Kinetics of the initial steps of rabbit psoas myofibrillar ATPases studied by tryptophan and pyrene fluorescence stopped-flow and rapid flow-quench. Evidence that cross-bridge detachment is slower than ATP binding.
AuthorsStehle R,Lionne C,Travers F,Barman T
JournalBiochemistry
PubMed ID10858300
A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies.
AuthorsRatner V,Kahana E,Eichler M,Haas E
JournalBioconjugate chemistry
PubMed ID12236801
Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state ... More
The small GTP-binding protein Rac promotes the dissociation of gelsolin from actin filaments in neutrophils.
AuthorsArcaro A
JournalJ Biol Chem
PubMed ID9422735
Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin ... More
Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments.
AuthorsYin HL, Iida K, Janmey PA
JournalJ Cell Biol
PubMed ID2831234
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 ... More
Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain.
AuthorsYarmola EG, Edison AS, Lenox RH, Bubb MR
JournalJ Biol Chem
PubMed ID11294839
We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS ... More
Alpha-adducin dissociates from F-actin and spectrin during platelet activation.
AuthorsBarkalow KL, Italiano JE, Chou DE, Matsuoka Y, Bennett V, Hartwig JH
JournalJ Cell Biol
PubMed ID12743105
'Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. alpha-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites ... More
The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen.
AuthorsBennett JS, Zigmond S, Vilaire G, Cunningham ME, Bednar B
JournalJ Biol Chem
PubMed ID10464255
'Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented ... More
An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.
AuthorsScita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, Di Fiore PP
JournalJ Cell Biol
PubMed ID11524436
'Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a ... More
Espins are multifunctional actin cytoskeletal regulatory proteins in the microvilli of chemosensory and mechanosensory cells.
AuthorsSekerková G, Zheng L, Loomis PA, Changyaleket B, Whitlon DS, Mugnaini E, Bartles JR
JournalJ Neurosci
PubMed ID15190118
'Espins are associated with the parallel actin bundles of hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction in mice and humans. Here, we report that espins are also concentrated in the microvilli of a number of other sensory cells: vomeronasal organ sensory neurons, ... More
A simple method for introducing a thiol group at the 5'-end of synthetic oligonucleotides.
AuthorsKumar A, Advani S, Dawar H, Talwar GP
JournalNucleic Acids Res
PubMed ID1886783
Intrastrand cross-linked actin between Gln-41 and Cys-374. II. Properties of cross-linked oligomers.
AuthorsKim E, Phillips M, Hegyi G, Muhlrad A, Reisler E
JournalBiochemistry
PubMed ID9922145
'Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s(o)20,w = 5.55 +/- 0.22 S), trimers (s(o)20,w = 6.93 +/- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment ... More
Pleckstrin homology domains interact with filamentous actin.
AuthorsYao L, Janmey P, Frigeri LG, Han W, Fujita J, Kawakami Y, Apgar JR, Kawakami T
JournalJ Biol Chem
PubMed ID10391917
'A fraction of Bruton''s tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcepsilonRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) ... More
Structure determination and characterization of Saccharomyces cerevisiae profilin.
'The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were ... More
Covalent modification of G-actin by pyridoxal 5'-phosphate: polymerization properties and interaction with DNase I and myosin subfragment 1.
AuthorsCombeau C, Carlier MF
JournalBiochemistry
PubMed ID1731881
'Pyridoxal 5''-phosphate (PLP), a lysine-specific reagent, has been used to modify G-actin. At pH 7.5, PLP reacted with 1.7-2 lysines on G-actin. Limited proteolytic digestion experiments indicated that, in agreement with previous works, essentially lysine-61 was modified in a 1:1 fashion by PLP, other lysines being much less reactive. A ... More
The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.
AuthorsMurphy CT, Spudich JA
JournalBiochemistry
PubMed ID10090768
'We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ... More
Biochemical characterization of the L-plastin-actin interaction shows a resemblance with that of alpha-actinin and allows a distinction to be made between the two actin-binding domains of the molecule.
'Actin interaction with L-plastin, a plastin/fimbrins isoform of the alpha-actinin family of molecules, is poorly characterized, from the biochemical point of view. Besides, molecular modeling of the T-isoform has recently provided a complete model of interaction with filamentous actin [Volkmann, N., DeRosier, D., Matsudaira, P., and Hanein, D. (2001) J. ... More
Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect.
