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Biomedical subjects

G Ashwell

Publications and source records attributed to G Ashwell.

At least 37 records · Page 2Linked to original sources
PubMed1986

Quantitative determination of intracellular, ferritin-associated radioactive iron by high-performance liquid chromatography and immunoprecipitation.

Antibodies raised against ferritin preparations of diverse origin provide an uncertain reagent for quantitation of the ferritin present in specific cell lysates. Utilizing K562 cells, a human leukemic cell line, techniques are described to resolve and to quantitate the ferritin-bound cytosolic iron. Processing the cell lysates by HPLC employing an anion-exchange or hydrophobic interaction column resulted in recovery of a single, ferritin-containing radioactive peak widely separated from the bulk of the non-ferritin-bound iron. Comparison of the yield obtained by chromatography with that by immunoprecipitation confirmed both the specificity and the quantitation of the antibody technique.

Binding Sites↗
PubMed1984

Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand-receptor complex.

Rat hepatocytes in monolayer culture rapidly internalized asialoglycoproteins and the receptors to which they are bound. Subsequent to endocytosis, the receptor-ligand complex is dissociated within an acidic endosome (Harford, J., K. Bridges, G. Ashwell, and R. D. Klausner, 1983, J. Biol. Chem. 258:3191-3197; Harford, J., A. W. Wolkoff, G. Ashwell, and R. D. Klausner, 1983, J. Cell Biol. 96:1824-1828). Here we show that addition of the proton ionophore monensin to the cells after dissociation has occurred results in intracellular rebinding of ligand molecules. With increasing time inside the cell, the ability of ligand to reassociate with receptor progressively decreases consistent with a segregation of receptor and ligand. The combination of colchicine and cytochalasin B appears to retard the process of segregation. In contrast, removal of sodium from the medium, while inhibiting degradation of ligand, does not affect the decrease in monensin-mediated rebinding. Nonetheless, both sodium deprivation and treatment with colchicine plus cytochalasin B result in the ligand remaining in a low density, nonlysosomal subcellular fraction. Thus, segregation, like dissociation, appears to occur in a pre-lysosomal endocytic compartment. Perturbation of the endocytic pathway by reduced temperature (18 degrees C) was also explored. Our data are consistent with two temperature-sensitive steps: receptor-ligand dissociation is inhibited and there is an independent temperature-sensitive step involved in delivery of ligand to lysosomes. This second effect was localized as being beyond the point in the pathway sensitive to sodium deprivation.

Animals↗
PubMed1984

Inhibition of the endocytic pathway for asialoglycoprotein catabolism.

Rat hepatocytes in primary culture bind, internalize and eventually degrade asialoglycoproteins. This process is mediated by a specific receptor in the hepatocyte plasma membrane. The endocytic pathway by which ligand molecules are translocated to lysosomes has been examined by the development of biological assays capable of distinguishing ligand populations at various points in the process. Inhibitors have been identified that perturb particular transitions that define the endocytic pathway. In the present paper, inhibition by the bacterial tripeptide leupeptin is compared to the effect of colchine plus cytochalasin B. The latter combination impedes intracellular segregation of ligand and receptor while leupeptin inhibits intralysosomal proteolysis. However, evidence is presented to indicate that the inhibitory effects colchine plus cytochaasin B consists of at least two components. One component is independent of the presence of ligand whereas the other is observed only when ligand is present together with the drugs.

Animals↗
PubMed1983

The binding site of chicken hepatic lectin.

The binding site of the chicken hepatic lectin involved in the clearance of N-acetylglucosamine-terminated serum glycoproteins was explored by a competitive binding assay using 3H-labeled agalacto-orosomucoid and various glycoproteins, polysaccharides, monosaccharides, and glycosides as inhibitors. The binding site is relatively small, involving a terminal nonreducing DGlcNAc structure with an equatorial N-acetamido group on carbon 2 and an equatorial hydroxyl group on carbon 4. Among the mono- and oligosaccharides tested, benzyl alpha DGlcNAc was the best inhibitor, being three times as effective as DGlcNAc; and in general, all alpha-anomeric glycosides were better than beta-glycosides. All oligosaccharides with terminal nonreducing beta DGlcNAc have almost the same inhibitory power, whereas those with nonreducing DGlc or DGal were relatively inactive. Among the serum and blood group glycoproteins, a Smith degraded human H substance with several exposed terminal nonreducing beta DGlcNAc residues was the most active and twice as effective as agalacto-orosomucoid and an A substance, Hog 75 10% precipitate. Almost all hog preparations, some with A or with H activity, were equally effective. A glycopeptide with terminal DGlcNAc was twice as active as one with terminal nonreducing DMan and DGlcNAc residues and almost three times as potent as one with terminal nonreducing DGal; a glycopeptide with terminal sialic acid was inactive. The slopes of the inhibition lines differed, reflecting the heterogeneity of the various determinant groups on the glycoproteins.

