Biomedical subjects

M A Geeves

Publications and source records attributed to M A Geeves.

At least 55 records · Page 3Linked to original sources

PubMed1997

Actin-tropomyosin activation of myosin subfragment 1 ATPase and thin filament cooperativity. The role of tropomyosin flexibility and end-to-end interactions.

Tropomyosin (Tm) bound to actin induces cooperative activation of actomyosin subfragment 1 (actin-S1) ATPase, observed as a sigmoid ATPase vs [S1] dependence. The activation is much steeper for gizzard muscle Tm (GTm) than for rabbit skeletal Tm (RSTm). To investigate if this greater cooperativity is due to increased communication between GTms along the thin filament, we studied effects of S1 binding on the state of actin-Tm using the fluorescence of pyrene-labeled Tm. Kinetic and equilibrium studies provided values for n, the apparent cooperative unit size [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273]. We report comparative studies of Tm-actin-S1 ATPase with values of n using GTm, RSTm, and 5aTm, a 1/7 shorter nonmuscle Tm from rat fibroblast cells [Pittenger, M. F., et al. (1994) Curr. Opin. Cell Biol., 6, 96]. 5aTm and GTm produce similar cooperative activation of actin-S1 ATPase and have similar n values that are 2-fold greater than RSTm, indicating a correlation between ATPase activation and n value. This appears to be due to the similarity of the C-terminal amino acid sequences of 5a and GTm which produce strong end-to-end interactions. The results are discussed in terms of a continuous flexible Tm strand on the actin filament.

Actin Cytoskeleton↗

PubMed1997

Dictyostelium discoideum myosin II: characterization of functional myosin motor fragments.

The transient kinetic properties of the recombinant myosin head fragments M761 and M781, which both lack the light chain binding domain (LCBD) and correspond to the first 761 and 781 residues of Dictyostelium discoideum myosin II, were compared with those of the subfragment 1-like fragment M864 and a shorter catalytic domain fragment M754. The properties of M761, M781, and M864 are almost identical in regard to nucleotide binding, nucleotide hydrolysis, actin binding, and the interactions between actin and nucleotide binding sites. Only the rate of the hydrolysis step was significantly faster for M761 and the affinity of M781 for actin significantly weaker than for M864. This indicates that the LCBD plays no major role in the biochemical behavior of the myosin head. In contrast, loss of the peptide between 754 and 761 produced several major changes in the property of M754 as documented previously [Woodward, S. K. A., Geeves, M. A., & Manstein, D. J. (1995) Biochemistry 34, 16056-16064]. We further show that C-terminal extension of M761 with one or two alpha-actinin repeats has very little effect on the behavior of the protein. The recombinant nature of M761 and the fact that it can be produced and purified in large amounts make it an ideal construct for systematic studies of the structure, kinetics, and function of the myosin motor.

Actins↗

PubMed1996

Myosin motors with artificial lever arms.

The myosin head consists of a globular catalytic domain and a light chain binding domain (LCBD). The coupling efficiency between ATP hydrolysis and myosin-induced actin movement is known to decline as the LCBD is truncated or destabilized. However, it was not clear whether the observed alteration in the production of force and movement reflects only the mechanical changes to the length of the LCBD or whether these changes also affect the kinetic properties of the catalytic domain. Here we show that replacement of the LCBD with genetically engineered domains of similar rigidity and dimensions produces functional molecular motors with unchanged kinetic properties. The resulting single-chain, single-headed motors were produced in Dictyostelium discoideum and obtained after purification from a standard peptone-based growth medium at levels of up to 12 mg/l. Their actin motility properties are similar or greater than those of native myosin. Rates of 2.5 and 3.3 microm/s were observed for motor domains fused to one or two of these domains, respectively. Their kinetic and functional similarity to the extensively studied myosin subfragment 1 (S1) and their accessibility to molecular genetic approaches makes these simple constructs ideal models for the investigation of chemo-mechanical coupling in the myosin motor.

Actin Cytoskeleton↗

PubMed1996

Bisphosphorylation of cardiac troponin I modulates the Ca(2+)-dependent binding of myosin subfragment S1 to reconstituted thin filaments.

We have reconstituted thin filaments comprising pyrene-labelled actin (pyr-actin), tropomyosin (Tm) and cardiac troponin (cTn). cTn was isolated in two defined phosphorylation states; completely dephosphorylated on all subunits and with only the cTnI subunit bisphosphorylated. The thin filament was saturated with cTn at a pyr-actin/Tm/cTn ratio of 7:1:1. The calcium-dependent binding of S1 to thin filaments was measured in a stopped-flow spectrophotometer and the dependence of the observed rate constant on [Ca2+] fitted to the Hill equation. The only significant difference between the two phosphorylation states of the filaments was a 0.36 decrease in the pCa50 on bisphosphorylation.

Actin Cytoskeleton↗

PubMed1996

Effects of hydrostatic pressure on fatiguing frog muscle fibres.