AuthorsValtorta F, Greengard P, Fesce R, Chieregatti E, Benfenati F
JournalJ Biol Chem
PubMed ID1317863
'Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that ... More
Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics.
AuthorsDe La Cruz EM, Pollard TD
JournalBiochemistry
PubMed ID7727403
'We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The ... More
Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein.
AuthorsLim RW, Furukawa R, Eagle S, Cartwright RC, Fechheimer M
JournalBiochemistry
PubMed ID9888821
'The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity ... More
The actin released from profilin--actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes.
AuthorsSouthwick FS, Young CL
JournalJ Cell Biol
PubMed ID2351690
'Chemoattractant stimulation of polymorphonuclear leukocytes is associated with a nearly two-fold rise in actin filament content. We examined the role of the actin monomer sequestering protein, profilin, in the regulation of PMN actin filament assembly during chemoattractant stimulation using a Triton extraction method. Poly-L-proline-conjugated Sepharose beads were used to assess ... More
Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein.
AuthorsShariff A, Luna EJ
JournalJ Cell Biol
PubMed ID2307703
'In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1-pyrenyl)iodoacetamide ... More
Functional analysis of rat acidic calponin.
AuthorsFujii T, Yabe S, Nakamura K, Koizumi Y
JournalBiol Pharm Bull
PubMed ID12033495
'Recombinant acidic calponin, a member of the calponin family, interacted with F-actin, but not with microtubules, desmin filaments, tropomyosin, calmodulin, S100 and phosphatidylserine (PS) vesicles with significant affinity. The bindings of acidic calponin to F-actin occurred in a concentration-dependent manner and were saturated at a molar ratio of about 1 ... More
Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.
AuthorsBlanchoin L, Pollard TD
JournalJ Biol Chem
PubMed ID10336448
'We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle ... More
Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide.
AuthorsBurtnick LD, Stewart DI, Clark ID, Smillie LB
JournalBiochemistry
PubMed ID3741838
'Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that ... More
Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.
AuthorsOstap EM, Pollard TD
JournalJ Cell Biol
PubMed ID8601584
'Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench ... More
A study of sensitized lanthanide luminescence in an engineered calcium-binding protein.
AuthorsClark ID, MacManus JP, Banville D, Szabo AG
JournalAnal Biochem
PubMed ID8489002
'In this study, the CD loop of the Ca(2+)-binding protein oncomodulin was replaced by a high-affinity, metal-binding sequence that was found to reverse the order of fill of the two sites in the protein. A cysteine was included at position 7 of this sequence, i.e., DKNADGCIEFEE. The cysteine allowed covalent ... More
Ca2+ regulation of gelsolin by its C-terminal tail.
AuthorsLin KM, Mejillano M, Yin HL
JournalJ Biol Chem
PubMed ID10862770
'Gelsolin is activated by Ca(2+) to sever actin filaments. Ca(2+) regulation is conferred on the N-terminal half by the C-terminal half. This paper seeks to understand how Ca(2+) regulates gelsolin by testing the "tail helix latch hypothesis," which is based on the structural data showing that gelsolin has a C-terminal ... More
Structural connectivity in actin: effect of C-terminal modifications on the properties of actin.
AuthorsCrosbie RH, Miller C, Cheung P, Goodnight T, Muhlrad A, Reisler E
JournalBiophys J
PubMed ID7858132
'In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in ... More
Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity.
AuthorsBenndorf R, Hayess K, Ryazantsev S, Wieske M, Behlke J, Lutsch G
JournalJ Biol Chem
PubMed ID8051180
'Characteristic features of mammalian small heat shock proteins are their rapid phosphorylation in response to stress and mitogenic signals and their ability to form multimeric particles of 200-700 kDa and large aggregates up to 5000 kDa. Recently, a chaperoning function and an actin polymerization-inhibiting activity were demonstrated for the recombinant ... More
Both isoforms of skeletal muscle subfragment 1 (S1A1 and S1A2) can induce actin polymerization with equal speed in the absence of ATP.
'The ability of myosin subfragment 1 to induce actin polymerization was reinvestigated using the DNase I inhibition assay, by electron microscopy after negative staining, cosedimentation, measurement of viscosity and the fluorescence increase of pyrenyl-labeled actin. Using these techniques we demonstrate that rabbit skeletal muscle myosin subfragment 1 containing either the ... More
Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms.
