scholarly journals Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin.

1990 ◽  
Vol 111 (2) ◽  
pp. 465-470 ◽  
Author(s):  
D H Schwyter ◽  
S J Kron ◽  
Y Y Toyoshima ◽  
J A Spudich ◽  
E Reisler
Keyword(s):  
Atpase Activity ◽  
Linear Dependence ◽  
Actin Filaments ◽  
Structural Integrity ◽  
Heavy Meromyosin ◽  
Dynamic Changes ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Coated Surface

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.

Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans

2001 ◽  
Vol 280 (4) ◽  
pp. C782-C788 ◽  
Author(s):  
Peter Höök ◽  
Vidyasagar Sriramoju ◽  
Lars Larsson
Keyword(s):  
Posttranslational Modifications ◽  
Actin Filaments ◽  
Mammalian Species ◽  
Type I ◽  
In Vitro Motility Assay ◽  
Sliding Speed ◽  
Myosin Heavy Chain Isoform ◽  
In Vitro Motility ◽  
Effects Of Aging

The effects of aging on the mechanical properties of myosin were measured in 87 fibers from muscles of humans ( n = 40), rats ( n = 21), and mice ( n = 26) using a single fiber in vitro motility assay. Irrespective of species, an 18–25% aging-related slowing in the speed of actin filaments was observed from 62 single fibers expressing the slow (type I) β-myosin heavy chain isoform. The mechanisms underlying the aging-related slowing of motility speed remain unknown, but it is suggested that posttranslational modifications of myosin by oxidative stress, glycation, or nitration play an important role. The aging-related slowing in the speed of actin filaments propelled by the type I myosin was confirmed in three mammalian species with an ∼3,400-fold difference in body size. Motility speed from human myosin was 3-fold slower than from myosin of the ∼3,400-fold smaller mouse and approximately twofold slower when compared with the ∼130-fold smaller rat, irrespective of age. A strong correlation was observed between the log values of actin sliding speed and body mass, suggesting that the effects of scaling is, at least in part, due to altered functional properties of the motor protein itself.

Sliding velocity of isolated rabbit cardiac myosin correlates with isozyme distribution

1992 ◽  
Vol 263 (2) ◽  
pp. H464-H472 ◽  
Author(s):  
H. Yamashita ◽  
S. Sugiura ◽  
T. Serizawa ◽  
T. Sugimoto ◽  
M. Iizuka ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Motility Assay ◽  
Cardiac Myosin ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Actin Cables

To investigate the relationship between the mechanical and biochemical properties of cardiac myosin, the sliding velocity of isolated cardiac myosin obtained from both euthyroid and hyperthyroid rabbits on actin cables was measured with an in vitro motility assay system. Ten rabbits (T) were treated with L-thyroxine to induce hyperthyroidism, and eight nontreated animals (N) were used as controls. Myosin was purified from the left ventricles of anesthetized animals. Myosin isozyme content was analyzed by the pyrophosphate gel electrophoresis method, and myosin adenosinetriphosphatase (ATPase) activity was determined on the same sample. Long well-organized actin cables of green algae, Nitellopsis, were used in the in vitro motility assay. Small latex beads were coated with purified cardiac myosin and introduced onto the Nitellopsis actin cables. Active unidirectional movement of the beads on the actin cables was observed under a photomicroscope, and the velocity was measured. The velocity was dependent on ATP concentrations, and the optimal pH for bead movement was approximately 7.0-7.5. The mean velocity was higher in T than in N (0.66 +/- 0.12 vs. 0.32 +/- 0.09 micron/s, P less than 0.01). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also higher in T than in N (0.691 +/- 0.072 vs. 0.335 +/- 0.072 microM Pi.mg-1.min-1, P less than 0.01, and 79 +/- 12 vs. 26 +/- 7%, P less than 0.01, respectively). The velocity of myosin closely correlated with both Ca(+2)-activated myosin ATPase activity (r = 0.87, P less than 0.01) and the percentage of alpha-myosin heavy chain (r = 0.87, P less than 0.01).