Animals↗
PubMed1983

Receptor-mediated endocytosis of transferrin in K562 cells.

Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.

Ammonium Chloride↗
PubMed1983

Intracellular dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. A pH-mediated nonlysosomal event.

The binding, internalization, and degradation of 125I-asialo-orosomucoid were studied in primary monolayer cultures of rat hepatocytes. Ligand entered the cell bound to the asialoglycoprotein receptor and subsequently dissociated from the receptor intracellularly. Rate coefficients for each of the transitions that constitute the endocytic pathway were computed. Subcellular fractionation on Percoll gradients revealed that prior to localization in lysosomes, 125I-asialo-orosomucoid resided in a fraction of slightly lower buoyant density than plasma membranes. Neither ammonium chloride (20 mM) nor leupeptin (0.1 mg/ml) affected ligand binding or internalization of prebound ligand. However, both reagents inhibited degradation of ligand by greater than 95%. Of the two, only ammonium chloride inhibited receptor-ligand dissociation. Ammonium chloride treatment resulted in the accumulation of ligand in the prelysosomal fraction. In contrast, exposure of cells to leupeptin led to accumulation of ligand within lysosomes. The results are interpreted in terms of pH-mediated dissociation of ligand-receptor complex within a nonlysosomal endocytic vesicle.

Animals↗
PubMed1983

Binding of apotransferrin to K562 cells: explanation of the transferrin cycle.

The binding of apotransferrin to the transferrin receptor on the surface of human leukemic K562 cells was found to be significantly less tight than that of the holoprotein, diferric transferrin. The finding that both ligands displayed linear Scatchard plots with similar receptor number (approximately equal to 150,000 per cell) and mutually inhibit each other's binding suggested that they bind to the same receptor. Both the dissociation and association rate of apotransferrin were markedly increased (28-fold and 15-fold, respectively) at pH 7.2 compared to pH 4.8. Using the values of these binding parameters, we propose a mechanism to account for the recycling of transferrin subsequent to internalization and residence within an acidic nonlysosomal organelle where iron is removed.

Apoproteins↗
PubMed1983

Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor.

Treatment of short-term monolayer cultures of rat hepatocytes with the proton ionophore, monensin, abolishes asialoglycoprotein degradation, despite little effect of the drug on either surface binding of ligand or internalization of prebound ligand. Centrifuging cell homogenates on Percoll density gradients indicates that, as a result of monensin treatment, ligand does not enter lysosomes but sediments instead in a lower density subcellular fraction that is likely an endocytic vesicle. Analyzing the degree of receptor association of intracellular ligand revealed that monensin prevents the dissociation of the receptor-ligand complex that normally occurs subsequent to endocytosis. The weak base, chloroquine, also blocks this intracellular dissociation. Evidence from sequential substitution experiments is presented, indicating that monensin and chloroquine act at the same point in the sequence of events leading to ligand dissociation. These data are discussed in terms of a pH-mediated dissociation of the receptor-ligand complex within a prelysosomal endocytic vesicle.

Animals↗
PubMed1982

Immunological approaches to the study of membrane receptors. A monoclonal antibody that inhibits the binding of asialoglycoproteins to the rat liver receptor.

The major polypeptide (43,000 daltons) of the rat liver receptor for asialoglycoproteins was isolated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Antibodies raised in a goat against this SDS-treated polypeptide exhibited marked cross-reactivity toward the SDS-denatured forms of the two other prominent polypeptides (54,000 and 64,000 daltons) of the receptor preparation. Monoclonal antibodies directed against the receptor were prepared using the spleen cells of mice immunized with the soluble, active receptor purified by affinity chromatography. The most extensively characterized of the monoclonal antibodies, designated D3-5D3, recognized the solubilized receptor and bound to the exterior surface of isolated rat hepatocytes. The binding of D3-5D3 to hepatocytes prevented subsequent binding of the ligand, 125I-asialo-orosomucoid. Conversely, occupation of the receptor with ligand inhibited binding of 125I-IgG prepared from D3-5D3 ascites fluid. The secondary structure of the receptor appears to be critical for recognition by D3-5D3, since denaturation of the receptor with 1% SDS, 5% beta-mercaptoethanol at 100 degrees C abolished antibody binding. Under less denaturing conditions (0.1% SDS, 25 degrees C), antigenic reactivity was retained by the receptor. Preparative electrophoresis using the latter conditions permitted the demonstration that D3-5D3 recognized a unique determinant that is present in each of the three polypeptides.