AuthorsKurzawa-Goertz SE, Perreault-Micale CL, Trybus KM, Szent-Györgyi AG, Geeves MA
JournalBiochemistry
PubMed ID9585566
'The striated muscle myosin of Placopecten moves actin faster in in vitro motility assays and has a higher actin-activated ATPase turnover rate than the myosin of the catch muscle. The heavy chain sequences of the two PlacoS1s are almost identical except at the surface loop 1 near the nucleotide binding ... More
Kinetics of actin elongation and depolymerization at the pointed end.
AuthorsWeber A, Northrop J, Bishop MF, Ferrone FA, Mooseker MS
JournalBiochemistry
PubMed ID3607031
'We measured the rate of elongation at the pointed filament end with increasing concentrations of G-actin [J(c) function] using villin-capped actin filaments of very small (actin/villin = 3, VA3) and relatively large size (actin/villin = 18, VA18) as nuclei for elongation. The measurements were made under physiological conditions in the ... More
Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin.
AuthorsJanmey PA, Lamb J, Allen PG, Matsudaira PT
JournalJ Biol Chem
PubMed ID1318302
'The polyphosphoinositides phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) inactivate the actin filament-severing proteins villin and gelsolin and dissociate them from monomeric and polymeric actin. A potential polyphosphoinositide- (PPI) binding site of human plasma gelsolin regulating filament severing has been localized to the region between residues 150-169 and to the ... More
Size distribution of linear and helical polymers in actin solution analyzed by photon counting histogram.
AuthorsTerada N, Shimozawa T, Ishiwata S, Funatsu T
JournalBiophys J
PubMed ID17172301
'Actin is a ubiquitous protein that is a major component of the cytoskeleton, playing an important role in muscle contraction and cell motility. At steady state, actin monomers and filaments (F-actin) coexist, and actin subunits continuously attach and detach at the filament ends. However, the size distribution of actin oligomers ... More
Probing the mechanism of ATP hydrolysis on F-actin using vanadate and the structural analogs of phosphate BeF-3 and A1F-4.
AuthorsCombeau C, Carlier MF
JournalJ Biol Chem
PubMed ID3182855
'The binding of phosphate analogs to F-ADP-actin filaments and their effect on the dynamics of the polymer have been investigated. Orthovanadate binds to F-actin with the same affinity as phosphate and, at low saturation levels, stabilizes the filament structure in a Pi-like fashion; at higher concentration, it promotes destabilization of ... More
Excimer fluorescence of pyrene-tropomyosin adducts.
AuthorsLin TI
JournalBiophys Chem
PubMed ID7115884
'Studies of the fluorescence of N-(1-pyrene)maleimide and N-(1-pyrenyl)iodoacetamide adducts of rabbit skeletal muscle tropomyosin revealed the presence of excimer fluorescence characterized by a broad emission band at 480 nm with a shoulder at 505 nm. Monomer fluorescence decay exhibited different lifetimes, viz., about 3, 22 and 87 ns for the ... More
Isolation of a Ca2+-dependent actin-fragmenting protein from brain, spinal cord, and cultured neurones.
AuthorsPetrucci TC, Thomas C, Bray D
JournalJ Neurochem
PubMed ID6304247
'Extracts of ox spinal cord and chicken brain were fractionated by ion-exchange chromatography and assayed for their ability to reduce the viscosity of muscle F-actin solutions. Two distinct peaks of activity were obtained, one of which was further purified by affinity chromatography on a DNAase-actin Sepharose column. Following molecular exclusion ... More
Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments.
AuthorsWohnsland F, Schmitz AA, Steinmetz MO, Aebi U, Vergéres G
JournalJ Biol Chem
PubMed ID10748210
'It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction ... More
GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro.
AuthorsBlader IJ, Cope MJ, Jackson TR, Profit AA, Greenwood AF, Drubin DG, Prestwich GD, Theibert AB
JournalMol Biol Cell
PubMed ID10069805
'Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified ... More
Tropomodulin caps the pointed ends of actin filaments.
AuthorsWeber A, Pennise CR, Babcock GG, Fowler VM
JournalJ Cell Biol
PubMed ID7798317
'Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining ... More
Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin.
