Rate Of ADP Dissociation Research Articles

The cardiomyocyte origins of diastolic dysfunction: cellular components of myocardial "stiffness".

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca2+ flux and Ca2+ levels in diastole, the troponin-tropomyosin complex role as a Ca2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

The Local Environment of Loop Switch 1 Modulates the Rate of ATP-Induced Dissociation of Human Cardiac Actomyosin.

Two isoforms of human cardiac myosin, alpha and beta, share significant sequence similarities but show different kinetics. The alpha isoform is a faster motor; it spends less time being strongly bound to actin during the actomyosin cycle. With alpha isoform, actomyosin dissociates faster upon ATP binding, and the affinity of ADP to actomyosin is weaker. One can suggest that the isoform-specific actomyosin kinetics is regulated at the nucleotide binding site of human cardiac myosin. Myosin is a P-loop ATPase; the nucleotide-binding site consists of P-loop and loops switch 1 and 2. All three loops position MgATP for successful hydrolysis. Loops sequence is conserved in both myosin isoforms, and we hypothesize that the isoform-specific structural element near the active site regulates the rate of nucleotide binding and release. Previously we ran molecular dynamics simulations and found that loop S291-E317 near loop switch 1 is more compact and exhibits larger fluctuations of the position of amino acid residues in beta isoform than in alpha. In alpha isoform, the loop forms a salt bridge with loop switch 1, the bridge is not present in beta isoform. Two isoleucines I303 and I313 of loop S291-E317 are replaced with valines in alpha isoform. We introduced a double mutation I303V:I313V in beta isoform background and studied how the mutation affects the rate of ATP binding and ADP dissociation from actomyosin. We found that ATP-induced actomyosin dissociation occurs faster in the mutant, but the rate of ADP release remains the same as in the wild-type beta isoform. Due to the proximity of loop S291-E317 and loop switch 1, a faster rate of ATP-induced actomyosin dissociation indicates that loop S291-E317 affects structural dynamics of loop switch 1, and that loop switch 1 controls ATP binding to the active site. A similar rate of ADP dissociation from actomyosin in the mutant and wild-type myosin constructs indicates that loop switch 1 does not control ADP release from actomyosin.

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation.

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450, restricting the flexibility of loop 1, was previously discovered in molecular dynamics simulations. Earlier it was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulate ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. To examine the hypothesis we expressed the wild type and mutant of the myosin head construct (1-843 amino acid residues) in differentiated C2C12 cells, and the kinetics of ATP-induced actomyosin dissociation and ADP release were characterized using stopped-flow spectrofluorometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. The flexibility of loop 1 does not affect the rate of ADP release from human cardiac actomyosin.

Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding.

Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the SH1-SH2 helix of the myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the SH1-SH2 helix modulate the affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C2C12 cells, and the duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. The mutant R694N showed a faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus indicating that electrostatic interactions within the SH1-SH2 helix region of human cardiac myosin modulate ADP release and thus, the duration of the strongly bound state of actomyosin.

Macromolecular Crowding Affects the Rate of ADP Release from Actomyosin

We have used macromolecular crowding agent Ficoll PM 70 to mimic the intracellular environment and study the effect of crowding on the kinetics of actomyosin dissociation and ADP release. Our previous studies of the heavily mutated D. discoideum myosin S1 construct showed that the effect of macromolecular crowding results in a more compact state of the myosin head. In the current study, we have used the wild-type rabbit skeletal myosin S1. Addition of 10% w/v Ficoll led to the faster rate of ATP induced actomyosin dissociation, in agreement with the excluded volume theory. The kinetics of ADP dissociation from actomyosin is best fitted to the two-exponential function. One component of the kinetics transient reflects ADP dissociation, which was slower than the rate of ADP dissociation without Ficoll in solution. Our data show that ADP binds actomyosin stronger in the presence of Ficoll. The rate of the second component of the transient does not depend on the concentration of nucleotides in solution. This component of the observed transient can be interpreted as the kinetics of myosin conformational change, leading to the opening of the active site for nucleotide to bind. We conclude that macromolecular crowding not only increases the chemical potential of solutes, but affects protein structural state due to change in the osmotic pressure.

Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.

Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator.

Effect of N-Terminal Extension of Cardiac Troponin I on the Ca(2+) Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils.

Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had a lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped-flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second-order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was 3-fold faster than that of wild-type myofibrils at low Ca(2+) concentrations. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentration, while the wild-type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.

Omecamtiv Mecarbil modulates the kinetic and motile properties of porcine β-cardiac myosin.

