N-Ethylmaleimide-modified actin filaments do not bundle in the presence of α-actinin

1995 ◽  
Vol 73 (1-2) ◽  
pp. 116-122 ◽  
Author(s):  
Aldo Milzani ◽  
Isabella DalleDonne ◽  
Roberto Colombo
Keyword(s):  
Polyethylene Glycol ◽  
Chemical Modification ◽  
Molecular Interactions ◽  
Binding Sites ◽  
Actin Filaments ◽  
Actin Assembly ◽  
Chemically Modified ◽  
C Terminus ◽  
Spatial Reorganization ◽  
Polyethylene Glycol 6000

We show that the modification of actin subdomain 1 by N-ethylmaleimide (NEM), which binds Cys-374 close to the C-terminus of the molecule, inhibits the α-actinin-induced bundling of actin filaments. This effect is not merely related to the block of Cys-374, since N-(1-pyrenyl)iodoacetamide (pyrene-IA) is unable to prevent bundling. Considering that NEM (but not pyrene-IA) influences actin assembly, we suggest that the inhibition of the actin – α-actinin interaction is due to the chemical modification of actin Cys-374 which, by inducing a marked spatial reorganization of actin monomers, is able to modify both the intra- and inter-molecular interactions of this protein. Finally, NEM-modified actin filaments form bundles in the presence of polyethylene glycol 6000 since, in this case, the side by side association of actin filaments does not depend on the accessibility of binding sites nor on the formation of chemical bonds.Key words: chemically modified actin, N-ethylmaleimide, pyrene-IA, Cys-374, actin bundles, α-actinin.

scholarly journals Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments.

1988 ◽  
Vol 106 (3) ◽  
pp. 805-812 ◽  
Author(s):  
H L Yin ◽  
K Iida ◽  
P A Janmey
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Assembly ◽  
Plasma Gelsolin ◽  
High Affinity ◽  
Binding Data ◽  
Actin Binding Domain

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.

scholarly journals A novel mode of capping protein-regulation by twinfilin

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Adam B Johnston ◽  
Denise M Hilton ◽  
Patrick McConnell ◽  
Britney Johnson ◽  
Meghan T Harris ◽  
...  
Keyword(s):  
Cell Morphology ◽  
Binding Sites ◽  
Actin Filaments ◽  
Protein Regulation ◽  
Actin Assembly ◽  
Binding Partner ◽  
In Vivo Binding ◽  
Capping Protein

Cellular actin assembly is controlled at the barbed ends of actin filaments, where capping protein (CP) limits polymerization. Twinfilin is a conserved in vivo binding partner of CP, yet the significance of this interaction has remained a mystery. Here, we discover that the C-terminal tail of Twinfilin harbors a CP-interacting (CPI) motif, identifying it as a novel CPI-motif protein. Twinfilin and the CPI-motif protein CARMIL have overlapping binding sites on CP. Further, Twinfilin binds competitively with CARMIL to CP, protecting CP from barbed-end displacement by CARMIL. Twinfilin also accelerates dissociation of the CP inhibitor V-1, restoring CP to an active capping state. Knockdowns of Twinfilin and CP each cause similar defects in cell morphology, and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity. Together, these observations define Twinfilin as the first ‘pro-capping’ ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle.

Hepatitis a Virus Concentration from Experimentally Contaminated Distilled, Tap, Waste and Seawater

1989 ◽  
Vol 21 (3) ◽  
pp. 255-258 ◽  
Author(s):  
Evangélos Biziagos ◽  
Jacques Passagot ◽  
Jean-Marc Crance ◽  
Robert Deloince
Keyword(s):  
Cell Culture ◽  
Polyethylene Glycol ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Tap Water ◽  
Virus Concentration ◽  
Beef Extract ◽  
Polyethylene Glycol 6000