AuthorsRosenfeld SS, Xing J, Cheung HC, Brown F, Kar S, Sweeney HL
JournalJ Biol Chem
PubMed ID9786863
'In this study, we have examined the mechanism of phosphorylation-dependent regulation in smooth muscle myosin through the use of structural and kinetic methodologies applied to several myosin fragments. Fluorescence anisotropy decay measurements demonstrate that regulatory light chain phosphorylation significantly reduces the rotational correlation time of regulatable myosin preparations, whereas minimally ... More
A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments.
AuthorsKuhlman PA, Hughes CA, Bennett V, Fowler VM
JournalJ Biol Chem
PubMed ID8626479
'Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating ... More
Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility.
AuthorsSkoble J, Portnoy DA, Welch MD
JournalJ Cell Biol
PubMed ID10931865
'The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are ... More
Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.
AuthorsEichinger L, Schleicher M
JournalBiochemistry
PubMed ID1591239
'Severin is a Ca(2+)-activated actin-binding protein that nucleates actin assembly and severs and caps the fast growing ends of actin filaments. It consists of three highly conserved domains. To investigate the domain structure of severin, we constructed genetically the N-terminal domain 1, the middle domain 2, and the tandem domains ... More
Intermolecular histone H4 interactions in core nucleosomes.
AuthorsChung DG, Lewis PN
JournalBiochemistry
PubMed ID3707931
'Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the ... More
Immunochemical probing of the N-terminal segment on actin: the polymerization reaction.
AuthorsDasGupta G, White J, Phillips M, Bulinski JC, Reisler E
JournalBiochemistry
PubMed ID2334693
'The N-terminal segment of actin contains a cluster of acidic residues which are implicated in macromolecular interactions of this protein. In this work, the interrelationship between the N-terminal segment and the polymerization of actin was studied by using affinity-purified antibodies directed against the first seven N-terminal residues on alpha-skeletal actin ... More
Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation.
'Actin polymerization occurs in amebae of Dictyostelium discoideum after chemotactic stimulation (Hall, A. L., A. Schlein, and J. Condeelis. 1988. J. Cell. Biochem. 37:285-299). When cells are lysed with Triton X-100 during stimulation, an actin nucleation activity is detected in lysates by measuring the rate of pyrene-labeled actin polymerization. This ... More
Analysis of the interactions of actin depolymerizing factor with G- and F-actin.
AuthorsHayden SM, Miller PS, Brauweiler A, Bamburg JR
JournalBiochemistry
PubMed ID8399168
'Chick actin depolymerizing factor (ADF) is an actin binding protein previously shown to rapidly depolymerize actin filaments in vitro, yielding a 1:1 complex of ADF and actin monomer. Here we show that ADF protects actin monomer from denaturation by EDTA by inhibiting the exchange of actin-bound nucleotide. Under low ionic ... More
Myosin subfragment 1 and structural elements of G-actin: effects of S-1(A2) on sequences 39-52 and 61-69 in subdomain 2 of G-actin.
AuthorsChen T, Haigentz M, Reisler E
JournalBiochemistry
PubMed ID1550820
'The effect of myosin on the structure of two sequences on G-actin, a loop between residues 39 and 52 and a segment between residues 61 and 69 from the NH2-terminus, was probed by limited proteolytic digestions of G-actin in the presence of the myosin subfragment 1 isozyme S-1(A2). Under the ... More
'Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share ... More
Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative.
AuthorsPinder JC, Gratzer WB
JournalBiochemistry
PubMed ID6215939
'The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes ... More
Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility.
AuthorsSkoble J, Auerbuch V, Goley ED, Welch MD, Portnoy DA
JournalJ Cell Biol
PubMed ID11581288
'The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein ... More
The tert-butyl hydroperoxide-induced oxidation of actin Cys-374 is coupled with structural changes in distant regions of the protein.
AuthorsDalleDonne I, Milzani A, Colombo R
JournalBiochemistry
PubMed ID10493817
'The susceptibility of monomeric actin to both methionine and cysteine oxidation when treated with the oxidizing agent tert-butyl hydroperoxide (t-BH) was investigated. The results show that no methionine residue was susceptible to oxidation by t-BH at concentrations of 1-20 mM, while Cys-374, one of the five cysteine residues of the ... More
Polymerization of actin and actin-like systems: evaluation of the time course of polymerization in relation to the mechanism.
