Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied the functions of contractile proteins in Acanthamoeba by a combination of structural, biochemical and physiological approaches. We used electron microscopy and image processing to determine the three-dimensional structure of actin and the orientation of the molecule in the actin filament. We measured the rate constants for actin filament elongation and re-evaluated the effect of MgCl2 on the filament nucleation process. In Acanthamoeba actin polymerization is regulated, at least in part, by profilin, which binds to actin monomers, and by capping protein, which both nucleates polymerization and blocks monomer addition at the 'barbed' end of the filament. To test for physiological functions of myosin-II, we produced a monoclonal antibody that inhibits the actin-activated ATPase. When microinjected into living cells, this active-site-specific antibody inhibits amoeboid locomotion. We expect that similar experiments can be used to test for the physiological functions of the other components of the Acanthamoeba contractile system.
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PMID:Actin and myosin function in acanthamoeba. 612 59

Profilin inhibits the rate of nucleation of actin polymerization and the rate of filament elongation and also reduces the concentration of F-actin at steady state. Addition of profilin to solutions of F-actin causes depolymerization. The same steady state concentrations of polymerized and nonpolymerized actin are reached whether profilin is added before initiation of polymerization or after polymerization is complete. The KD for formation of the 1:1 complex between Acanthamoeba profilin and Acanthamoeba actin is in the range of 4 to 11 microM; the KD for the reaction between Acanthamoeba profilin and rabbit skeletal muscle actin is about 60 to 80 microM, irrespective of the concentrations of KCl or MgCl2. The critical concentration of actin for polymerization and the KD for the actin-profilin interaction are independent of each other; therefore, a change in the critical concentration of actin alters the amount of actin bound to profilin at steady state. As a consequence, the presence of profilin greatly amplifies the effects of small changes in the actin critical concentration on the concentration of F-actin. Profilin also inhibits the ATPase activity of monomeric actin, the profilin-actin complex being entirely inactive.
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PMID:The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin. 646 52

Human platelets were obtained in the fully resting state by treating discoid populations with 1.5 mM tetracaine and in the activated state by treatment with 2 microM A-23187. After gel filtration or washing, respectively, platelet suspensions were lysed with 1% Triton X-100 at pH 6.8. The precipitates from resting platelets viewed by negative staining appeared predominantly granular with a few very short microfilaments. They contained polypeptides of 250, 100, 45, 38, 36.5, and 35 Kdaltons, and three small polypeptides including one with the mobility of profilin on SDS gels. Precipitates from activated platelets lacked this low molecular weight band and contained a major band at 200 Kdaltons with the mobility of myosin; these precipitates had significant K+, Ca++ ATPase activity absent from the precipitate of resting platelets. As seen in negative staining, precipitates from activated platelets contained microfilaments arranged as nets or bundles. The granular resting precipitates were transformed in vitro into microfilament bundles by washing the precipitates in buffer at higher pH (7.6) in the presence of 5 X 10(-5) M calcium chloride.
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PMID:Platelet activation and microfilament bundling. 719 75

Two beta-actin mutants, one with proline 38 replaced with alanine (P38A) and the other with cysteine-374 replaced with serine (C374S), as well as the wild-type beta-actin, were expressed in the yeast, S. cerevisiae, purified to homogeneity, and analyzed in vitro for polymerizability and interaction with DNase I, myosin, and profilin. Both mutations interfered with the polymerization of the actin, and with its interaction with myosin. The C374S mutation had the most pronounced effect; it reduced the polymerizability of the actin, abolished its binding to profilin, and filaments containing this mutation moved at reduced rates in the in vitro 'motility assay'. The ATPase activity measured in solutions containing myosin subfragment 1 was similar for both the mutant and wild-type actins.
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PMID:Mutations in beta-actin: influence on polymer formation and on interactions with myosin and profilin. 835 91

