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)

Band 3 protein is a major erythrocyte transmembrane glycoprotein. We compared the degradation of band 3 protein by calpain I (a cytoplasmic, micromolar-Ca2(+)-requiring thiol proteinase) in the cells from old individuals (greater than 70 years old) to that in the cells from young ones (20-30 years old). In the young, little degradation of band 3 protein occurred in calpain-treated erythrocyte ghosts. In the old, significant band 3 protein degradation was found in erythrocyte ghosts treated similarly. The difference between young and old in the susceptibility of band 3 protein to calpain was retained in membrane vesicles (membranes stripped of peripheral proteins by NaOH) and in chymotrypsin-generated 60 kDa fragment (CH-60). The isolated N-terminal cytoplasmic 43 kDa fragment was degraded by calpain to a similar extent in old and in young. The separated 17 kDa membrane domain of the CH-60 and the trypsin-generated C-terminal 55 kDa membrane-spanning fragment were not degraded by calpain I in the young, nor in the old. Thus the N-terminal cytoplasmic domain is the domain degraded by calpain I. Enhanced sensitivity in the old is observed in intact band 3 protein and in CH-60, the isolated cytoplasmic domain being equally susceptible in young and old. The observed age-related enhanced sensitivity to calpain is consistent with the presence of modifications in band 3 protein and alterations in the association with the calpain-calpastatin system. Band 3 protein has several important functions, with modifications in the protein having implications for altered cell behaviour in the old individual.
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PMID:Band 3 protein degradation by calpain is enhanced in erythrocytes of old people. 201 84

In both the endemic and sporadic forms of pemphigus foliaceus (PF), antiepidermal autoantibodies against desmoglein I are present. Desmoglein I is a highly insoluble 160-kD transmembrane glycoprotein of the desmosomal core. The detailed immunochemical characterization of the epitope(s) recognized by the PF autoantibodies is hampered by its large molecular weight and the insolubility of desmoglein I in nondenaturing buffers. This study was designed to identify alternative methods that could yield soluble immunoreactive PF antigen (Ag) from normal human epidermis. The presence of PF Ag in human epidermis and in its soluble or insoluble fractions was monitored by indirect immunofluorescence, immunoadsorption of PF sera, and immunoprecipitation of radiolabeled fractions. The PF Ag from trypsin-resistant, radiolabeled cell envelope preparations was cleaved by papain and immunoprecipitated by PF sera. A 50-kD peptide, isoelectric at pH 5.5-5.8, was immunoprecipitated by sera from all patients with endemic PF (n = 15) or idiopathic PF (n = 4), and by two of four pemphigus vulgaris sera, but by no control sera (n = 7). This study shows that a significant fraction of the PF Ag is insoluble, trypsin-resistant, and is associated with the cornified cell envelope fraction, but an Ag fragment can be obtained in a small molecular weight, soluble, and immunoreactive form by papain digestion. This 50-kD papain fragment is more amenable to detailed chemical and immunologic characterization than the native molecule.
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PMID:Pemphigus foliaceus antigen: characterization of a keratinocyte envelope associated pool and preparation of a soluble immunoreactive fragment. 247 34