AuthorsFrieden C, Goddette DW
JournalBiochemistry
PubMed ID6661414
'The time course of protein polymerization of the nucleation--elongation type is examined by using a general computer-simulation solution. For a simple nucleation--elongation scheme, it is shown that the half-time of polymerization is not necessarily a good measure of the nucleus size as has been previously suggested [Oosawa, F., & Kasai, ... More
Cross-link between cys 374 and cys 10 of actin abolishes polymerizability and allows study of the properties of the "F-actin monomer".
AuthorsHeintz D, Faulstich H
JournalBiochemistry
PubMed ID8555183
'Actin cross-linked between cys 374 and cys 10 via a disulfide-containing bridge, c-A, is completely unpolymerizable even in the presence of phalloidin. Upon the addition of dithiothreitol, c-A polymerizes with high yield, indicating that denaturation due to the modification was almost absent. In the present study we show that cross-linked ... More
Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex.
AuthorsWeed SA, Karginov AV, Schafer DA, Weaver AM, Kinley AW, Cooper JA, Parsons JT
JournalJ Cell Biol
PubMed ID11018051
'Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its ... More
Actin polymerization. The effect of brevin on filament size and rate of polymerization.
AuthorsDoi Y, Frieden C
JournalJ Biol Chem
PubMed ID6480587
'Fluorescent probes covalently bound to actin or to the actin binding protein, brevin, have been utilized to provide information about actin filaments formed in the presence of brevin as well as about the effect of brevin on the rate of polymerization. At actin to brevin ratios of 10:1 to 100:1, ... More
Twinfilin is required for actin-dependent developmental processes in Drosophila.
AuthorsWahlström G, Vartiainen M, Yamamoto L, Mattila PK, Lappalainen P, Heino TI
JournalJ Cell Biol
PubMed ID11724820
'The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A ... More
Regulation of G protein-coupled receptor kinase 5 (GRK5) by actin.
AuthorsFreeman JL, De La Cruz EM, Pollard TD, Lefkowitz RJ, Pitcher JA
JournalJ Biol Chem
PubMed ID9685424
'G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a ... More
Actin and light chain isoform dependence of myosin V kinetics.
AuthorsDe La Cruz EM, Wells AL, Sweeney HL, Ostap EM
JournalBiochemistry
PubMed ID11087368
'Recent studies on myosin V report a number of kinetic differences that may be attributed to the different heavy chain (chicken vs mouse) and light chain (essential light chains vs calmodulin) isoforms used. Understanding the extent to which individual light chain isoforms contribute to the kinetic behavior of myosin V ... More
Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons.
AuthorsPodolski JL, Steck TL
JournalJ Biol Chem
PubMed ID3335517
'We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X ... More
Interactions among red cell membrane proteins.
AuthorsPodgorski A, Alster P, Elbaum D
JournalBiochemistry
PubMed ID3126812
'Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy ... More
Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility.
Authors Bear James E; Svitkina Tatyana M; Krause Matthias; Schafer Dorothy A; Loureiro Joseph J; Strasser Geraldine A; Maly Ivan V; Chaga Oleg Y; Cooper John A; Borisy Gary G; Gertler Frank B;
JournalCell
PubMed ID12086607
'Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded ... More
Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization.
AuthorsCooper JA, Walker SB, Pollard TD
JournalJ Muscle Res Cell Motil
PubMed ID6863518
'The fluorescence of pyrene-labelled actin is much higher after polymerization. We have characterized in detail the polymerization properties of pyrene actin and report that native and pyrene actin are identical using the following criteria: (1) the time course of polymerization; (2) the elongation rate constants; (3) the intrinsic viscosity; and ... More
Kinetics of myosin subfragment-1-induced condensation of G-actin into oligomers, precursors in the assembly of F-actin-S1. Role of the tightly bound metal ion and ATP hydrolysis.
AuthorsFievez S, Pantaloni D, Carlier MF
JournalBiochemistry
PubMed ID9305975
'In a low ionic strength buffer and in the absence of free ATP, the interaction of G-actin (G) with myosin subfragment-1 (S1) leads to the formation of arrowhead-decorated F-actin-S1 filaments, through a series of elementary steps. The initial formation of GS and G2S complexes is followed by their condensation into ... More
Metal ions modulate the effect of doxorubicin on actin assembly.
AuthorsColombo R, Dalle Donne I, Milzani A
JournalCancer Biochem Biophys
PubMed ID2268851
'Doxorubicin (DXR) exhibits a significant activity in many human malignant neoplasms but, unfortunately, produces many undesirable cellular troubles which mainly lead to a severe dose-dependent cardiomyopathy. Many Authors had suggested that doxorubicin interacts with actin and affects the intracellular ion composition; following this reasoning, we studied the effect of doxorubicin ... More
Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.