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 actin is a useful model for studying the properties of monomeric actin under polymerization conditions. Addition of salt, for example, produced fluorescence changes possibly reflecting conformational transitions but did not lead to the development of phalloidin binding capacity. Cross-linking of the two cysteine residues also caused a decrease in the nucleotide exchange rate by a factor of ca. 3, an effect that was fully reversed by the addition of KCl. Cross-linked actin inhibits DNase I to the same extent as G-actin and binds thymosin beta 4 and profilin as shown by cross-linking studies. Capping capacity for the barbed end of the filament was not observed, although it might have been expected from the fact that both ends of the cross-link are anchored to subdomain 1. Using the 61-FITC derivative of c-A we showed that c-A is able to bind to myosin S1 with a KD in the microM range. In agreement with this, c-A shows actomyosin ATPase activity with a Kapp comparable to that of F-actin, but a Vmax decreased by a factor of ca. 11. The c-A myosin S1 complex provides the hitherto smallest model of actomyosin, which appears promising for crystallization and X-ray analysis.
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PMID:Cross-link between cys 374 and cys 10 of actin abolishes polymerizability and allows study of the properties of the "F-actin monomer". 855 83

Effects of ADP-ribosylation of skeletal muscle alpha-actin by Clostridium perfringens iota toxin and by turkey erythrocyte ADP-ribosyltransferase A on profilin-regulated nucleotide exchange and ATPase activity were compared. ADP-ribosylation of actin at Arg 177 by Clostridium perfringens iota toxin increased the nucleotide dissociation rate from 2.2 x 10(-3) s-1 to 4.5 x 10(-3) s-1 without affecting the profilin-induced stimulation of nucleotide exchange. In contrast, ADP-ribosylation of actin at Arg95/Arg372 induced by turkey erythrocyte transferase decreased the nucleotide dissociation rate to 1.5 x 10(3) s-1 and inhibited the profilin-induced stimulation of nucleotide exchange. Whereas toxin-induced ADP-ribosylation at Arg177 blocked actin ATPase, basal G-actin ATPase was not altered by ADP-ribosylation at Arg95/Arg372 but inhibited profilin effects on actin ATPase.
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PMID:ADP-ribosylation of actin by Clostridium perfringens iota toxin and turkey erythrocyte ADP-ribosyltransferase A: effects on profilin-regulated nucleotide exchange and ATPase activity. 897 27

The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants showed defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p formed complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localized to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton.
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PMID:Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis. 908 82

Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 microM under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 microM under physiological conditions. The PAcov polymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.
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PMID:Filament assembly from profilin-actin. 1003 10

We have investigated the effects of profilin on nucleotide binding to actin and on steady state actin polymerization. The rate constants for the dissociation of ATP and ADP from monomeric Mg-actin at physiological conditions are 0.003 and 0.009 s-1, respectively. Profilin increases these dissociation rate constants to 0.08 s-1 for MgATP-actin and 1.4 s-1 for MgADP-actin. Thus, profilin can increase the rate of exchange of actin-bound ADP for ATP by 140-fold. The affinity of profilin for monomeric actin is found to be similar for MgATP-actin and MgADP-actin. Continuous sonication was used to allow study of solutions having sustained high filament end concentrations. During sonication at steady state, F-actin depolymerizes toward the critical concentration of ADP-actin [Pantaloni, D., et al. (1984)J. Biol. Chem. 259, 6274-6283], our analysis indicates that under these conditions a significant number of filaments contain terminal ADP-actin subunits. Addition of profilin to this system increases the polymer concentration and increases the steady state ATPase activity during sonication. These data are explained by the fast exchange of ATP for ADP on the profilin-ADP-actin complex, resulting in rapid ATP-actin regeneration. An important function of profilin may be to provide the growing ends of filaments with ATP-actin during periods when the monomer cycling rate exceeds the intrinsic nucleotide exchange rate of monomeric actin.
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PMID:Impact of profilin on actin-bound nucleotide exchange and actin polymerization dynamics. 1005 48

Profilin and beta/gamma-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with N-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and P(i) release from the actin subunit as it assumes its stable conformation in the helical filament.
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PMID:A cross-linked profilin-actin heterodimer interferes with elongation at the fast-growing end of F-actin. 1184 98


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