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Biomedical subjects

D Branton

Publications and source records attributed to D Branton.

At least 91 records · Page 5Linked to original sources
PubMed1980

Analytical characterization of beetroot vacuole membrane.

Vacuoles from beetroot (Beta vulgaris L. var. esculenta Gürke) isolated by a mechanical procedure were osmotically lysed to separate the membrane and sap components for analysis. Approximately 62% of the vacuole proteins, 70% of the nondialyzable carbohydrates and almost all of the phospholipids and sterols were recovered in the membrane fraction. The vacuole membrane had a phospholipid protein ratio of 0.68 and a sterol:phospholipid ratio of 0.21. 17 complex polar lipids including phosphatides and glycolipids have been tentatively identified. Phosphatidylcholine (54%) and phosphatidylethanolamine (24%) were the most prominent phosphoglycerides besides phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid (1, 4, 5, and 12%, respectively). A putative sulfoglycoside and two major ceramide glycoside-like lipids, resembling those of animal lysosomes, were identified by thin-layer chromatography. High-resolution SDS-acrylamide gel electrophoresis of the polypeptides from the vacuole revealed 15 major bands with apparent molecular weights ranging from 91,000 to 12,000. Selective elution experiments delineated those polypeptides that were peripheral membrane proteins or sap proteins adsorbed to the membrane, and those that exhibited hydrophobic interactions with the lipid core. Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification.

Cell Fractionation↗
PubMed1979

Retention of lipid asymmetry in membranes on polylysine-coated polyacrylamide beads.

Phosphatidylcholine-specific exchange protein from calf liver was used to study the asymmetry and transmembrane movement of phosphatidylcholine in rat erythrocyte membranes isolated on polylysine-coated beads. While confirming previously published results for sealed ghosts, we found that for membranes attached to beads, where the cytoplasmic surface is exposed, about 36% of the total phosphatidylcholine is readily available for exchange, while the remaining 64% is exchangeable at a much slower rate. This indicates that the relative transbilayer asymmetry of phosphatidylcholine is largely maintained when red cell membranes are isolated on beads. On the other hand, transmembrane movement of phosphatidylcholine is decreased in membranes attached to cationized beads: the half-time for equilibration of phosphatidylcholine between the two monolayers of the membrane is 8 h for membranes on beads, compared to 1.5 h for sealed ghosts. Our results indicate that polylysine-derivatized beads are a useful tool for studying asymmetric properties of biological membranes.

Acrylamides↗
PubMed1979

Identification by peptide analysis of the spectrin-binding protein in human erythrocytes.

One-dimensional and two-dimensional peptide-mapping techniques are used to identify the protein which gives rise to the 72,000 dalton alpha-chymotryptic fragment previously shown to be the membrane attachment site for spectrin. Peptide maps of the 72,000 dalton fragment are very different from maps of Bands 1, 2, 2.9, 3, 3.1, 4.1, and 4.2 and very similar to maps of the apparently closely homologous polypeptides, Bands 2.1, 2.2, 2.3, and 2.6. Limited proteolysis of erythrocyte membranes is shown to generate Band 3', another polypeptide which has been associated with spectrin-binding activity. Peptide maps of Band 3' are very similar to maps of Band 2.1, suggesting that Band 3' is also a proteolytic fragment of Band 2.1. It is concluded that Band 2.1 and possibly some or all of the other, related polypeptides which electrophorese in the 2 region is (are) the spectrin-binding protein(s) of the human erythrocyte.

Carrier Proteins↗
PubMed1979

Purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1.

Two peripheral proteins of the human erythrocyte membrane that are capable of forming a stable complex with spectrin have been purified. The proteins, band 2.1 (Mr 210,000) and band 4.1 (Mr 82,000), are water soluble and exist as monomers in solution. Both exhibit strong, specific binding to purified spectrin molecules as determined by cosedimentation in sucrose gradients and both enhance binding to spectrin-depleted, inside-out vesicles that have been stripped of bands 2.1 and 4.1. Rotary replicas of bound material reveal site-specific associations among native, but not heat-denatured, molecules.

Binding Sites↗
PubMed1978

The shape of spectrin molecules from human erythrocyte membranes.

Purified spectrin dimers and tetramers have been directly visualized by low-angle shadowing. The 9-S heterodimer is an asymmetric flexible molecule about 1000 A in length, its constituent monomer polypeptides forming two strands which in many molecules are individually visible, lying partially separated from one another or twisting round each other in a loose double helix. The 12-S tetramer is formed by the end-to-end association of two heterodimers, without overlap. The protein bears no physical resemblance to myosin.

Erythrocyte Membrane↗
PubMed1978

Coupling polylysine to glass beads for plasma membrane isolation.

Solid glass beads for use in isolating cell membranes were coated with a stable, covalently attached layer of polylysine. The optimal conditions for coating the bead surface were established and the beads were tested by measuring the attachment of human erythrocyte plasma membranes. When compared to other beads, such as those with absorbed polylysine or protamine, none retained red-cell membranes as well as glass beads with covalently linked polylysine.

Adsorption↗
PubMed1978

Membrane isolation on polylysine-coated glass beads. Asymmetry of bound membrane.