AuthorsSmith SK, Farnbach AR, Harris FM, Hawes AC, Jackson LR, Judd AM, Vest RS, Sanchez S, Bell JD
JournalJ Biol Chem
PubMed ID11294854
'Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin ... More
Actin filament annealing in the presence of ATP and phalloidin.
AuthorsKinosian HJ, Selden LA, Estes JE, Gershman LC
JournalBiochemistry
PubMed ID8241122
'The re-formation of actin filaments after fragmentation by sonication in the presence of phalloidin and ATP has been found to follow second-order kinetics. The data are described by a model in which the rate of actin filament annealing is proportional to the square of the number concentration of actin filaments ... More
Subunit flow in F-actin under steady-state conditions. Application of a novel method to determination of the rate of subunit exchange of F-actin at the terminals.
AuthorsSuzuki N, Mihashi K
JournalBiophys Chem
PubMed ID2526660
'We developed a novel method to determine the subunit exchange rates of F-actin at its terminals under quasi-steady-state conditions by using a powerful fluorescent probe, N-(1-pyrenyl)iodoacetamide. The applicability of the method was checked with regard to both theoretical and experimental aspects. We determined the rates of subunit exchange of F-actin ... More
Kinetic characterization of a monomeric unconventional myosin V construct.
AuthorsTrybus KM, Krementsova E, Freyzon Y
JournalJ Biol Chem
PubMed ID10488077
'An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 ... More
High affinity binding of brain myosin-Va to F-actin induced by calcium in the presence of ATP.
AuthorsTauhata SB, dos Santos DV, Taylor EW, Mooseker MS, Larson RE
JournalJ Biol Chem
PubMed ID11517216
'Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its ... More
Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants.
AuthorsButt E, Immler D, Meyer HE, Kotlyarov A, Laass K, Gaestel M
JournalJ Biol Chem
PubMed ID11383510
'Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for ... More
Inhibition of actin filament depolymerization by the Dictyostelium 30,000-D actin-bundling protein.
AuthorsZigmond SH, Furukawa R, Fechheimer M
JournalJ Cell Biol
PubMed ID1328254
'We have studied the effect of the Dictyostelium discoideum 30,000-D actin-bundling protein on the assembly and disassembly of pyrenyl-labeled actin in vitro. The results indicate that the protein is a potent inhibitor of the rate of actin depolymerization. The inhibition is rapid, dose dependent, and is observed at both ends ... More
Separation and characterization of the two functional regions of troponin involved in muscle thin filament regulation.
AuthorsSchaertl S, Lehrer SS, Geeves MA
JournalBiochemistry
PubMed ID8519745
'Mild proteolytic cleavage of the troponin complex yields TnT1, the N-terminal fragment of troponin T, and TnT2IC, a complex of the C-terminal fragment of troponin T (TnT2) with troponin I (TnI) and troponin C (TnC) [Morris, E. P., & Lehrer, S. S. (1984) Biochemistry 23, 2214-2220]. Both TnT1 and TnT2IC ... More
Actin's view of actomyosin interface.
AuthorsMiller CJ, Cheung P, White P, Reisler E
JournalBiophys J
PubMed ID7787100
'Actomyosin interactions were examined by using yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84. In the in vitro motility experiments, actin filaments of D24A/D25A or E99A/E100A mutants moved in the presence of 0.7% methylcellulose at the velocities of wild-type actin. Without methylcellulose, these ... More
Profilin-actin complexes directly elongate actin filaments at the barbed end.
AuthorsPring M, Weber A, Bubb MR
JournalBiochemistry
PubMed ID1737036
'We demonstrate that the profilin-G-actin complex can elongate actin filaments directly at the barbed end but cannot bind to the pointed end. During elongation, the profilin-actin complex binds to the barbed filament end, whereupon profilin is released, leaving the actin molecule behind. This was first proposed by Tilney [Tilney, L. ... More
An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein.
AuthorsFreeman NL, Chen Z, Horenstein J, Weber A, Field J
JournalJ Biol Chem
PubMed ID7890691
'The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We ... More
Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer.
