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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
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↗
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↗
PubMed1977

Interpreting the results of freeze-etching.

Morphological data obtained by freeze-fracturing and other low temperature techniques must be interpreted in terms of molecular organization and function. Interpretation is aided by physical and biochemical approaches. Physical approaches such as rotary replication and ultralow temperature fracturing can improve resolution and preserve molecular arrangements which are difficult or impossible to observe with standard freeze-etching techniques. Biochemical approaches such as dissociation-reconstitution experiments can establish the molecular parameters underlying electron-microscopically visible forms. Both approaches are illustrated by investigations of the human erythrocyte membrane.

Erythrocyte Membrane↗
PubMed1977

Intramembrane particle aggregation in erythrocyte membranes and band 3-lipid recombinants.

The low pH-induced aggregation of intramembrane particles in human erythrocyte membranes was studied in native membranes and in a reconstituted model system. A significant difference in such aggregation was found when samples of freshly prepared ghosts were compared to ghosts receiving pretreatments that removed most of spectrin-actin from underneath the membrane. All conditions effective in aggregating particles are equally effective in precipitating extracted mixtures of spectrin and actin. In Band 3-lipid recombinants, the pH-induced aggregation of particles was duplicated only in samples containing spectrin-actin that equilibrated with these recombinants in sucrose gradients. Therefore, it was proposed that spectrin-actin components, through their associations with the underlying intramembrane particles, could impede particle lateral mobility and also determine particle redistribution in erythrocyte membrane.

Actins↗
PubMed1976

Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation.

Physicochemical properties of mixtures of spectrin and actin extracted from human erythrocyte ghosts have been correlated with ultrastructural changes observed in freeze-fractured erythrocyte membranes. (1) Extracted mixtures of spectrin and actin have a very low solubility (less than 30 mug/ml) near their isoelectric point, pH 4.8. These mixtures are also precipitated by low concentrations of Ca2+, Mg2+, polylysine or basic proteins. (2) All conditions which precipitate extracts of spectrin and actin also induce aggregation of the intramembrane particles in spectrin-depleted erythrocyte ghosts. Precipitation of the residual spectrin molecules into small patches on the cytoplasmic surface of the ghost membrane is thought to be the cause of particle aggregations, implying an association between the spectrin molecules and the intramembrane particles. (3) When fresh ghosts are exposed to conditions which precipitate extracts of spectrin and actin, only limited particle aggregation occurs. Instead, the contraction of the intact spectrin meshwork induced by the precipitation conditions compresses the lipid bilayer of the membrane, causing it to bleb off particle-free, protein-free vesicles. (4) The absence of protein in these lipid vesicles implies that all the proteins of the erythrocyte membrane are immobilized by association with either the spectrin meshwork or the intramembrane particles.

Actins↗
PubMed1976

Reconstitution of intramembrane particles in recombinants of erythrocyte protein band 3 and lipid: effects of spectrin-actin association.

The integral membrane protein Band 3 of the human erythrocyte, either purified or in a crude Triton X-100 extract of ghosts, was combined with egg lecithin in a cholate solution. During dialysis to remove cholate, lipid bilayer vesicles formed in which Band 3 existed as a dimer and in which intramembrane particles indistinguishable from those in the native membrane were exposed by freeze-fracturing. The recombinant vesicles were stable in both high and low salt concentrations, sedimented at a density that increased in prportion to their protein content, and bound spectrin-actin extracted from erythrocyte ghosts. When spectrin-actin was associated with the vesicles, the behavior of the recombinant intramembrane particles simulated that of the erythrocyte ghost intramembrane particles: they were dispersed at pH 7.6 and aggregrated at pH 5-5.5. Thus, some of the characteristics of the native membrane have been reconstituted in the recombinant.

Actins↗
PubMed1976

Freeze-fracture autoradiography: feasibility.

We have shown that the combination of freeze-fracture with electron microscope autoradiography can be developed into a technique for correlating the molecular structure of the biological membrane with its chemical and functional characteristics. Within the limits of electron microscope autoradiographic resolution, FARG has the potential to detect the relative distribution of molecules in each half of the membrane and within the plane of the membrane. The use of radioisotopic labels in combination with freezing techniques requires minimal perturbation of the system being studied and may be suitable for the examination of substances which would be extracted or would diffuse during the normal fixation and embedding procedures used in standard electron microscope autoradiography.

Autoradiography↗
PubMed1974

Intramembrane particle aggregation in erythrocyte ghosts. I. The effects of protein removal.

We have used freeze-etching and SDS-polyacrylamide gel electrophoresis to study the conditions under which the intramembrane particles of the human erythrocyte ghost may be aggregated. The fibrous membrane protein, spectrin, can be almost entirely removed from erythrocyte ghosts with little or no change in the distribution of the particles. However, after spectrin depletion, particle aggregation in the plane of the membrane may be induced by conditions which cause little aggregation in freshly prepared ghosts. This suggests that the spectrin molecules form a molecular meshwork which limits the translational mobility of the erythrocyte membrane particles.

Blood Proteins↗