We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector of cardiac muscle myosin, on the kinetic and "in vitro" motility properties of the porcine ventricular heavy meromyosin (PV-HMM). Omecamtiv Mecarbil increases the equilibrium constant of the hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as determined by quench flow, but the maximal rates of both the hydrolysis step and tryptophan fluorescence increase are unchanged by the drug. OM also increases the amplitude of the fast phase of phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production in muscle by 4-fold. These results suggest a mechanism in which hydrolysis of M-ATP to M-ADP-Pi occurs both before and after the recovery stroke, but rapid acceleration of phosphate dissociation by actin occurs only on post-recovery stroke A-M-ADP-Pi. One of the more dramatic effects of OM on PV-HMM is a 14-fold decrease in the unloaded shortening velocity measured by the in vitro motility assay. The increase in flux through phosphate dissociation and the unchanged rate of ADP dissociation (AM-ADP → AM + ADP) by the drug produce a higher duty ratio motor in which a larger fraction of myosin heads are strongly bound to actin filaments. The increased internal load produced by a larger fraction of strongly attached crossbridges explains the reduced rate of in vitro motility velocity in the presence of OM and predicts that the drug will produce slower and stronger contraction of cardiac muscle.

The Regulation of Actomyosin ATPase in Cardiac Muscle by the N-Terminal Extension of Cardiac Troponin T

Cardiac troponin T (cTnT) and cardiac troponin I (cTnI) both have an amino-terminal variable region that plays a role in modifying the overall protein conformation and functions in the calcium-dependent regulation of cardiac muscle function. Previous studies have showed that in comparison with wild type controls, transgenic mouse hearts over-expressing cTnT lacking the N-terminal variable region (cTnT-ND) had an increased rate of relaxation. In contrast, transgenic mouse heart over-expressing cTnI lacking the N-terminal extension (cTnI-ND) had lower sensitivity to calcium activation of ATPase, resulting enhanced ventricular relaxation and Frank-Starling response. Recently, we demonstrated that the second order mant-dATP binding rate of cardiac myofibrils containing cTnI-ND was three-fold as fast as that of wild type myofibrils in low [Ca2+]. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentrations, while the wild type controls were not significantly affected. These results from native cardiac myofibrils under physiological conditions indicate that the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase. In the present study, we employed the same techniques in order to investigate which step(s) of the ATPase cycle is regulated by the N-terminal variable region of cTnT. mant-ATP binding, mant-ADP/ADP dissociation, and phosphate releasing rate of cTnT-ND myofibrils are examined and the results will provide novel information dissecting the function of the two troponin subunits and their posttranslational modifications.

Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1

Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin were high (Kactin = 4 μM). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s(-1), respectively). Physiological concentrations of free Mg(2+) modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.

Nucleotide Release Sequences in the Protein Kinase SRPK1 Accelerate Substrate Phosphorylation

Protein kinases are essential signaling enzymes that transfer phosphates from bound ATP to select amino acids in protein targets. For most kinases, the phosphoryl transfer step is highly efficient, while the rate-limiting step for substrate processing involves slow release of the product ADP. It is generally thought that structural factors intrinsic to the kinase domain and the nucleotide-binding pocket control this step and consequently the efficiency of protein phosphorylation for these cases. However, the kinase domains of protein kinases are commonly flanked by sequences that regulate catalytic function. To address whether such sequences could alter nucleotide exchange and, thus, regulate protein phosphorylation, the presence of activating residues external to the kinase domain was probed in the serine protein kinase SRPK1. Deletion analyses indicate that a small segment of a large spacer insert domain and a portion of an N-terminal extension function cooperatively to increase nucleotide exchange. The data point to a new mode of protein kinase regulation in which select sequences outside the kinase domain constitute a nucleotide release factor that likely interacts with the small lobe of the kinase domain and enhances protein substrate phosphorylation through increases in ADP dissociation rate.

Direct Visualization of Walking Myosin V Molecules by High-Speed AFM

Facilitated by the processivity of myosin V, numerous single-molecule studies have demonstrated that it moves hand over hand with a ∼36 nm advance for every ATP hydrolysis. However, these results are obtained using optical microscopy and therefore the molecule itself is invisible in the observations. The motor has been visualized using electron microscopy but the obtained images are static. Thus, the detailed structural dynamic of walking myosin V and its functional mechanism are not yet fully described. Here, we directly visualized walking M5-HMM (tail-truncated myosin V) using high-speed AFM with nanometer and sub-second spatiotemporal resolution. The molecular movies provided not only corroborative visual evidence for established and speculated behaviors but also revealed unappreciated ones. First, it was cleared that the forward movement is driven not by bending but by rotation of the leading head. The leading head often showed ‘foot-stomping-like brief detachment and rebinding followed by forward step. In ADP, the coiled-coil tail of the two-headed bound molecule occasionally unwound, which was followed by the leading lever-arm swing, indicating the presence of intramolecular tension responsible for the powerstroke. From these observations, it is concluded that tension for forward movement can be produced without transitioning through a weak-binding ADP-Pi-bound state. Moreover, in low concentrations of ADP, the leading head showed conformational switching between the straight and sharp bend conformations, from which the rate of ADP dissociation from the leading head was estimated to be 0.1/s. This is straightforward evidence that the ATPase cycle is essentially stalled at the leading head. Thus, dynamic visualization of functioning M5-HMM by high-speed AFM lead to a comprehensive understanding of how it operates to function.