AuthorsSun M, Oakes JL, Ananthanarayanan SK, Hawley KH, Tsien RY, Adams SR, Yengo CM
JournalJ Biol Chem
PubMed ID16377637
'The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding ... More
Biochemical characterization of ezrin-actin interaction.
AuthorsYao X, Cheng L, Forte JG
JournalJ Biol Chem
PubMed ID8636161
'The highly related actin isoforms are thought to have different functions. We recently demonstrated a polarized distribution of actin isoforms in gastric parietal cells and association of gastric ezrin with the cytoplasmic beta-actin isoform (Yao, X., Chaponnier, C., Gabbiani, G., and Forte, J. G. (1995) Mol. Biol. Cell. 6, 541-557). ... More
NH2-terminal truncation of skeletal muscle troponin T does not alter the Ca2+ sensitivity of thin filament assembly.
AuthorsFisher D, Wang G, Tobacman LS
JournalJ Biol Chem
PubMed ID7592713
'To investigate how Ca2+ binding to troponin C regulates muscle contraction, the Ca(2+)-sensitive properties of thin filament assembly were studied as the tropomyosin binding, NH2-terminal region of troponin T was progressively shortened. Troponin complexes were prepared that contained skeletal muscle troponin C, troponin I, and either intact troponin T (TnT) ... More
Gain-of-function mutations conferring actin-severing activity to human macrophage cap G.
AuthorsSouthwick FS
JournalJ Biol Chem
PubMed ID7814409
'Nonmuscle cell motility requires marked changes in the consistency and shape of the peripheral cytoplasm. These changes are regulated by a gel-sol transformation of the actin filament network, and actin filament-severing proteins are responsible for network solation. Macrophage Cap G, unlike all other proteins in the gelsolin family, caps but ... More
Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.
AuthorsPalmgren S, Ojala PJ, Wear MA, Cooper JA, Lappalainen P
JournalJ Cell Biol
PubMed ID11604420
'Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to ... More
Nebulin as an actin zipper. A two-module nebulin fragment promotes actin nucleation and stabilizes actin filaments.
AuthorsChen MJ, Shih CL, Wang K
JournalJ Biol Chem
PubMed ID8376391
'Nebulin is a family of giant muscle proteins (700-900 kDa) that interact with actin to form composite thin filaments in the skeletal muscle sarcomere. This modular protein is composed predominantly of repeating sequence modules of 31-38 residues. To understand the minimum size and number of sequence modules that are required ... More
Identification of reactive vicinal cysteines in Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases.
AuthorsRojas MC, Encinas MV, Kemp RG, Latshaw SP, Cardemil E
JournalBiochim Biophys Acta
PubMed ID8329445
'Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases (EC 4.1.1.49/32) have been labeled with N-(1-pyrenyl)-iodoacetamide. Reagent incorporation was completely prevented by the presence of the respective nucleoside diphosphate plus MnCl2. Under appropriate conditions, 2 mol of reagent per mol of enzyme subunit were incorporated. The fluorescence ... More
Mechanism of myosin subfragment-1-induced assembly of CaG-actin and MgG-actin into F-actin-S1-decorated filaments.
AuthorsFievez S, Carlier MF, Pantaloni D
JournalBiochemistry
PubMed ID9305976
'The kinetics and mechanism of myosin subfragment-1-induced polymerization of G-actin into F-actin-S1-decorated filaments have been investigated in low ionic strength buffer and in the absence of free ATP. The mechanism of assembly of F-actin-S1 differs from salt-induced assembly of F-actin. Initial condensation of G-actin and S1 into oligomers in reversible ... More
The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin.
AuthorsCriddle AH, Geeves MA, Jeffries T
JournalBiochem J
PubMed ID3911945
'A pyrene label attached to Cys-374 of actin has been shown to be a useful probe for monitoring the interaction of actin with myosin subfragments [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38]. We report that the presence of this label decreases the affinity of actin for myosin subfragment ... More
Interaction of profilin with G-actin and poly(L-proline).
AuthorsPerelroizen I, Marchand JB, Blanchoin L, Didry D, Carlier MF
JournalBiochemistry
PubMed ID8031780
'The interaction of bovine spleen profilin with ATP- and ADP-G-actin and poly(L-proline) has been studied by spectrofluorimetry, analytical ultracentrifugation, and rapid kinetics in low ionic strength buffer. Profilin binding to G-actin is accompanied by a large quenching of tryptophan fluorescence, allowing the measurement of an equilibrium dissociation constant of 0.1-0.2 ... More
Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides.