The concentration of cell-culture-adapted hepatitis A virus (HAV) from experimentally contaminated distilled, drinking, waste and seawater was performed by using a filter adsorption-elu-tion method in the following conditions: HAV seeded in water was adsorbed at pH 4.0 to two nitrocellulose membranes (1.2 and 0.45 µm porosity for distilled and tap water or 8.0 and 3.0 µm porosity for waste and seawater), then eluted by 3% beef-extract at pH 8.5 and further concentrated by polyethylene glycol 6000 precipitation. Thus, HAV in 5 to 50 liters of seeded waters was concentrated approximately 1,700 to 17,000 fold with greater than 70% recovery of the initial virus added to the samples.

scholarly journals Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

1994 ◽  
Vol 126 (2) ◽  
pp. 433-443 ◽  
Author(s):  
A McGough ◽  
M Way ◽  
D DeRosier
Keyword(s):  
Image Analysis ◽  
Smooth Muscle ◽  
Binding Sites ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Outer Face ◽  
Actin Binding Domain

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol

2010 ◽  
Vol 146 (3) ◽  
pp. 318-325 ◽  
Author(s):  
Boris N. Novikov ◽  
Janet K. Grimsley ◽  
Rory J. Kern ◽  
James R. Wild ◽  
Melinda E. Wales
Keyword(s):  
Polyethylene Glycol ◽  
Chemical Modification ◽  
Organophosphorus Hydrolase

scholarly journals Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

2002 ◽  
Vol 13 (4) ◽  
pp. 1190-1202 ◽  
Author(s):  
Hélène Defacque ◽  
Evelyne Bos ◽  
Boyan Garvalov ◽  
Cécile Barret ◽  
Christian Roy ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Membrane Surface ◽  
Latex Bead ◽  
Actin Assembly ◽  
Wild Type ◽  
Membrane Surfaces ◽  
Organelle Membrane

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process ( Defacque et al., 2000b ). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

scholarly journals Phosphated Avocado Seed: A Renewable Biomaterial for Preparing a Flame Retardant Biofiller

2021 ◽  
Author(s):  
DAVID Zuluaga-Parra ◽  
L.F Ramos-deValle ◽  
Saul Sanchez ◽  
J.R. Torres-Lubián ◽  
J.A. Rodríguez-Gonzalez ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Chemical Modification ◽  
Flame Retardant ◽  
Photoelectron Spectroscopy ◽  
Morphological Changes ◽  
Ethylene Vinyl Acetate ◽  
Chemically Modified ◽  
Air Atmosphere ◽  
Thermal Stability Of ◽  
Avocado Seed

Abstract The cellulose and starch present in the avocado seed can be chemically modified to obtain biofillers with fire retarding characteristics. The resulting composites could be used as substitute of the corresponding halogenated composites. For this, the avocado seed was first washed, dehydrated and pulverized, and thereafter, chemically modified with phosphoric acid in the presence of urea. This was studied using infrared spectroscopy, nuclear magnetic resonance and X-Ray photoelectron spectroscopy, in order to determine the resulting chemical structure and confirm the presence of the proposed functional groups. In addition, scanning electron microscopy and elemental analysis were used, respectively, to establish the resulting morphological changes, as well as the elements present on the surface of the modified material. Thermogravimetric analysis was also carried out in order to establish the thermal stability of the material and predict the effect on the flame retardancy due to the mentioned chemical modification. Further tests established that the obtained modified structure and morphology of the avocado seed was highly dependent on the method used to dehydrate the pulverized avocado seed. It was also determined that chemical modification greatly increased the thermal stability of the avocado seed in air atmosphere. The flame-retardant effect of the modified avocado seed was assessed in polyethylene/ethylene-vinyl-acetate (PE/EVA) composites via cone calorimeter tests. These results showed that the modified avocado seed decreased the peak of the heat release rate (pHRR) by 50% and the total heat released (THR) by 15%. This phosphated avocado seed could be a good option as a renewable biofiller for polymer composites with enhanced flame-retardant properties.

Sign in / Sign up

Export Citation Format

Share Document