Animals↗
PubMed1982

Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes.

The endocytosis leading to degradation of 125I-labeled asialo-orosomucoid specifically bound to the surface of freshly isolated hepatocytes was monitored as a function of time at 37 degrees C. Experimental values were determined for the rates of internalization, dissociation of the receptor-ligand complex, and degradation of the labeled ligand. Compartmental analysis and computer modeling revealed that the data were compatible with dissociation of ligand from receptor preceding ligand degradation. The rate coefficient for internalization was calculated to be an order of magnitude greater than that for receptor--ligand dissociation. Ligand internalization did not result in concomitant depletion in the total number of cell surface receptors. Our data are taken to indicate that ligand remains associated with the receptor after internalization, that the complex is dissociated prior to degradation, and that new, unoccupied receptors are promptly returned to the cell surface from an internal pool.

Animals↗
PubMed1981

Molecular size of the hepatic receptor for asialoglycoproteins determined in situ by radiation inactivation.

Radiation inactivation was used to determine the functional molecular size of the rat liver membrane protein which binds desialylated glycoproteins. Purified plasma membranes from rat liver were irradiated with high energy electrons from a linear accelerator and then assayed for 125I-asialo-orosomucoid binding activity. Target size analysis of the data revealed that increasing doses of ionizing radiation from 1-48 megarads resulted in a monoexponential decay in binding activity due to a decrease in the number of available binding sites; dissociation and binding affinity were unaffected. The molecular weight of the rat binding protein, determined in situ by target analysis, was 104,000 +/- 17,000; that of the rabbit binding protein was 109,000 +/- 5,000. Comparison of the value obtained by irradiation of the intact rat plasma membrane with that of the purified receptor revealed the latter to have an apparent molecular weight of 148,000 +/- 16,000. Evidence is presented to indicate that the observed increase in target size was a response to the presence of Triton X-100 used in the solubilization and assay procedure. In contrast to the size of the ligand binding functional unit, the antireceptor antibody binding site was estimated to be 30,000 +/- 2,000.

Animals↗
PubMed1980

Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes.

Freshly isolated rat hepatocytes attained maximal ability to bind, internalize, and degrade 125I-asialo-orosomucoid after 5 h in suspension culture at 37 degrees C. Comparison of the number and distribution of the asialoglycoprotein binding sites of these cells revealed that 5% (6.7 x 10(4) receptors/cell) were on the external cell membrane with an average residency time of slightly less than 3 min. The remaining 95% were located intracellularly, as determined with detergent-solubilized hepatocytes. Binding of ligand (asialo-orosomucoid) was time-dependent, saturable, and dissociable. The dissociation constant for the single high affinity binding site was calculated to be 3.4 x 10(-8) M. The amount of asialo-orosomucoid metabolized by these cells over a period of 3 h at 37 degrees C was reduced 50% by the inclusion of 1 mM cycloheximide in the incubation medium. However, even in the absence of protein synthesis, 34 times more asialo-orosomucoid was metabolized than could be bound by the cell surface receptors, or twice the total capacity of the intact hepatocyte. These results provide clear evidence for the stability of the binding receptor under conditions where the ligand is being continually destroyed and support the previously proposed recycling hypothesis (Tanabe, T., Pricer, W.E., Jr., and Ashwell, G. (1979) J. Biol. Chem. 254, 1038-1043).

Animals↗
PubMed1980

Reconstitution of the hepatic asialoglycoprotein receptor with phospholipid vesicles.

A solubilized detergent-free preparation of the hepatic binding protein specific for asialoglycoproteins associates spontaneously with small unilamellar lipid vesicles. This process is independent of the phase transition of the lipid and effectively restores the specific binding activity of the receptor protein. The insensitivity of the resulting lipid-protein complex to ionic strength provides evidence for a hydrophobic interaction. There is a perturbation of the lipid phase transition concomitant with addition of the protein. Circular dichroism studies indicate that the protein undergoes a conformational change on association with lipid. Binding of specific ligand produces further physical changes in the receptor as indicated by alterations in the tryptophan fluorescence quenching pattern.

Animals↗