Kinetic Analysis of Human α and β Cardiac Myosin Motor Domains

We report the first kinetic analysis of recombinant human and cardiac myosin subfragments-1 (S1). The two isoforms show significant differences; the kinetics of recombinant β-S1 are similar to β-S1 from mammalian tissues whereas α-S1 has more in common with fast skeletal muscle myosins. The second order rate constant of ATP binding is ∼twice as fast for α-S1 (2.3 vs. 1.3 µM1s−1) for both S1 and actoS1. The apparent maximum rate constants of ATP binding to S1 and actoS1 (kmax) are similar for both α and β (∼160 s−1 α- & β-S1, ∼1400 s−1 for α- & β-actoS1). The affinities of ADP for S1 (α, 2.8; β, 0.5 µM) and for AM (α, 152; β, 21 µM) are much tighter for β-S1. The ADP dissociation rate constants are also much slower for β-S1 (2.7 vs. 0.9 s−1 for S1 and >1200 vs. 84 s−1 for actoS1). The ADP release rate constant from AMD is of the right order to limit the shortening velocity of the β isoform. For α-S1 the ADP release is too fast to measure and faster than kmax of ATP induced dissociation of actoS1 and therefore not limiting the contraction velocity. The affinity of both S1s for actin is tight (< 30 nM) and ADP weakens this more than 50 fold to 2.4 µM for α-S1 and less than 5 fold for β-S1. Calculated cycle durations at physiological 5mM ATP predict that both and spend ∼20% of their contractile time actin-bound, but have greatly different cycle times of ∼6 vs. ∼84 ms, respectively. These isoforms are coexpressed in human heart so their diverse kinetics likely lead to interesting dynamics in their cooperative contributions to cardiac contraction.

Mechanism of Regulation of Native Cardiac Muscle Thin Filaments by Rigor Cardiac Myosin-S1 and Calcium

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P(i) and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P(i) by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P(i) for thin filaments such as would be expected by a simple "steric blocking" of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.

Temperature Dependence of MgATP and MgADP Affinity of Fast and Slow Rat Myosin Isoforms: An in Vitro Motility Assay Approach

It has been suggested that the rate of ADP release from acto-myosin and the rate of acto-myosin dissociation by ATP may play different roles to define unloaded shortening velocity of slow and fast myosins and tha their role might change with temperature (Nytray et al. 2006; Iorga et al. 2007). In this study, the in vitro motility assay (IVMA) approach was used to study the effect of MgATP and MgADP on actin sliding velocity (Vf) on slow and fast skeletal myosin isoforms at different temperatures. The velocity of actin filaments sliding on pure slow (myosin 1) and pure fast (myosin 2B) myosin isoforms from the rat was determined in a range of [MgATP] (0.01-2mM) and in the presence or absence of 2mM MgADP. Experiments were performed at 20, 25 and 35 °C. The rate constants of ADP release and ATP binding to acto-myosin were calculated. The inhibition of Vf by MgADP was greater in slow then fast isoforms and the rate constant of ADP release was higher in fast than slow isoform. The results suggest that, in slow isoforms, the maximum velocity could be limited by the rate of ADP dissociation from actomyosin. The ADP inhibitory effect decreased and the rate constant of ADP release increased in each isoform with temperature accounting for the increase in Vf. No differences were found between isoforms and among temperatures in the rate constant of ATP binding. The possibility that the rate of acto-myosin dissociation induced by ATP could play a role in defining Vf in fast isoforms will be discussed.

Myosin 5A Walking Mechanism: The Structural Basis of Slow ADP Dissociatin from the Lead Head

Using electron microscopy and image averaging, we have observed myosin 5a walking along actin filaments in the presence of low concentrations of ATP. Most molecules are attached with 13 actin subunits between heads but ∼10% are bound with 11 or 15 subunit spacings. Most lead heads are in the pre-powerstroke conformation, but some post-powerstroke lead heads are observed, especially at smaller separations where there is less strain in the myosin. Post-powerstroke lead heads have the converter at the front of the motor domain with its lever bent strongly backwards. Lead heads attached at the 13 subunit spacing are 98 % in pre-powerstroke state, tethered there by the trail head. However, heads spaced by 11 subunits are more evenly distributed (60:40) pre- to post-powerstroke. No post-powerstroke lead heads are seen in heads spaced by 15 actin subunits. These results are consistent with an energy difference of 10 kJ/mole between the pre- and post-power stroke conformations at 11 and 13 actin subunit separation. The post-powerstoke lead head is a new attached state of myosin: the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. The rate of ADP dissociation from lead heads measured by stopped-flow fluorescence is >30 fold slower than from trail heads. The slower rate can be explained by a mechanism in which ADP only dissociates from the post-powerstroke state. ADP dissociation from the lead head is therefore inhibited by an unfavorable equilibrium between the pre-and post-powerstroke conformations. Supported by NIH EB00209.

