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D Schubert

Publications and source records attributed to D Schubert.

At least 127 records · Page 7Linked to original sources
PubMed1987

Reconstitution of the erythrocyte anion transport system: recent progress.

The anion transport system of the human erythrocyte membrane was reconstituted in phosphatidylcholine vesicles by a new procedure. The reconstituted system shows all major properties of the sulfate transport system found in the erythrocyte membrane (Scheuring, U., K. Kollewe, W. Haase, and D. Schubert: J. Membrane Biol. 90, 123-135 (1986)). Modifications of the reconstitution method have now led to increased values of the internal volumes of the protein-lipid vesicles and of the average turnover number of the transport protein (which is now larger than in the erythrocyte membrane). Gel filtration on Sephacryl S-1000 has allowed the isolation of vesicles of a narrow size distribution.

Anions↗
PubMed1986

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane.

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. The sample was dialyzed overnight against detergent-free buffer. Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30 degrees C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100 mM, inhibition of the flux by H2DIDS and flufenamate (approx. KI-values at 30 degrees C: 0.1 and 0.7 microM, respectively), and "right-side-out" orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H2DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

A chick neural retina adhesion and survival molecule is a retinol-binding protein.

A 20,000-D protein called purpurin has recently been isolated from the growth-conditioned medium of cultured embryonic chick neural retina cells (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 101:1071-1077). Purpurin is a constituent of adherons and promotes cell-adheron adhesion by interacting with a cell surface heparan sulfate proteoglycan. It also prolongs the survival of cultured neural retina cells. This paper shows that purpurin is a secretory protein that has sequence homology with a human protein synthesized in the liver that transports retinol in the blood, the serum retinol-binding protein (RBP). Purpurin binds [3H]retinol, and both purpurin and chick serum RBP stimulate the adhesion of neural retina cells, although the serum protein is less active than purpurin. Purpurin and the serum RBP are, however, different molecules, for the serum protein is approximately 3,000 D larger than purpurin and has different silver-staining characteristics. Finally, purpurin supports the survival of dissociated ciliary ganglion cells, indicating that RBPs can act as ciliary neurotrophic factors.

Amino Acid Sequence↗
PubMed1986

Protein complexity of central nervous system cell lines.

The total extracellular proteins and the most abundant 960 intracellular proteins of clonal CNS nerve and glial cell lines were examined by quantitative 2-dimensional acrylamide gel electrophoresis. While less than 0.2% of the intracellular proteins differ among the 5 nerve and 4 glial cell lines studied, over 65% of the extracellular proteins vary in distribution between the 2 major classes of CNS cells. These data indicate that the phenotypic complexity of nerve and glia populations is similar and that most of the protein complexity is in extracellular molecules.

Cell Line↗
PubMed1985

Induction of the proto-oncogene fos by nerve growth factor.

Nerve growth factor (NGF) causes the differentiation of PC12 cells to sympathetic neuron-like cells and also induces a rapid but transient expression of fos mRNA and protein. fos mRNA transcripts can be detected 5 min after the addition of NGF, are maximally abundant after 30 min, and then their levels decrease. fos protein synthesis parallels the expression of fos mRNA, and the induced fos proteins are located in the nucleus. cAMP, epidermal growth factor, the phorbol ester phorbol 12-myristate 13-acetate, and K+ depolarization also induce the fos gene. Growth of PC12 cells in the presence of dexamethasone, which induces differentiation into chromaffin-like cells, is not accompanied by fos expression. We propose that while fos gene induction is associated with the differentiation of PC12 cells to sympathetic nerve, its enhanced expression is primarily involved in the anabolic responses induced by NGF and many growth factors.

Animals↗
PubMed1985

Ontogeny of electrically excitable cells in cultured olfactory epithelium.

A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuron-specific enolase but not GFAP. These round cells originate by means of the differentiation of the GFAP-positive flat cell to a round cell, followed by the division of the round cell. Therefore, neuron-like cells can be derived from cells that synthesize GFAP.

Action Potentials↗
PubMed1985

Isolation of a cell-surface receptor for chick neural retina adherons.

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion. In solution they increase the rate of cell-cell aggregation. Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process. We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it. Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation. An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface. This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion. These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).

Animals↗
PubMed1985

Cell-substratum adhesion in chick neural retina depends upon protein-heparan sulfate interactions.

Embryonic chick neural retina cells in culture release complexes of proteins and glycosaminoglycans, termed adherons, which stimulate cell-substratum adhesion when adsorbed to nonadhesive surfaces. Two distinct retinal cell surface macromolecules, a 170,000-mol-wt glycoprotein and a heparan sulfate proteoglycan; are components of adherons that can independently promote adhesion when coated on inert surfaces. The 170,000-mol-wt polypeptide contains a heparin-binding domain, as indicated by its retention on heparin-agarose columns and its ability to bind [3H]heparin in solution. The attachment of embryonic chick retinal cells to the 170,000-mol-wt protein also depends upon interactions between the protein and the heparan sulfate proteoglycan, since heparan sulfate in solution disrupts adhesion of chick neural retina cells to glass surfaces coated with the 170,000-mol-wt protein. This adhesion is not impaired by chondroitin sulfate or hyaluronic acid, which indicates that inhibition by heparan sulfate is specific. Polyclonal antisera directed against the cell surface heparan sulfate proteoglycan also inhibit attachment of retinal cells to the 170,000-mol-wt protein, which suggests that cell-adheron binding is mediated in part by interactions between cell surface heparan sulfate proteoglycan and 170,000-mol-wt protein contained in the adheron particles. Previous studies have indicated that this type of cell-substratum adhesion is tissue-specific since retina cells do not attach to muscle adherons. Schubert D., M. LaCorbiere, F. G. Klier, and C. Birdwell, 1983, J. Cell Biol. 96:990-998.

