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)

Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [32P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [32P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain.
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PMID:Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C. 301 73

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome-associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1-anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.
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PMID:The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. 326 Feb 38

The human transferrin receptor could be fluorographically detected after immunoprecipitation from a leukemic T-cell line labeled with [3H]palmitic acid. The label was found ony in association with the human transferrin receptor and not in association with two other major plasma membrane glycoproteins, demonstrating that the incorporation of radioactivity was not due to metabolism of the palmitate. Treatment of sodium dodecyl sulfate-polyacrylamide gels containing the [3H]palmitate-labeled transferrin receptor with hydroxylamine, prior to fluorography, resulted in release of a substantial fraction of the label from the molecule. In addition, at least part of the label released from immunoprecipitates of the transferrin receptor by treatment with hydroxylamine was identified as palmitohydroxamate, providing further evidence that the labeled fatty acid is covalently bound to the receptor. A proteolytic fragment (Mr = 70,000) derived from the portion of the transferrin receptor exposed on the cell surface can be obtained by trypsin digestion of intact or Nonidet P-40-solubilized cells. When cells were labeled with [3H]palmitic acid, none of the radioactivity could be detected in the tryptic fragment. Thus, the bound palmitate appears to be associated with the region of the molecule that is in close proximity to the plasma membrane.
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PMID:Covalent binding of fatty acid to the transferrin receptor in cultured human cells. 626

HeLa cells were found to have a single class of non-interacting receptors specific for transferrin. Both apotransferrin and diferric transferrin competed equally with 125I-diferric transferrin for receptor binding. Transferrin binding was temperature-dependent and reversible. Binding of transferrin to cells exhibited a KD of 27 nM with a maximum binding capacity of 1.8-3.7 x 10(6) molecules/cell. Cells grown in the presence of diferric transferrin or in the presence of ferric ammonium citrate exhibited a concentration- and time-dependent decrease in 125I-diferric transferrin binding. The decrease in binding activity reflected a reduction in receptor number rather than an alteration in ligand receptor affinity. Growth of cells in saturating concentrations of apotransferrin did not cause a decrease in receptor number. When iron-treated cells were removed to media free of ferric ammonium citrate, the receptor number returned to control values by 40 h. When receptors were removed with trypsin, cells grown and maintained in ferric ammonium citrate-supplemented media demonstrated a rate of receptor reappearance 47% that of control cells grown in ferric ammonium citrate-free media. Cells grown in media supplemented with diferric transferrin or ferric ammonium citrate exhibited an increase in cytosolic iron content. The transferrin receptor number returned to normal after cells were removed to unsupplemented media, despite persistent elevation of cytosolic iron content. Increased iron content did not appear to be the sole factor determining receptor number.
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PMID:Regulation of HeLa cell transferrin receptors. 628 49

Two fragments, each corresponding to approximately half of the ovotransferrin (OTf) molecule and containing an iron-binding site were produced by digestion with affinity bound trypsin and were purified by isoelectric focusing and gel filtration chromatography. The immunologically distinct "half-molecules" individually have little ability to bind to transferrin receptors on chick embryo red blood cells or to donate iron to them. Combining them, however, leads to both binding and iron donation approaching that found for holo-OTf. Furthermore, similar amounts of radiolabeled iron can be extracted into the putative heme fraction from Fe2OTf and from the various combined half-molecules. These findings conflict with those reported by Keung and Azari ( (1982) J. Biol. Chem. 257, 1184-1188) for subtilisin-derived half-molecules of OTf examined in a similar system. They found that each half-molecule appeared to bind at a level of approximately one-third that of Fe2OTf and that the half-molecules competed with each other for binding sites. In contrast, our equilibrium binding studies, in the presence of 2,4-dinitrophenol to prevent iron removal, led to the determination of 4.79 X 10(4) binding sites/cell for Fe2OTf, 4.44 X 10(4) for the NH2-terminal half-molecules in the presence of excess COOH-terminal half-molecules and 4.17 X 10(4) for COOH-terminal half-molecules in the presence of NH2-terminal half-molecules; apparent binding constants were estimated to be 3.29 X 10(6), 1.19 X 10(6), and 0.67 X 10(6) M-1 for these same samples. Problems associated with equilibrium binding studies in which a narrow range of concentrations of ligand is used and/or iron is being removed are discussed. Labeled combined half-molecules were half as effective as labeled Fe2OTf in competition with unlabeled Fe2OTf. These findings are consistent with the lower apparent binding constant found in the equilibrium binding studies. Equimolar apo-OTf had no effect on binding of either Fe2OTf or the combined half-molecules. It seems apparent from our studies that the NH2- and COOH-terminal half-molecules each contain a recognition region both of which are necessary for binding to the transferrin receptor and iron donation to the chick embryo red blood cell.
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PMID:Physiological levels of binding and iron donation by complementary half-molecules of ovotransferrin to transferrin receptors of chick reticulocytes. 631 14