Unidirectional Movement Of Fascin-induced Actin Bundle On Heavy Meromyosin In An In Vitro Motility Assay

2008 ◽  
Vol 40 (Supplement) ◽  
pp. S297
Author(s):  
Hideyo Takatsuki ◽  
Kevin M. Rice ◽  
Shinichi Asano ◽  
Devashish Desai ◽  
Madhukar Kolli ◽  
...  
Keyword(s):  
Motility Assay ◽  
Heavy Meromyosin ◽  
In Vitro Motility Assay ◽  
Actin Bundle ◽  
In Vitro Motility ◽  
Unidirectional Movement

The in vitro motility assay parameters of actin filaments from Mytilus edulis exposed in vivo to copper ions

2009 ◽  
Vol 491 (1-2) ◽  
pp. 32-38 ◽  
Author(s):  
Natalia N. Vikhoreva ◽  
Petr G. Vikhorev ◽  
Maria A. Fedorova ◽  
Ralf Hoffmann ◽  
Alf Månsson ◽  
...  
Keyword(s):  
Mytilus Edulis ◽  
Actin Filaments ◽  
Copper Ions ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

scholarly journals Acceleration of the sliding movement of actin filaments with the use of a non-motile mutant myosin in in vitro motility assays driven by skeletal muscle heavy meromyosin

PLoS ONE ◽  
2017 ◽  
Vol 12 (7) ◽  
pp. e0181171 ◽  
Author(s):  
Kohei Iwase ◽  
Masateru Tanaka ◽  
Keiko Hirose ◽  
Taro Q. P. Uyeda ◽  
Hajime Honda
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
In Vitro Motility

scholarly journals A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments

2000 ◽  
Vol 350 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Wu BING ◽  
Adam KNOTT ◽  
Steven B. MARSTON
Keyword(s):  
Actin Filaments ◽  
Isometric Force ◽  
Critical Concentration ◽  
Isometric Tension ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
Simple Method ◽  
In Vitro Motility ◽  
Muscle Tropomyosin

We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the invitro motility assay. Immobilized α-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of α-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing α-actinin concentration. The concentration of α-actinin needed to stop all filaments from moving (0.8µg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the ‘index of retardation’ as the concentration of α-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized α-actinin on motility in the presence of 10mM Pi we found that the index of retardation was 0.62±0.07 (n = 3) times that in the absence of Pi. This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to Pi. In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9–3.4-fold greater than that of actin with all tropomyosin isoforms.

scholarly journals Myosin-gelsolin cooperativity in actin filament severing and actomyosin motor activity

2020 ◽  
Author(s):  
Venukumar Vemula ◽  
Tamas Huber ◽  
Marko Usaj ◽  
Beáta Bugyi ◽  
Alf Mansson
Keyword(s):  
Motor Activity ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Cellular Motility ◽  
In Vitro Motility Assay ◽  
Cooperative Effects ◽  
In Vitro Motility ◽  
Filament Dynamics

AbstractActin is a major intracellular protein with key functions in cellular motility, signalling and structural rearrangements. Its dynamic behavior with actin filaments (F-actin) polymerising and depolymerising in response to intracellular changes, is controlled by actin-binding proteins (ABPs). Gelsolin is one of the most potent filament severing ABPs. However, myosin motors that interact with actin in the presence of ATP also produce actin filament fragmentation through motor induced shearing forces. To test the idea that gelsolin and myosin cooperate in these processes we used the in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin (5 nM) at very low [Ca2+] (free [Ca2+] ∼6.8 nM) appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM [MgATP], an effect that was increased at increased HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. As further support of myosin-gelsolin cooperativity we observed reduced sliding velocity of the HMM propelled filaments in the presence of gelsolin. Overall, the results corroborate ideas for cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity, leading among other effects to enhanced F-actin severing of possible physiological relevance.

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