Animals↗
PubMed1985

Isolation of an adhesion-mediating protein from chick neural retina adherons.

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.

Animals↗
PubMed1984

Formation and properties of tetramers of band 3 protein from human erythrocyte membranes in planar lipid bilayers.

Lipid bilayer experiments were performed in the presence of solubilized band 3 protein from human red cell membranes. Band 3 protein increased the conductance of the lipid membranes by several orders of magnitude. Membrane conductance was found to be dependent on the fourth power of protein concentration. This shows that four band 3 subunits form an ion permeable pathway in the lipid bilayer membranes. It also shows that, in the membranes, the protein molecules undergo an association equilibrium which involves at least the monomer and the tetramer of the protein, relaxation towards equilibrium being rapid on the time scale of the experiment. The increase in bilayer conductance induced by the band 3 tetramer could be inhibited by pretreatment of the protein with several SH-reagents (pCMB, pCMBS, DTNB) which also inhibit water transport across the human red cell membrane. Other SH-reagents which do not influence water transport (iodoacetamide, N-ethylmaleimide) did not show any influence on the band 3 induced conductance increase. A band 3-mediated exchange of anions comparable to that in the erythrocyte membrane did not occur in the system studied by us. Our results suggest that, in the human erythrocyte membrane, a pore formed by the band 3 tetramer could be the pathway responsible for the protein-mediated part of water transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

An ultracentrifuge study on the self-association of glucose dehydrogenase from Bacillus megaterium.

The self-association of glucose dehydrogenase (beta-D-glucose:NAD(P) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium was studied by analytical ultracentrifugation. The pH and composition of the buffer used were such that, owing to a reversible partial dissociation of the tetrameric enzyme, enzyme activity was reduced. It was found that under these conditions the protein exists in a monomer/dimer/tetramer association equilibrium.

Bacillus megaterium↗
PubMed1984

Phospholipases elevate cyclic AMP levels and promote neurite extension in a clonal nerve cell line.

Phospholipases A2 and C increase choline acetyltransferase (CAT) activity and promote neurite extension in PC12 cells. Since these phospholipases also elevate intracellular cyclic AMP, they probably stimulate CAT and neurite outgrowth through their ability to increase cyclic AMP levels. Other possible modes of action of phospholipases such as the release of arachidonic acid or the production of lysolipids are less likely to play a role in this system.

Animals↗
PubMed1983

The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100.

Stable noncovalent dimers of band 3 protein from human erythrocyte membranes, in which state the protein is thought to exist after solubilization by the nonionic detergent Triton X-100, do not occur when purified batches of the detergent are used. Instead, the protein is in a monomer/dimer/tetramer association equilibrium. The stable dimers do appear, however, when the detergent has been 'aged'. They thus seem to be artifacts.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

The state of association of band 3 protein of the human erythrocyte membrane in solutions of nonionic detergents.

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was solubilized and purified in aqueous solutions of two nonionic detergents: Ammonyx-LO (dimethyl laurylamine oxide) and C12E9 (nonaethylene glycol lauryl ether). The state of association of the purified protein was studied by analytical ultracentrifugation. Band 3 protein solubilized and studied in solutions of Ammonyx-LO was found to be in a monomer/dimer/tetramer association equilibrium. Band 3 protein freshly prepared in C12 E9 showed the same behaviour; however, during aging the protein was converted into stable noncovalent dimers. The conversion was retarded by the presence of beta-mercaptoethanol or by treatment of the samples with iodoacetamide; it seems to be due to oxidation of the protein by degradation products of the detergent. It is concluded that a monomer/dimer/tetramer association equilibrium is the native state of association of band 3 protein solubilized by nonionic detergents. Since nonionic detergents are assumed not to interfere with protein-protein interactions among membrane proteins, the results strongly support the claim that, in the erythrocyte membrane, band 3 is in a monomer/dimer/tetramer association equilibrium (Dorst, H.-J. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1605-1618).

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

A role for adherons in neural retina cell adhesion.

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion; they also stimulate the rate of cell-cell aggregation. Adheron-stimulated adhesion is tissue specific, and the spontaneous aggregation of neural retina cells is inhibited by monovalent Fab' fragments prepared from an antiserum against neural retina adherons. Therefore cell surface antigenic determinants shared with adherons are involved in normal cell-cell adhesions. The particles from the heterogeneous neural retina population contain many proteins and several glycosaminoglycans. The adherons migrate as a symmetrical 12S peak on sucrose gradients and are predominantly 15-nm spheres when examined by electron microscopy. Finally, the specific activity of neural retina adherons increases from embryonic days 7 through 12 and then declines. These results suggest that glycoprotein particles may be involved in some of the adhesive interactions between neural retina cells and between the cells and their environment.

Animals↗