AuthorsSchafer DA, Jennings PB, Cooper JA
JournalJ Cell Biol
PubMed ID8858171
'Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, ... More
Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes.
AuthorsSiegel DL, Branton D
JournalJ Cell Biol
PubMed ID3882722
'Band 4.9 (a 48,000-mol-wt polypeptide) has been partially purified from human erythrocyte membranes. In solution, band 4.9 polypeptides exist as trimers with an apparent molecular weight of 145,000 and a Stokes radius of 50 A. Electron microscopy shows that the protein is a three-lobed structure with a radius slightly greater ... More
Induction of the polymerization of actin from the actin:thymosin beta 4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin.
AuthorsBallweber E, Hannappel E, Niggemeyer B, Mannherz HG
JournalEur J Biochem
PubMed ID8055911
'Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin ... More
Local environmental change from the G- to F-form of the actin molecule detected on anisotropy decay measurement.
AuthorsSasaki Y, Tsunomori F, Yamashita T, Horie K, Ushiki H, Ishikawa R, Kohama K
JournalJ Biochem (Tokyo)
PubMed ID7822235
'The fluorescence intensity has been reported to increase 10 to 25 times when N-(1-pyrene)-iodoacetamide (PIAA)-conjugated actin polymerizes from the G- to the F-form. To elucidate the molecular mechanism underlying this process, we measured the time-averaged anisotropy of PIAA-actin in both the G- and F-forms. The anisotropy ratio of PIAA-G-actin (0.137 ... More
Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin.
AuthorsDasGupta G, White J, Cheung P, Reisler E
JournalBiochemistry
PubMed ID2252908
'The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by ... More
Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.
'To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we ... More
Calcium dependence of villin-induced actin depolymerization.
AuthorsWalsh TP, Weber A, Davis K, Bonder E, Mooseker M
JournalBiochemistry
PubMed ID6525347
'"Cutting" of actin filaments by villin was evaluated from the time course of filament depolymerization. Depolymerization was initiated by diluting polymerized actin, labeled with a fluorescent probe on either lysine-374 or cysteine-375, to a concentration well below the critical into a medium containing free villin and various concentrations of calcium ... More
A re-examination of the interaction of vinculin with actin.
AuthorsWilkins JA, Lin S
JournalJ Cell Biol
PubMed ID3005334
'Vinculin prepared by published procedures (i.e., Feramisco, J. R., and K. Burridge, 1980, J. Biol. Chem., 255:1194-1199) contains contaminants that have been shown by Evans et al. (Evans, R. R., R. M. Robson, and M. H. Stromer, 1984, J. Biol. Chem., 259:3916-3924) to reduce the low-shear viscosity of F-actin solutions. ... More
Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors.
AuthorsSimard JR, Getlik M, Grütter C, Pawar V, Wulfert S, Rabiller M, Rauh D,
JournalJ Am Chem Soc
PubMed ID19572644
'Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less ... More
Ca-dependent binding of actin to gelsolin.
AuthorsKhaitlina S, Hinssen H
JournalFEBS Lett
PubMed ID12067717
'Ca(2+) of 0.3-1.0 microM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca-free conformation, and binding of one actin monomer to the N-terminal half of gelsolin. On the other hand, gelsolin-induced enhancement of pyrene actin fluorescence was observed only above 50 microM Ca(2+), ... More
A biomimetic motility assay provides insight into the mechanism of actin-based motility.
AuthorsWiesner S, Helfer E, Didry D, Ducouret G, Lafuma F, Carlier MF, Pantaloni D
JournalJ Cell Biol
PubMed ID12551957
'Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the ... More
Characterization of the functional domains on the C-terminal region of caldesmon using full-length and mutant caldesmon molecules.
AuthorsWang Z, Horiuchi KY, Chacko S
JournalJ Biol Chem
PubMed ID8567684
'A series of C-terminal deletion mutants of chicken gizzard smooth muscle caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locations of the functional domains of CaD involved in the regulation of actomyosin ATPase and the binding of actin, tropomyosin, ... More
Maleimidobenzoyl-G-actin: structural properties and interaction with skeletal myosin subfragment-1.
AuthorsBettache N, Bertrand R, Kassab R
JournalBiochemistry
PubMed ID2271579
'We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt--and myosin subfragment 1 (S-1)-induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region ... More