The transferrin receptor is a 180 000-dalton protein which can be dissociated to two 90 000-dalton polypeptides under reducing conditions. It can be labelled by lactoperoxidase-catalysed iodination on the cell surface at 0 degree C. Trypsin digestion of labelled cells at 0 degree C can be used to degrade those receptors on the cell surface; they release a 70 000-dalton soluble fragment which binds to transferrin. When cells are labelled at 0 degree C, then warmed to 37 degrees C, the labelled receptors enter the cells and become trypsin resistant. These receptors enter the cells, probably via coated pits, with a half-life of approximately 5 min. Since there is about three times as much receptor inside cells as on the surface, this means that transit through the cell to the cell surface takes approximately 21 min, if all receptors are on the same cycling pathway.
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PMID:Transferrin receptor and its recycling in HeLa cells. 632 61

Rat monoclonal antibodies against mouse transferrin receptor have been used to isolate and characterize the mouse receptor molecule. The molecule is a dimeric glycoprotein of Mr 200 000 resembling its human homolog of Mr 190 000. Receptor molecules prepared from different lymphoid cell populations show structural differences which can be explained by variations in the carbohydrate moiety of the molecule. Both the antibody-binding site and the transferrin-binding site are located on tryptic fragments of Mr 80 000 on the extracellular part of the molecule. After trypsin treatment, these fragments are partially retained at the cell surface, probably non-covalently bound to one intact receptor subunit, but they are released at higher trypsin concentrations. The soluble fragments retain their ability to bind transferrin and appear to exist as dimers. In this fragment, there are no disulfide bonds present. Disulfide bonds are located near the plasma membrane. Studies using a cleavable cross-linker indicated the presence of cross-linking sites at the intramembranous or the cytoplasmic part of the molecule.
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PMID:Structural characteristics of the mouse transferrin receptor. 632 90

Periportal and perivenous rat liver parenchymal cells were isolated according to the digitonin-collagenase perfusion method. Affinities and maximal specific binding of a conjugate of glutathione S-transferase and the alpha 2-macroglobulin receptor-associated protein (GST-39kDaP), of lactoferrin and of transferrin to freshly isolated periportal parenchymal cells in vitro were not significantly different from values obtained with perivenous cells. It is concluded that the receptors for these three ligands show a zonally homogeneous expression in rat liver. The zonal homogeneity in binding observed for GST-39kDaP is at variance with the 1.5-fold higher periportal over perivenous binding of trypsin-activated alpha 2-macroglobulin. Since GST-39kDaP as well as trypsin-activated alpha 2-macroglobulin are ligands for the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein, it is suggested that GST-39kDaP can bind to (an) additional receptor(s) with a higher perivenous expression. The zonal homogeneity observed with lactoferrin, an inhibitor of ligand binding to the lipoprotein remnant receptor, may indicate zonal homogeneity of the lipoprotein remnant receptor. The observed zonal homogeneity of the transferrin receptor suggests an equal and essential need for iron by parenchymal cells across the rat liver acinus in vivo.
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PMID:Zonal distribution of receptor binding of trypsin-activated alpha 2-macroglobulin, alpha 2-macroglobulin receptor-associated protein, lactoferrin and transferrin on rat liver parenchymal cells. 754 60

