The limiting rate of the ATP‐mediated dissociation of actin from rabbit skeletal muscle myosin subfragment 1

Millar, N C; Geeves, Michael A.

doi:10.1016/0014-5793(83)80954-5

Cited by 84 publications

(85 citation statements)

References 19 publications

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“…2A shows the fluorescence transients observed at 15°C when 0.5 M actin-S1 is mixed with 20 M ATP in the stopped-flow fluorimeter. The observed transient for pso S1 was well described by a single exponential equation, as has been previously reported (28), with an observed rate constant (k obs ) of 103 s Ϫ1 and amplitude of 77%. For sol S1, the transient in Fig.…”

Section: Resultssupporting

confidence: 83%

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Iorga

Adamek

Geeves

2007

Journal of Biological Chemistry

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Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the ␤-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actinmyosin dissociates at up to ϳ1000 s ؊1, a very similar rate constant to MHC-2, but 10 -15% of the complex must go through a slow isomerization (ϳ20 s ؊1 ) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actinmyosin⅐ADP and provide evidence of three closely related actinmyosin⅐ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (ϳ20 s ؊1 ), and a third complex that releases ADP too slowly (ϳ6 s ؊1 ) to be on the main ATPase pathway. The role of these actin-myosin⅐ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes (1-6). During the myosin ATPase cycle, the myosin motor domain (or cross-bridge) undergoes a series of conformational changes coupled to the binding of nucleotide and actin, which results in a translocation of the myosin cargo domain relative to the actin track (7). The sequence of molecular events in the actin-myosin cross-bridge cycle appears essentially the same for all myosins so far studied. The different mechanochemical properties of each myosin are attributed to a modulation of the rates and equilibrium constants of individual molecular events to match each myosin to its physiological role.The most widely studied myosins are the vertebrate, striatedmuscle myosins, which define the class II myosins. This class of myosins have a dimerization domain that forms a long coiled-coil and which can assemble further to form the backbone of the bipolar thick myosin filament. Of the class II myosin, the myosin heavy chain 2 (MHC-2) 4 isoform found primarily in white, anaerobic, fast-contracting, skeletal muscle has been most thoroughly described at both the molecular and physiological levels. In adult, mammalian, muscle tissue the MHC-2 is found as various isoforms (e.g. 2a, 2b, 2x) and it has been established that the essential mechanical properties of the muscle fiber contraction (e.g. maximum velocity of shortening, force per cross-bridge) are properties of the MHC present in the t...

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Section: Resultssupporting

confidence: 83%

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Iorga

Adamek

Geeves

2007

Journal of Biological Chemistry

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“…The unbinding of ATP is very slow, Յ7 ϫ 10 Ϫ4 s Ϫ1 , and we interpret this as being due to the slow reversal of the hydrolysis step. Our value for the release of P i and detachment of the rear head is high, Ͼ1,500 s Ϫ1 , but the ATP-induced detachment of myosin from actin is thought to occur at Ϸ5,000 s Ϫ1 (43). The second-order rate constant for ADP rebinding of 0.25 M Ϫ1 ⅐s Ϫ1 is a little smaller than …”

Section: Discussionmentioning

confidence: 70%

Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism

Schief

Clark

Crevenna

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

116

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The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K M (the [ATP] required for half-maximal speed) was 28 ؎ 1 M, and the maximum speed was 904 nm͞s. Pi behaved as a competitive inhibitor with K I ‫؍‬ 9 ؎ 1 mM. ADP behaved approximately as a competitive inhibitor with K I ‫؍‬ 35 ؎ 2 M. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and͞or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P i is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P i before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P i release from the other.crossbridge cycle ͉ motor protein ͉ chemomechanical coupling K inesin is a motor protein that couples the free energy derived from the hydrolysis of ATP into mechanical work used to drive cellular motility. Two properties of kinesin are essential for its function. First, kinesin undergoes directed motion. It moves toward the plus-or fast-growing end of a microtubule as it transports membrane-bounded organelles toward the periphery of neurons and other cells (1) where the plus ends of microtubules are usually located (2). And second, kinesin is processive. An individual kinesin molecule can move up to several microns along a microtubule without dissociating (3). Processivity ensures that even a small vesicle with just one or two motors on its surface will spend a large fraction of its time attached to and moving along a microtubule (as opposed to diffusing in the cytoplasm).The directed and processive motion of kinesin is tightly coupled to the hydrolysis of ATP. High-precision tracking of kinesin-coated beads reveals that kinesin takes 8-nm steps (4) from one tubulin dimer to the next along a path that is parallel to the axis of the microtubule (5). Direct measurement of the ATPase rate and its correlation to the speed of movement indicates that in standard motility assays where the load is low, kinesin hydrolyses only one ATP per 8-nm step (6). A stoichiometry of 1 step per ATP implies that each cycle of ATP hydrolysis [the binding of ATP to kinesin's nucleotide-binding pocket, its hydrolysi...

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“…In particular, the velocity of L2 (ϩ4) was considerably lower than that of the wild type as mentioned above. In general, velocity depends on the time of strongly bound state with actin, which is determined by ADP dissociation rate from acto-myosin⅐ADP complex and actomyosin dissociation rate by ATP binding (32,33). Thus, it is anticipated that the slow movement of L2 (ϩ4) is due to reduction in either of these 2 rates or in both.…”

Section: Loop 3 Mutantsmentioning

confidence: 99%

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

Ito

Yamaguchi

Yanase

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected Vmax and Kapp of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected Kapp and the velocity, although it did not affect Vmax. Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.actin ͉ ATPase ͉ motility ͉ cytoplasmic streaming ͉ molecular engineering C ytoplasmic streaming in characean algal cells is extremely fast, and this streaming is brought about by the movement of myosin-coated organelles along actin filament bundles fixed inside the cell (1). Myosin purified from Chara corallina could translocate actin filaments in the in vitro motility assay at a velocity comparable to that of the cytoplasmic streaming (approximately 50 m s Ϫ1 ) (2, 3). This velocity is about 10 times faster than that of the fast skeletal muscle myosin and the Chara myosin is the fastest motor protein known so far. We have cloned cDNA of the Chara myosin heavy chain (4) and succeeded in expressing functional motor domain (5, 6). The velocity of the expressed motor domain measured by in vitro motility assay was comparable to that of the native Chara myosin if we consider the difference in the lever arm length. Relevant steps in actomyosin ATPase cycle to the sliding velocity are ADP release from actomyosin and ATP reassociation resulting in actin-myosin dissociation. Kinetic analyses of Chara myosin motor domain revealed that both the ADP release and the ATP-induced dissociation from actin were very fast (6). Time spent in the strongly bound state with actin, which was estimated from these rates, was less than 1 ms. This very short strongly bound state, together with a large step size (7), are probably the reason for the very high velocity of Chara myosin. Actin-activated ATPase activity of expressed Chara motor domain was approximately 500 s Ϫ1 head Ϫ1

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The limiting rate of the ATP‐mediated dissociation of actin from rabbit skeletal muscle myosin subfragment 1

Cited by 84 publications

References 19 publications

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

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