In this study we have used complementary biochemical and immunological techniques to establish that the lymphoma GP85 membrane glycoprotein is a transmembrane protein with a cytoplasmic domain that binds directly to ankyrin, a molecule known to link the membrane to the cytoskeleton. The evidence supporting our conclusion that the GP85 is a transmembrane glycoprotein is as follows: (a) GP85 can be surface-labeled with Na 125I and contains wheat germ agglutinin-binding sites, indicating that it has an extracellular domain; (b) GP85 can be phosphorylated by intracellular kinases, indicating that it has an intracellular domain; and (c) GP85 can be successfully incorporated into phospholipid vesicles, indicating the existence of a hydrophobic domain in the molecule. Further studies show that GP85 displays immunological cross-reactivity with the lymphocyte Pgp-1 (differentiation-specific) membrane glycoprotein, and with the erythrocyte anion transport membrane protein, band 3. Immunocytochemical studies indicate that an ankyrin-like protein accumulates underneath the lymphoma GP85 cap structure, suggesting an association of the ankyrin-like protein and GP85. This relationship has been further confirmed by the following results of binding and reconstitution experiments: (a) purified GP85 binds directly to an ankyrin-Sepharose column; (b) purified GP85 inserts into phospholipid vesicles in both the normal (right side out) and reversed (inside out) orientation (and with only the reversed configuration permits binding of ankyrin to GP85); and (c) cleavage of GP85 with trypsin yields a 40-kD peptide fragment that is part of the cytoplasmic domain and contains the ankyrin binding site(s). Based on these findings, we suggest that the lymphoma GP85 transmembrane glycoprotein contains a cytoplasmic domain that is directly involved in linking ankyrin to the cytoskeleton. This transmembrane linkage may play a pivotal role in receptor capping and cell activation in lymphocytes.
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PMID:Mouse T lymphoma cells contain a transmembrane glycoprotein (GP85) that binds ankyrin. 296 10

A protein with an Mr of 145,000 (p145) was detected by antibodies to phosphotyrosine by Western blot (immunoblot) analysis. This protein was phosphorylated on tyrosine in a gastric carcinoma cell line. In cells that were metabolically labeled with 32Pi, this protein was phosphorylated on tyrosine and serine. p145 is a cysteine-rich transmembrane glycoprotein. The extracellular domain could be labeled by 125I under nonpermeating conditions and was cleaved by mild trypsin treatment of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed a shift of p145 mobility to an apparent Mr of 190,000. After immunoprecipitation with phosphotyrosine antibodies, p145 displayed a strong associated protein kinase activity in vitro, becoming phosphorylated on tyrosine. There was no immunological cross-reaction between p145 and known tyrosine kinases. Both in vivo and in vitro tyrosine phosphorylations were unaffected by the addition of known growth factors. However, p145 was rapidly dephosphorylated in vivo when cells were exposed to low pH, a condition that is known to dissociate ligands from their receptors. These data suggest that p145 is associated with a protein tyrosine kinase activity which, in the tumor cell line studied, is activated by an as yet unidentified factor.
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PMID:p145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line. 321 Nov 49

In this study we have used (phorbol-12-O-tetradecanoylphorbol 13-acetate) and its biologically inactive analogue, 4 alpha-phorbol 12,13-didecanoate), to investigate platelet protein phosphorylation with special emphasis on the properties of a membrane protein-cytoskeleton (transmembrane) complex during platelet activation. Our data indicate that phorbol-12-O-tetradecanoylphorbol 13-acetate (but not 4 alpha-phorbol 12,13-didecanoate) induces both a specific platelet shape change and the preferential phosphorylation of a 180-kDa protein (presumably due to the activation of protein kinase C on the cytoplasmic side of the membrane). Further analysis reveals that the 180-kDa protein can be iodinated by lactoperoxidase and is sensitive to trypsin treatment, indicating exposure of this protein on the outer cell surface. The 180-kDa protein has also been found to contain wheat germ agglutinin-binding sites. All evidence indicates that the 180-kDa polypeptide is a transmembrane glycoprotein and, most importantly, that this protein is found to be preferentially accumulated into a specific membrane-cytoskeleton complex during activation via phorbol-12-O-tetradecanoylphorbol 13-acetate treatment. We believe that the observed phosphorylation of this protein may be closely related to the formation of a complex between several membrane proteins and the cytoskeleton during the initial stages of platelet activation.
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PMID:Phorbol ester-induced phosphorylation of a transmembrane glycoprotein (GP 180) in human blood platelets. 404 78

