glycosylated domain of the cholecystokinin receptor protein.
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PMID:Biochemical characterization of a subtype pancreatic cholecystokinin receptor and of its agonist binding domain. 158 23

Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface.
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PMID:Solubilization of a functionally active platelet-activating factor receptor from rabbit platelets. 165 81

The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.
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PMID:Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein. 190 25

To identify Chlamydia trachomatis genes involved in attachment to host cells, a chlamydial genomic library was screened on the basis of binding characteristics by two methods. In the whole-cell screen, individual recombinant Escherichia coli clones were assayed for adherence to eukaryotic cells. In the membrane-binding screen, each recombinant colony of E. coli was treated with CHCl3 and assayed for binding to purified, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized, 35S-labeled eukaryotic membrane material. Initial screening with McCoy cells was refined by using HEC-1B cells, a human endometrial epithelial cell line, which discriminate among recombinants adhering to McCoy cells. Some recombinants demonstrate significantly greater adherence to HEC-1B cells than to McCoy cells and appear, by transmission electron microscopy, to associate with electron-dense areas of the epithelial cell plasma membrane, resembling coated pits. Recombinants positive by one or both screening methods were examined by Southern and Western (immunoblot) analyses, which revealed the presence of chlamydial sequences inserted in the plasmids and the expression of novel 18-, 28-, and approximately 82 kDa, and perhaps of 18 Maxicell analysis of selected recombinants confirmed that the proteins of 28 and approximately 82 kDa, and perhaps of 18 kDa, are plasmid encoded. Antiserum generated against the recombinant approximately 82-kDa protein reacted in Western analysis with a similar-sized protein from C. trachomatis serovar E elementary bodies (EB) and reticulate bodies, serovar L2 EB, and C. psittaci EB. E. coli JM109(pPBW58) contains a 6.7-kb plasmid insert which encodes proteins of all three sizes. Under a number of different conditions in the whole-cell attachment assay--i.e., at 4 degrees C, in Ca(2+)- and Mg(2+)-free medium, in the presence of trypsin or dextran sulfate, and with rabbit aortic endothelial cells--the binding specificity of JM109(pPBW58) parallels that of C. trachomatis EB. Finally, the adherence phenotype of E. coli JM109(pPBW58) correlates directly with the presence of the recombinant plasmid; the phenotype is lost concurrently with loss of the recombinant plasmid, and the into E. coli JM109. The role of the 18-, 28-, and approximately 82-kDa proteins in mediating attachment, whether they act in concert as a complex or individually, has yet to be determined.
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PMID:Recombinant Escherichia coli clones expressing Chlamydia trachomatis gene products attach to human endometrial epithelial cells. 193 59

Prostaglandin (PG) D2 binding activity was retained at the highest level in the P2 fraction prepared from porcine temporal cortex with the use of buffer containing mannitol and quinacrine. Then, the activity in this fraction was solubilized with maximal recovery by 10 mM CHAPS. The specific PGD2 binding time-dependently increased and was saturated at around 70 nM. Scatchard plots were fitted to a straight line with a Kd value of 20 nM and Bmax of 120 fmol/mg protein. The binding sites showed high specificity for PGD2. In addition, heat and trypsin treatments remarkably decreased the binding activity. These results suggest that the specific binding protein for PGD2 can be solubilized from these membranes.
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PMID:Solubilization of prostaglandin D2 binding protein from porcine temporal cortex. 198 14

[3H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [3H]-cocaine with a high affinity site of 170 fmole/mg protein (Kd 16.7 nM). The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [3H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear.
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PMID:Characterization of a cocaine binding protein in human placenta. 215 65

The production of interleukin (IL 1) by normal human peripheral blood monocytes purified by Ficoll-Hypaque density sedimentation, Percoll-gradient sedimentation, and plastic adherence can be detected as early as 30 min intracellularly, and extracellularly within 1 hr after stimulation with lipopolysaccharide (LPS). Production of mRNA coding for the isoelectric point 7.0 species of IL 1 was also detected as early as 1 hr after LPS stimulation and reached a maximum level at 6 hr. Cell-associated IL 1 activity could be extracted with CHAPS detergent from every cell fraction (i.e., membranes, cytosol, and particulates), but was present mainly (greater than 95%) in the cytosol of LPS-activated monocytes and the myelomonocytic cell line, THP-1. The apparent m.w. of IL 1 activity on high pressure liquid chromatography gel filtration in every cell fraction was approximately 23,000 daltons, with a minor peak at 31,000 daltons, whereas the IL 1 activity in the culture supernatants was 17,000 daltons. Western blotting analysis of LPS-stimulated monocyte extracts showed two forms of IL 1 corresponding to 31,000 daltons and 25,000 daltons. Exposure of viable cells to trypsin and plasmin released biologically active 23,000 dalton IL 1 only from IL 1-producing cells such as activated monocytes and IL 1-producing Ebstein-Barr virus B lymphocyte cell lines. Consequently, biologically active IL 1 is presumably exposed on the outer surface of cell membranes. Furthermore, IL 1 release by human monocytes in plasminogen-depleted fetal calf serum was considerably decreased. Conversely, supplementation of plasminogen-depleted serum with purified plasminogen restored the IL 1 production, suggesting that plasmin or plasmin-like factors may be involved in the regulation of the release of IL 1 from IL 1-producing cells. In conclusion, the results suggest that IL 1 is rapidly produced, is pooled in the cytosol, and in part is processed by enzymes, is transferred to the plasma membranes, and is then released from the cells. Tissue plasminogen activator and serum enzymes such as plasmin may therefore be involved in the release of IL 1 from IL 1-producing cells.
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PMID:Intracellular localization of human monocyte associated interleukin 1 (IL 1) activity and release of biologically active IL 1 from monocytes by trypsin and plasmin. 242 Aug 74

A method is developed for purification of the protein of the Ca2+-activated K+ channel from outer renal medulla of pig kidney. The response of this K+ channel to physiological concentrations of Ca2+ is important for regulation of transtubular NaCl transport. In reconstituted vesicles direct addition of calmodulin doubles Ca2+ activation with sufficient affinity (K1/2 0.1 nM) for chromatographic purification of the protein. For purification luminal plasma membrane vesicles are isolated on metrizamide density gradients and solubilized in CHAPS. The fraction of soluble protein retained on calmodulin-Sepharose 4B columns in the presence of Ca2+ and eluted by EGTA is 0.7%. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into phospholipid vesicles. It distributes on two bands of 51 and 36 kDa after gel electrophoresis in SDS. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation of the channel. Phosphorylation from cAMP-dependent protein kinase strongly stimulates Ca2+-activated K+ channel activity and labels the 51 kDa band suggesting that this protein is involved in regulation of K+ channel opening.
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PMID:Purification of Ca2+-activated K+ channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla. 243 63


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