Nerve growth factor potentiates actomyosin adenosinetriphosphatase (original) (raw)
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Polymerization of G-actin by myosin subfragment 1
Journal of Biological Chemistry
The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar ratios. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mga+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:l ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:l acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(l-pyreny1)iodoacetamide.
Isolation and purification of actomyosin ATPase from mammalian brain
Journal of Neuroscience Methods, 1986
A simple technique for the isolation and purification of mammalian brain actomyosin, based on extraction of whole brains in low ionic-strength buffer, is described. The final preparation of brain actomyosin is obtained in good yield, has relatively high K+-EDTA and Ca2+-ATPase activities, and is substantially free of other ATPases and tubulin. The preparation is useful for initial enzymatic studies and/or as an enrichment step toward purification of the individual protein components. The Mg 2 +-ATPase and K+-EDTA ATPase activities are strongly inhibited by the sulfhydril blocking reagent, pHMB. Interaction between the actin and myosin components can be demonstrated. Brain actomyosin had a distinct electrophoretic profile and enzymatic activity when compared with smooth muscle actomyosin from the aorta.
Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments * □ S
The regulation of the conformational dynamics of cellular actin structures is poorly understood. Results: Myosin and tropomyosin stabilize the conformation of formin-nucleated flexible actin filaments. Conclusion: Actin-binding proteins can play a central role in the establishment of the conformational properties of actin filaments. Significance: Our results add to our understanding of the mechanisms regulating the conformational and functional versatility of the actin cytoskeleton.
Role of sequence 18-29 on actin in actomyosin interactions
Biochemistry, 1993
Affinity-purified polyclonal antibodies prepared against a synthetic peptide corresponding to sequence 18-29 from the N-terminus of rabbit a-skeletal actin reacted with G-and F-actin. Epitope mapping experiments with thrombin and hydroxylamine cleaved actin, and immunochemical assays verified the specificity of antibodies for the 18-29 sequence on actin. The binding of up to 0.5 mol of IgG per mole of actin did not affect the rigor binding of myosin subfragment 1 (S-1) to actin. Similarly, the binding of IgG to actin was not changed by a complete saturation of actin by S-1. In contrast to this, the weak act&-1 interactions in the presence of ATP were strongly inhibited by the 18-29 antibodies. At 25 OC, the act&-1 ATPase activity was inhibited by IgG stronger than the binding of S-lBATPTS to actin. Thus, at this temperature, a catalytic inhibition of the act&-1 system appears to account at least in part for the antibody effect. Act&-1 ATPase activities at 25 OC were inhibited also by Fab(18-29). At 5 OC, the act&-1 ATPase activity and the binding of S-1.ATP to actin were inhibited approximately to the same extent by IgG(18-29). These results are discussed in terms of S-1 binding sites on actin and the possible role of sequence 18-29 in actomyosin interactions.
The Journal of biological chemistry, 1991
Polymerization of G-actin to F-actin was indicated by an increase in light-scattering intensity after the addition of a heptapeptide (Ile-Arg-Ile-Cys(MT)-Arg-Lys-Gly-OEt), an analog of the actin-binding S-site on S-1 heavy chain. The half-maximal concentration of the heptapeptide which induced an increase in the light-scattering intensity at 25 degrees C was about 110 microM, which was in the range of the dissociation constant of this peptide with F-actin. The polymerization of G-actin to F-actin by binding of the heptapeptide was further demonstrated by ultracentrifugal separation, Pi liberation, and electron microscopy. The polymerization of G-actin was induced only by the heptapeptide, but not by fragments of the heptapeptide. The well known acceleration of polymerization of G-actin by the myosin head may be due to the binding of G-actin with the S-site on the myosin head.
Actomyosin interactions in the presence of ATP and the N-terminal segment of actin
Biochemistry - BIOCHEMISTRY-USA, 1992
The binding of myosin subfragment 1 (S-1) to actin in the presence of ATP and the acto-S-1 ATPase activities of acto-S-1 complexes were determined at 5 degrees C under conditions of partial saturation of actin, up to 90%, by antibodies against the first seven N-terminal residues on actin. The antibodies [Fab(1-7)] inhibited strongly the acto-S-1 ATPase and the binding of S-1 to actin in the presence of ATP at low concentrations of S-1, up to 25 microM. Further increases in S-1 concentration resulted in a partial and cooperative recovery of both the binding of S-1 to actin and the acto-S-1 ATPase while causing only limited displacement of Fab(1-7) from actin. The extent to which the binding and the ATPase activity were recovered depended on the saturation of actin by Fab(1-7). The combined amounts of S-1 and Fab binding to actin suggested that the activation of the myosin ATPase activity was due to actin free of Fab. Examination of the acto-S-1 ATPase activities as a function of S-1 bound to actin at different levels of actin saturation by Fab(1-7) revealed that the antibodies inhibited the activation of the bound myosin. Thus, the binding of antibodies to the N-terminal segment of actin can act to inhibit both the binding of S-1 to actin in the presence of ATP and a catalytic step in ATP hydrolysis by actomyosin. The implications of these results to the regulation of actomyosin interaction are discussed.
Isolation and polymerization of brain actin
Journal of Neurobiology, 1975
The studies presented here confirm earlier reports that an actin-like protein is abundant in brain. However, when the traditional procedures for isolating muscle actin are applied to brain, many different proteins are extracted. Tubulin, a major protein in brain with properties similar
Actin's view of actomyosin interface
Biophysical Journal
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/E1 OOA mutants moved in the presence of 0.7% methylcellulose at the velocities of wild-type actin. Without methylcellulose, these mutant filaments, but not the D80/D81 or E83/K84 filaments, dissociated from the assay surface upon addition of ATP. Measurements of myosin subfragment-1 (S1) binding to D24A/D25Aand E99A/E1 OA-polymerized actins in the presence of ATP revealed a threeand twofold decrease in their binding constant, respectively, compared with wild-type actin. In contrast to this, all monomeric actins had the same binding affinity for Si. The rates and extents of polymerization of D24A/D25A and E99A/E1 OA actins by Si were reduced in comparison to wild-type actin. The local structure of subdomain-2 on actin, as probed by subtilisin cleavage, was not altered for either mutant. A twofold decrease in nucleotide exchange was detected for the D24A/D25A mutant actin. These results demonstrate the involvement of the D24/D25 and E99/E1 00 residues in the weak binding of myosin to actin and reveal that residues D80/D81 and E83/K84 do not modulate actomyosin interactions.