Trophoblast-like BeWo cells form well-polarized epithelial monolayers, when cultured on permeable supports. Contrary to other polarized cell systems, in which the transferrin receptor is found predominantly on the basolateral cell surface, BeWo cells express the transferrin receptor at both apical and basolateral cell surfaces (Cerneus, D.P., and A. van der Ende. 1991. J. Cell Biol. 114: 1149-1158). In the present study we have addressed the question whether BeWo cells use a different sorting mechanism to target transferrin receptors to the cell surface, by examining the biosynthetic and transcytotic pathways of the transferrin receptor in BeWo cells. Using trypsin and antibodies to detect transferrin receptors at the cell surface of filter-grown BeWo cells, we show that at least 80% of newly synthesized transferrin receptor follows a direct pathway to the basolateral surface, demonstrating that the transferrin receptor is efficiently intracellularly sorted. After surface arrival, pulse-labeled transferrin receptor equilibrates between apical and basolateral cell surfaces, due to ongoing transcytotic transport in both directions. The subsequent redistribution takes over 120 min and results in a steady state distribution with 1.5-2.0 times more transferrin receptors at the basolateral surface than at the apical surface. By monitoring the fate of surface-bound 125I-transferrin, internalized either from the apical or basolateral surface transcytosis of the transferrin receptor was studied. About 15% of 125I-transferrin is transcytosed in the basolateral to apical direction, whereas 25% is transcytosed in the opposite direction, indicated that the fraction of receptors involved in transcytosis is roughly twofold higher for the apical receptor pool, as compared to the basolateral pool. Upon internalization, both apical and basolateral receptor pools become redistributed on both surfaces, resulting in a twofold higher number of transferrin receptors at the basolateral surface. Our results indicate that in BeWo cells bidirectional transcytosis is the main factor in surface distribution of transferrin receptors on apical and basolateral surfaces, which may represent a cell type-specific, post-endocytic, sorting mechanism.
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PMID:Bidirectional transcytosis determines the steady state distribution of the transferrin receptor at opposite plasma membrane domains of BeWo cells. 837 59

Neisseria gonorrhoeae is capable of iron utilization from human transferrin in a receptor-mediated event. Transferrin-binding protein 1 (Tbp1) and Tbp2 have been implicated in transferrin receptor function, but their specific roles in transferrin binding and transferrin iron utilization have not yet been defined. We utilized specific gonococcal mutants lacking Tbp1 or Tbp2 to assess the relative transferrin-binding properties of each protein independently of the other. The apparent affinities of the wild-type transferrin receptor and of Tbp1 and Tbp2 individually were much higher than previously estimated for the gonococcal receptor and similar to the estimates for the mammalian transferrin receptor. The binding parameters of both of the mutants were distinct from those of the parent, which expressed two transferrin-binding sites. Tbp2 discriminated between ferrated transferrin and apotransferrin, while Tbp1 did not. Results of transferrin-binding affinity purification, and protease accessibility experiments were consistent with the hypothesis that Tbp1 and Tbp2 interact in the wild-type strain, although both proteins were capable of binding to transferrin independently when separated in the mutants. The presence of Tbp1 partially protected Tbp2 from trypsin proteolysis, and Tbp2 also protected Tbp1 from trypsin exposure. Addition of transferrin to wild-type but not mutant cells protected Tbp1 from trypsin but increased the trypsin susceptibility of Tbp2. These observations indicate that Tbp1 and Tbp2 function together in the wild-type strain to evoke binding conformations that are distinct from those expressed by the mutants lacking either protein.
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PMID:Binding and surface exposure characteristics of the gonococcal transferrin receptor are dependent on both transferrin-binding proteins. 863 22


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