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D Branton

Publications and source records attributed to D Branton.

At least 55 records · Page 3Linked to original sources
PubMed1982

A stable, high capacity, F-actin affinity column.

A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds 125I-heavy meromyosin and 125I-tropomyosin. The associations between the F-actin affinity matrix and the iodinated F-actin binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

Actins↗
PubMed1982

Mapping functional sites on biological macromolecules.

Electron microscopy of macromolecules dried from glycerol and rotary shadowed from a low angle can reveal the structure of individual molecules, or groups of molecules, with remarkable clarity. We used this technique to examine the interaction of the red blood cell cytoskeletal proteins spectrin, a 500,000 dalton protein which is long (750 A) and flexible;actin, a 43,000 dalton protein capable of polymerizing into double helical filaments; and band 4.1, an 82,000 dalton globular protein. By examining binary and ternary complexes of these molecules, the binding sites for actin, band 4.1 and a fourth protein ankyrin, which links the cytoskeleton to the membrane, have been mapped along the length of the spectrin molecule. These findings, which have enabled us to construct a model of the red cell cytoskeleton, show that low angle shadowing is a powerful but simple method for investigating associations among macromolecules.

Actins↗
PubMed1981

Assembly units of clathrin coats.

Clathrin, a polypeptide of molecular weight (MW) 180,000, is the main constituent of the polygonal network that forms the coat of coated pits and vesicles; these vesicles play a part in intracellular transport between membranous organelles. This function involves specific recognition of target membranes as well as fusion and fission events that must be coordinated with the assembly, partial disassembly or reorganization of the clathrin coats. To understand these interactions on a molecular level, information about the structure of clathrin and the interactions of clathrin with itself and other proteins is required. Here we show that purified clathrin coats dissociate reversibly into triskelions, structures composed of three usually bent, rather flexible legs irradiated from a centre. We have determined the molecular weight of these triskelions and conclude that they contain trimers of clathrin together with about three light molecular weight polypeptide chains.

Animals↗
PubMed1981

The binding of clathrin triskelions to membranes from coated vesicles.

We have developed a quantitative method to study the interaction of radiolabeled clathrin triskelions with membranes from brain coated vesicles. Clathrin triskellions do not bind to coated vesicles unless these are stripped of their clathrin coat. The binding of triskelions is of high affinity (KD congruent to 2 x 10(-9) M), is saturable and depends on vesicle concentration. Triskelions bind to protein-sensitive structures of the stripped vesicles. Polypeptides of about 110,000 daltons appear to be involved in the binding structure. The association of triskelions to stripped vesicles was confirmed by electron microscopy.

Cell-Free System↗
PubMed1981

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane.

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.

Actins↗
PubMed1981

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity.

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre-extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat-denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat-denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X-100 extraction of isolated D. discoideum membranes results in a Triton-insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.

Actins↗
PubMed1980

Reassociation of ankyrin with band 3 in erythrocyte membranes and in lipid vesicles.

The binding of human erythrocyte ankyrin (band 2.1) to the erythrocyte membrane has been characterized by reassociating purified ankyrin with ankyrin-depleted inside-out vesicles. Ankyrin reassociates at high affinity with a limited number of protease-sensitive sites located only on the cytoplasmic side of the erythrocyte membrane. Depleting the vesicles of band 4.2 does not affect their binding capacity. A 45,000-dalton polypeptide derived from the cytoplasmic portion of band 3 competitively inhibits the binding of ankyrin to inside-out vesicles. Although the bulk of band 3 molecules appear to have the potential for binding ankyrin, nly a fraction of the band 3 molecules in native membranes or in reconstituted liposomes actually provides accessible high affinity ankyrin binding sites.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1980

Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin.

Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin. Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.

Amino Acids↗
PubMed1980

The effect of endogenous proteases on the spectrin binding proteins of human erythrocytes.

We have demonstrated that in human erythrocyte ghosts endogenous proteolytic activity is responsible for the digestion of the spectrin binding proteins (bands 2.1 to 2.6). The pH optimum, cofactor requirements and inhibitor sensitivity have been established. Our results indicate that proteolysis of bands 2.1 to 2.6 and the formation of 3', a fragment containing an active spectrin binding site, can occur through two enzymatic pathways: a cascade of consecutive proteolytic cleavages of the spectrin binding proteins inhibited by phenylmethylsulfonyl fluoride or a Ca2+-stimulated, phenylmethylsulfonyl fluoride-insensitive, EDTA-inhibited cleavage of band 2.1 to band 2.3, followed by digestion to band 3' by phenylmethylsulfonyl fluoride-inhibitable enzymes. These findings may provide the techniques necessary to prevent proteolysis of the spectrin binding proteins during purification and reconstitution experiments and provide insight into how they are formed in vivo.

Calcium↗
PubMed1980

Transbilayer mapping of membrane proteins using membranes isolated on polylysine-coated polyacrylamide beads.

Erythrocyte and HeLa cell plasma membranes were isolated on polylysine-coated polyacrylamide beads and the transbilayer disposition of their proteins was investigated. When membranes of intact erythrocytes were isolated on beads and then labelled by lactoperoxidase-catalysed iodination, their labelling pattern was similar to that of inside-out vesicles in solution. When the membranes of intact HeLa cells were isolated on beads and then labelled by galactose oxidase-[3H]borohydride treatment, no glycoprotein or glycolipid sugars were accessible. On the other hand, when the HeLa cell membranes were isolated on beads and then labelled by the lactoperoxidase-catalysed iodination, all of the major membrane proteins were iodinated. These experiments confirmed for HeLa cell membranes what had previously been shown for erythrocyte membranes: when the membranes of intact cells are isolated on beads, the accessibility of their surfaces to enzymatic probes is the same as would be expected of inside-out vesicles in suspension. Double-label experiments, in which the HeLa cell membranes were labelled first on the intact HeLa cells and again after isolation on beads, identified several proteins which may span the membrane.

Borohydrides↗
PubMed1980

Spectrin-actin associations studied by electron microscopy of shadowed preparations.

By shadowing specimens dried onto mice sheets we have obtained clear images of actin crosslinked by spectrin, an actin-binding protein found in erythrocytes. We conclude that spectrin dimers possess a single binding site for F actin. Tetramers formed by head-to-head association of two dimers possess two actin binding sites, one at each tail. Polymerizing G actin in the presence of spectrin tetramers or mixing preformed F actin with spectrin tetramer plus bad 4.1 results in an extensively crosslinked network of actin filaments. When G actin is polymerized in the presence of spectrin at spectrin:actin mole ratios close to that present on the erythrocyte membrane, large amorphous protein networks are formed. These networks are clusters of spectrin around 25 nm diameter structures which may be actin protofilaments. These networks are similar to the cytoskeletal network seen after erythrocyte membranes are extracted with detergent, and may represent the first in vitro assembly of a cytoskeletal complex resembling that of the native cell both biochemically and structurally.

Actins↗
PubMed1980

Structural comparison of several actin-binding macromolecules.

The cytoskeletal components, macrophage actin-binding protein and filamin, were dried from glycerol and examined by low-angle rotary shadowing electron microscopy. Both are elongate, flexible molecules whose general morphologi is similar to that of erythrocyte spectrin. Neither actin-binding protein nor filamin binds to spectrin-depleted erythrocyte membranes.

Actins↗