Erythrocyte membranes isolated on polylysine-coated glass beads exhibit many of the properties of the native membrane. Gel electrophoresis indicates that all major protein components of the membrane are retained during membrane isolation. The membrane integrity and accessibility of selected components was tested using non-penetrating probes. In general, membranes on beads displayed accessibility properties typical of inside-out vesicles. The accessibility of membrane acetylcholinesterase to assay reagents, as well as membrane accessibility to the actions of neuraminidase, trypsin and galactose oxidase-NaB3H4 demonstrated that the protoplasmic surface of membrane isolated on beads was exposed, while the extracellular surface was inaccessible. The differential accessibility of the membrane surfaces demonstrates the feasibility of investigating asymmetry of membranes isolated on cationic glass beads.

Acetylcholinesterase↗
PubMed1978

Spectrin binding and the control of membrane protein mobility.

Transmembrane proteins of the human erythrocyte show restricted in-plane mobility. Many of the restrictions on mobility are attributable to the molecules of spectrin which are located on the protoplasmic surface of the erythrocyte membrane. These molecules are elongate, form end-to-end heterodimer associations, and bind selectively to protein (or proteins) accessible on inside-out, but not right-side out, membrane vesicles.

Actins↗
PubMed1978

Actin--membrane interactions: association of G-actin with the red cell membrane.

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.

Actins↗
PubMed1978

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study.

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.

Erythrocyte Membrane↗
PubMed1977

Membrane polypeptides co-induced with the bacterial bioluminescent system.

Electrophoretic analysis of membrane proteins and electron microscopy of freeze-fracture replicas reveal that certain polypeptides and intramembrane particles are absent in Beneckea harveyi harvested prior to luminescence induction but present after induction. The polypeptides and particles are also absent in mutants which fail to synthesize the luminescent system. These correlations implicate membrane polypeptides in the bacterial bioluminescent system.

Cell Membrane↗
PubMed1977

Lateral mobility of human erythrocyte integral membrane proteins.

Fluorescein isothiocyanate-labelled integral membrane proteins are mobile in the membranes of human erythrocytes that have fused (and haemolysed) by Sendai virus or polyethylene glycol. Minimum diffusion coefficients are of the order of 10(-11) cm2 s-1 at 37 degrees C. This mobility is reduced several-fold at room temperature, not detected at 0 degrees C, and is significantly greater in fresh than in aged blood. Mobility was assessed by observing the spread of fluorescence on labelled cells which had been fused with unlabelled cells; neither intramembrane particle aggregation nor spectrin release occurred during this process.

Adenosine Triphosphate↗
PubMed1977

Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin.

A specific association between spectrin and the inner surface of the human erythrocyte membrane has been examined by measuring the binding of purified [32P]spectrin to inside out, spectrin-depleted vesicles and to right side out ghost vesicles. Spectrin was labeled by incubating erythrocytes with 32Pi, and eluted from the ghost membranes by extraction in 0.3 mM NaPO4, pH 7.6. [32P]Spectrin was separated from actin and other proteins and isolated in a nonaggregated state as a So20,w = 7 S (in 0.3 mM NaPO4) or So20,w = 8 S (in 20 mM KCl, 0.3 mM NaPO4) protein after sedimentation on linear sucrose gradients. Binding of [32P]spectrin to inverted vesicles devoid of spectrin and actin was at least 10-fold greater than to right side out membranes, and exhibited different properties. Association with inside out vesicles was slow, was decreased to the value for right side out vesicles at high pH, or after heating spectrin above 50 degrees prior to assay, and was saturable with increasing levels of spectrin. Binding to everted vesicles was rapid, unaffected by pH or by heating spectrin, and rose linearly with the concentration of spectrin. Scatchard plots of binding to inverted vesicles were linear at pH 7.6, with a KD of 45 microng/ml, while at pH 6.6, plots were curvilinear and consistent with two types of interactions with a KD of 4 and 19 microng/ml, respectively. The maximal binding capacity at both pH values was about 200 microng of spectrin/mg of membrane protein. Unlabeled spectrin competed for binding with 50% displacement at 27 microng/ml. [32P]Spectrin dissociated and associated with inverted vesicles with an identical dependence on ionic strength as observed for elution of native spectrin from ghosts. MgCl2, CaCl2 (1 to 4 mM) and EDTA (0.5 to 1 mM) had little effect on binding in the presence of 20 mM KCl, while at low ionic strength, MgCl2 (1 mM) increased binding and inhibited dissociation to the same extent as 10 to 20 mM KCl. Binding was abolished by pretreatment of vesicles with 0.1 M acetic acid, or with 0.1 microng/ml of trypsin. The periodic acid-Schiff-staining bands were unaffected by trypsin digestion which destroyed binding; mild digestion, which decreased binding only 50%, converted Band 3 almost completely to a membrane-bound 50,000-dalton fragment resistant to further proteolysis. These experiments suggest that attachment of spectrin to the cytoplasmic surface of the membrane results from a selective protein-protein interaction which is independent of erythrocyte actin. A direct role of the major sialoglycoprotein or Band 3 as a membrane binding site appears unlikely.

Binding Sites↗
PubMed1977

Rotary replication for freeze-etching.

Rotary replication has been adapted to freeze-etching and evaluated using T4 polyheads, erythrocyte ghosts, and chloroplast membranes. Conventional electron microscopy, electron diffraction, and optical diffraction and filtering indicate that platinum-carbon rotary replication renders radially symmetrical contrast and 25 A resolution to freeze-etched specimens so as to clarify subunit structure not normally evident in unidirectional shadow replicas.

Carbon↗
PubMed1977

Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

Cell Count↗