Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

scholarly journals A novel 36,000-dalton actin-binding protein purified from microfilaments in Physarum plasmodia which aggregates actin filaments and blocks actin-myosin interaction.

1982 ◽  
Vol 93 (3) ◽  
pp. 604-614 ◽  
Author(s):  
S Ogihara ◽  
Y Tonomura
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Actin Binding ◽  
Cyclic Contraction ◽  
Actin Binding Protein ◽  
Gel Layer ◽  
Skeletal Myosin ◽  
Myosin Interaction ◽  
Physarum Plasmodia ◽  
Triton X

In the plasmodia of Physarum polycephalum, which show a cyclic contraction-relaxation rhythm of the gel layer, huge aggregates of entangled actin microfilaments are formed at about the onset of the relaxation (R. Nagai, Y. Yoshimoto, and N. Kamiya. 1978. J. Cell Sci. 33:205-225). By treating the plasmodia with Triton X-100, we prepared a demembranated cytoskeleton consisting of entangled actin filaments and found that the actin filaments hardly interact with rabbit skeletal myosin. From the cytoskeleton we purified a novel actin-binding protein which binds stoichiometrically to actin and makes actin filaments curled and aggregated. It also inhibits the ATPase activity as well as the superprecipitation of reconstituted rabbit skeletal muscle actomyosin. This protein has a polypeptide molecular weight of 36,000 and binds 7 mol of actin/mol 36,000 polypeptide.

scholarly journals Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring.

1990 ◽  
Vol 111 (3) ◽  
pp. 1089-1105 ◽  
Author(s):  
J B Gorlin ◽  
R Yamin ◽  
S Egan ◽  
M Stewart ◽  
T P Stossel ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Hydrophobic Interactions ◽  
Binding Protein ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Leaf Spring ◽  
Cross Linking ◽  
Membrane Glycoproteins ◽  
Actin Binding Protein ◽  
Physical Measurements

Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin filaments. The large size of the leaves, their interruption by two hinges and flexible actin-binding site, facilitate cross-linking of widely dispersed actin filaments.

THE DISTRIBUTION OF GP lb AND THE STABILITY OF THE PLASMA MEMBRANE ARE DEPENDENT UPON AN INTACT MEMBRANE SKELETON

1987 ◽  
Author(s):  
J K Boyles ◽  
JE B Fox ◽  
M C Berndt
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Protein ◽  
Protein A ◽  
Cell Aggregation ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Gold Particles ◽  
Actin Binding Protein ◽  
The Stability

Platelets are know to have a cytoskeleton of actin filaments. We have presented evidence that they also have a membrane skeleton linked to the cytoskeletal filaments and that the membrane skeleton is linked to GP Ib-IX on the plasma membrane via actin-binding protein. In the current study, electron microscopy of thick (0.2 ym) epoxy sections was used to identify the distribution of GP lb. After various treatments, platelets were fixed and incubated with affinity-purified GP lb antibody and colloidal gold-labeled Protein-A. The entire cell surface was covered with a network of short intersecting chains of relatively evenly spaced gold particles. This was true of platelets in blood dripped directly from a vein into fixative, of washed discoid platelets, and of platelets activated by thrombin, ionophore, or cold under conditions in which aggregation did not occur. This pattern was not affected by the size of the gold label, the immunocytochemical protocol, or the fixative. The number of gold particles per cell was between 10,000 and 20,000, indicating a 1:1 ratio of label to GP lb. The distribution of GP lb was not affected by a level of cyto-chalasin B sufficient to disrupt the actin filaments of the platelet cytoskeleton. Proteolysis of actin-binding protein is known to be induced by treatment of platelets with dibucaine and by platelet activation (with either ionophore or thrombin) under conditions in which cell aggregation occurs. These same treatments caused GP lb to cluster. They also produced platelets with unstable membranes that vesiculated when the cells were subjected to shear force during centrifugation or osmotic-ally stressed during fixation. These studies show that both the distribution of GP lb and membrane stability are dependent upon the integrity of actin-binding protein and the membrane skeleton. In the high-shear environment of the blood vessel, the membrane skeleton and its linkage to GP Ib-IX and the cytoskeleton may be essential for proper platelet function.

scholarly journals Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.

1980 ◽  
Vol 87 (3) ◽  
pp. 841-848 ◽  
Author(s):  
J H Hartwig ◽  
J Tyler ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Average Length ◽  
Binding Protein ◽  
Actin Binding ◽  
Branch Points ◽  
Heavy Meromyosin ◽  
Actin Binding Protein ◽  
Protein Dimers ◽  
Protein Molecules

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.

scholarly journals Recycling of platelet phosphorylation and cytoskeletal assembly.

1984 ◽  
Vol 98 (1) ◽  
pp. 8-15 ◽  
Author(s):  
A C Cox ◽  
R C Carroll ◽  
J G White ◽  
G H Rao
Keyword(s):  
Protein Phosphorylation ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Binding Protein ◽  
Actin Binding ◽  
Dibutyryl Camp ◽  
Actin Binding Protein ◽  
Fibrinogen Binding ◽  
Triton X ◽  
Subsequent Addition

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.

scholarly journals Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1400-1413 ◽  
Author(s):  
R Niederman ◽  
P C Amrein ◽  
J Hartwig
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Molar Ratio ◽  
Computer Assisted ◽  
Three Dimensional Structure ◽  
Actin Binding Protein ◽  
Critical Point Drying

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

scholarly journals Effect of actin-binding protein on the sedimentation properties of actin.

1982 ◽  
Vol 94 (1) ◽  
pp. 51-55 ◽  
Author(s):  
S Rosenberg ◽  
A Stracher
Keyword(s):  
Actin Filaments ◽  
Human Platelet ◽  
Binding Protein ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Protein ◽  
Cell Biol

Actin and actin-binding protein (ABP) have recently been purified from human platelet cytoskeletons (S. Rosenberg, A. Stracher, and R.C. Lucas, 1981, J. Cell Biol. 91:201-211). Here, the effect of ABP on the sedimentation of actin was studied. When ABP was added to preformed F-actin filaments, it bound until a maximum ratio of 1:9 (ABP:actin, mol:mol) was reached. however, when actin was polymerized in the presence of ABP, two and a half times more ABP was able to bind to the actin- that is, every 3.4 actin monomers were now bound by an ABP dimer. ABP was not able to induce the sedimentation of actin under nonpolymerizing conditions but was able to reduce the time and concentration of actin required for sedimentation under slow polymerizing conditions. ABP, therefore, exerts its effect of G-actin by either nucleating polymerization or by cross-linking newly formed oligomers into a more sedimentable form.

scholarly journals The Plant-Specific Actin Binding Protein SCAB1 Stabilizes Actin Filaments and Regulates Stomatal Movement in Arabidopsis

2011 ◽  
Vol 23 (6) ◽  
pp. 2314-2330 ◽  
Author(s):  
Yang Zhao ◽  
Shuangshuang Zhao ◽  
Tonglin Mao ◽  
Xiaolu Qu ◽  
Wanhong Cao ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Actin Binding ◽  
Stomatal Movement ◽  
Actin Binding Protein

scholarly journals Identification of membrane proteins mediating the interaction of human platelets

1980 ◽  
Vol 86 (1) ◽  
pp. 77-86 ◽  
Author(s):  
D Phillips ◽  
L Jennings ◽  
H Edwards
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Membrane Surface ◽  
Direct Interaction ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Insoluble Residue ◽  
Membrane Glycoproteins ◽  
Actin Binding Protein ◽  
Activated Platelets

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

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