T200 glycoprotein, a major cell surface component of murine hematopoietic cells, is a phosphorylated transmembrane glycoprotein. Two distinct regions of the molecule can be defined by radiolabeling with a variety of metabolic precursors or by lactoperoxidase-catalyzed iodination, in combination with protease treatments, immunoprecipitation techniques, and peptide "mapping" analysis. A relative protease-resistant domain, which is exposed on the cell surface and contains the antigenic site recognized by a monoclonal anti-T200 antibody known to react with the exterior cell surface, contains most if not all of the mannose-containing oligosaccharide units of the glycoprotein and all of the amino acid residues labeled by lactoperoxidase-catalyzed iodination of intact viable cells. This protease-resistant fragment migrates with an apparent molecular weight of approximately 100,000 in sodium dodecyl sulfate-polyacrylamide gels. The remaining portion of the molecule contains a region, extensively digested by trypsin, which is exposed on the cytoplasmic side of the plasma membrane and contains phosphoserine residues which can be labeled with 32PO4 in vivo. A 125I-labeled tryptic peptide derived from this region of the molecule was obtained if membrane preparations from cells disrupted by nitrogen cavitation were labeled by lactoperoxidase-catalyzed iodination.
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PMID:Disposition of T200 glycoprotein in the plasma membrane of a murine lymphoma cell line. 735 47

Glucosidase I, the first enzyme in the N-linked oligosaccharide processing pathway, cleaves the distal alpha 1,2-linked glucose residue from the Glc3-Man9-GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent lambda clones were isolated which allowed the construction of a full-length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92-kDa protein contains a single N-glycosylation site of the Asn-Xaa-Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N-terminus (amino acids 38-58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far. Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1-deoxynojirimycin or N,N-dimethyl-deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93-kDa form, indicating that it carries a high-mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an approximately 4-kDa polypeptide from the membrane-associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type-II transmembrane glycoprotein, which contains a short cytosolic tail of approximately 37 amino acids, followed by a single transmembrane domain and a large C-terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.
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PMID:Cloning and expression of glucosidase I from human hippocampus. 763 46

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.
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PMID:Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1. 1086 13

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.
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PMID:Identification of N-linked glycosylation sites in human nephrin using mass spectrometry. 1721 72

The objective of this study was to evaluate the potential of P(0) protein, a cell adhesion molecule from peripheral nerve myelin, as a targeting ligand for liposomes. To evaluate binding characteristics and identify possible binding domains, cell-interaction studies were carried out with P(0) protein reconstituted into liposomes (P(0) liposomes) under various conditions. P(0) liposomes with intact P(0) protein were tested after endoglycosidase F treatment (cleaves the carbohydrate moiety) or trypsin digestion (removes the hydrophobic portion, residues 1-79, leaving the carbohydrate portion intact). The cellular uptake was quantitated using radioactive lipids in the liposome bilayer and a liposome-entrapped water soluble compound (inulin). The presence of intact P(0) protein in the liposome bilayer increased the rate of interaction of liposomes 3-4 times with M21 melanoma and HTB-11 neuroblastoma cells (cells of neuroectodermal origin), two times with Caki-1 renal carcinoma cells, and marginally with 3T3 fibroblasts (mesodermal origin). This binding was inhibited by anti-chick P(0) antibodies and Fab fragments. A control transmembrane glycoprotein, glycophorin A., when reconstituted in liposomes had no effect on the binding of liposomes with M21 cells. The results indicate that P(0) protein plays a specific role in the binding of liposomes to cells. Removal of the N-asparagine linked carbohydrate from the P(0) protein in the liposomes resulted in an increase of their association with M21 cells five times that of control liposomes (no protein) and two times that of the non-endoglycosidase F-treated P(0) liposomes. To further characterize the binding domains of P(0) reconstituted into liposomes, competition studies were carried out in the presence of synthetic P(0)-peptides. The competition studies indicated that both the extracellular (residues 90-96) and intracellular (residues 201-207) domain of P(0) protein may be involved in the interaction with cell membranes. The results suggest that P(0) protein is capable of mediating specific heterophilic interactions with various cell lines and targeting to cells of neuroectodermal origin may be achieved.
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PMID:Targeting liposomes through immunoglobulin superfamily domains: P0 protein as a model. 1956 84


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