The Effect of Rigor Myosin Upon the pCa of Calcium Binding to Native Cardiac Thin Filaments

We have used double mixing stopped-flow fluorescence to measure the effect of calcium on the kinetics of the dissociation of the hydrolysis product deoxymantADP (mdADP) from cardiac myosin-mdADP and cardiac myosin-mdADP-Pi by native cardiac thin filaments. Increasing the calcium concentration from pCa > 7 to pCa < 4 increased the rate of dissociation of mdADP from cardiac myosin-S1-mdADP-Pi ∼100 fold from 0.5 s−1 to 50 s−1 while the rate of dissociation of mdADP from cardiac myosin-S1-ADP increased only ∼10 fold from 15 s−1 to 150 s−1. Rigor myosin-S1 bound to the thin filaments increased the apparent pCa of mdADP dissociation myosin-S1-mdADP-Pi from 0.12 to 0.79 uM. The change in pCa is similar to the increase in the rate of ADP dissociation but is much less than the acceleration in the rate of rate of product dissociation from myosin-ADP-Pi, ∼ 100 fold. These results indicate that slow dissociation of phosphate limits the rate of ADP dissociation from acto(thinfilaments)myosin-ADP-Pi and that there are different mechanisms for the calcium regulation of dissociation of the two products of myosin ATP hydrolysis, ADP and phosphate. These results support a mechanism in which phosphate dissociation from actomyosin-ADP-Pi is the step of the hydrolysis cycle that is principally regulated by calcium and do not support a mechanism such as the three state mechanism in which the regulation is a result different distributions of thin filament states in presence and absence of bound calcium that occur prior to myosin binding. This work is supported by a NIH HL84604.

ADP but Not Pi Dissociation Contributes to Rate Limitation for Escherichia coli Rho

To define the molecular mechanism by which ATP hydrolysis powers the 5'-->3' travel of homohexameric Escherichia coli transcription termination factor Rho along RNA, rates for association and dissociation of non-RNA substrates and products were measured. Rapid mix/chemical quench and stopped-flow spectrofluorometry measurements were carried out with Rho and [gamma-(32)P]ATP, mantADP, or fluorescently tagged E. coli phosphate-binding protein. The results indicate that the P(i) off-rate is not rate limiting, but at approximately 90 s(-1), the ADP dissociation rate is comparable to the 30 s(-1) k(cat). Previous results indicate that the chemistry step of ATP hydrolysis by Rho is at least 10-fold faster than the overall catalytic cycle. The as yet unmeasured RNA dissociation step, which could be associated with a protein conformation change, might also be a rate-limiting factor.

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell's waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell's waltzer fibroblasts.

Differences in the Mechanisms of Calcium Regulation of the Acceleration of ADP Dissociation from Myosin-ADP and Myosin-ADP-Pi by Native Cardiac Thin Filaments

We have used double mixing stopped-flow fluorescence to measure the kinetics of the dissociation of the hydrolysis products deoxymantADP (mdADP) from cardiac myosin-mdADP and cardiac myosin-mdADP-Pi by native cardiac thin filaments. Increasing the calcium concentration increases the rate of dissociation of mdADP from cardiac myosin-S1-ADP-Pi ∼100 fold from 0.5 s−1 at pCa > 7 to 50 s−1 at pCa < 4. Increasing the calcium concentration increases the rate of dissociation of mdADP from cardiac myosin-S1-ADP-Pi only 10 fold from 15 s−1 at pCa > 7 to 150 s−1 at pCa < 4. These results indicate that slow dissociation of phosphate limits the rate of ADP dissociation from acto(thinfilaments)myosin-ADP-Pi and that there are different mechanisms for the calcium regulation of dissociation of the two products of myosin ATP hydrolysis, ADP and phosphate. These results support a mechanism in which the step of the hydrolysis cycle that is principally regulated by calcium is phosphate dissociation from actomyosin-ADP-Pi and do not support a mechanism such as the three state mechanism in which the regulation is a result different distributions of thin filament states in presence and absence of bound calcium that occur prior to myosin binding. This work is supported by a NIH HL84604.