Effects of increased hydrostatic pressure (range 0.1-10 MPa) on isometric twitch and tetanic contractions of single, intact, frog muscle fibres were examined at 4, 11 and 21 degrees C and at different stages of fatigue. Twitch tension was potentiated by pressure at all temperatures, but the extent of potentiation was more pronounced at higher temperatures (34% MPa-1 at 21 degrees C, compared to 8% MPa-1 at 4 degrees C). Tetanic tension was depressed by pressure at 4 degrees C (approximately 0.7% MPa-1) but was potentiated by pressure at 21 degrees C (approximately 0.4% MPa-1). The effect of hydrostatic pressure on the tetanic tension was dependent on the fatigue status of the muscle fibre: during the early stages of fatigue (when tetanic tension was depressed by < 20%), high pressure produced a tension depression (as in an unfatigued muscle fibre), whilst during the later stages of fatigue high pressure induced a significant potentiation of tetanic. Our results support the suggestion that excitation-contraction coupling and contractile activation are impaired during late fatigue. Pressure-effects were basically similar to caffeine-effects under a variety of conditions, suggesting that an enhancement of Ca2+ release may be contributory to potentiation of twitch tension and, in severely, fatigued muscle, potentiation of tetanic tension. In the rested state and during early fatigue the main effect of pressure is an inhibition of the crossbridge cycle.

Animals↗

PubMed1996

A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using microgram quantities of protein.

The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is <<1 microM at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a Kd below 0.1 microM. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for Kd's as low as 10 nM and we demonstrate that the Kd's can be estimated using only microgram quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.

Actins↗

PubMed1995

Separation and characterization of the two functional regions of troponin involved in muscle thin filament regulation.

Mild proteolytic cleavage of the troponin complex yields TnT1, the N-terminal fragment of troponin T, and TnT2IC, a complex of the C-terminal fragment of troponin T (TnT2) with troponin I (TnI) and troponin C (TnC) [Morris, E. P., & Lehrer, S. S. (1984) Biochemistry 23, 2214-2220]. Both TnT1 and TnT2IC bind tightly to the tropomyosin.actin (Tm.actin) thin filament and influence the interaction of myosin subfragment 1 (S1) with Tm.actin. TnT1 does not affect the rate of S1 binding to Tm.actin but does increase the cooperativity with which S1 "turns on" Tm.actin, monitored by the excimer fluorescence of a pyrene label attached to Cys 190 of Tm [Geeves, M.A., & Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. The apparent cooperative unit size of Tm.actin is increased from 6 to 9 by TnT1 and to 12 by whole troponin. In contrast, TnT2IC has no effect on the cooperativity of Tm.actin but does make the apparent S1-binding rate constant, kapp, Ca(2+)-sensitive; i.e., in the absence of Ca2+, kapp is reduced 2-3-fold by both TnT2IC and whole troponin. Thus, the N- and C-terminal regions of TnT appear to act independently in modulating effects of S1 binding to the Tm.actin thin filament that are important in regulation.

Actins↗

PubMed1995

Kinetic characterization of the catalytic domain of Dictyostelium discoideum myosin.

The myosin head consists of a globular motor or catalytic domain that contains both the catalytic and actin binding sites, and a neck region which consists of a 8.5 nm alpha-helix that emerges from the globular part of the heavy chain and is stabilized by the binding of the essential and regulatory light chains. High levels of M754, a recombinant polyhistidine-tagged catalytic domain-like fragment of myosin II, were produced in Dictyostelium discoideum and purified using a rapid extraction protocol and metal chelate chromatography. Approximately 1.2 mg of homogeneous, functional protein was obtained per gram of cells. Kinetic analysis of M754 showed that the recombinant protein still has all the typical properties of a myosin ATPase. However, the removal of the light chain domain does have a pronounced effect on enzymatic activity. Nucleotide on-rates are 7-16-fold slower for M754 than for a myosin head fragment that includes the neck region. In contrast, the rate of ATP binding and dissociating the actin-bound catalytic domain is 10-fold increased. Overall the results indicate that the truncation of the heavy chain affects the nucleotide binding site and the communication between the nucleotide and actin binding sites. Furthermore, it seems that the nucleotide site of M754 is not fully formed but binding to actin or ATP stabilizes the structure in general and the nucleotide binding site in particular.

Adenosine Diphosphate↗

PubMed1995

Kinetics of nucleotide binding to pyruvate carboxylase.

The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl Coenzyme A↗

PubMed1995

Characterization of the equilibrium between blocked and closed states of muscle thin filaments.

We recently proposed a three-state model for the regulation of actomyosin interaction by tropomyosin and troponin (Tm.Tn). In this model, the thin filament exists in rapid equilibrium between the following three states: blocked, which cannot bind myosin significantly; closed, which can bind myosin weakly to form the A-state; open, which can bind both to form the A-state and isomerize to the strongly bound R-state. In this study, we demonstrate that the equilibrium between the blocked and closed states is calcium sensitive with an equilibrium constant, KB, of 0.3 and > or = 10 at pCa 8.9 and 4.6, respectively. The pCa dependence of KB is typical of that for calcium binding to thin filaments with a mid-point at pCa 5.6 and a Hill coefficient of 1.8. KB is independent of ionic strength over the range 0.4-0.06 M but increases dramatically below 0.05 M to > or = 10 at 0.01 M suggesting loss of the blocked state at low ionic strength. The blocked state also has reduced occupancy at high temperatures. KB, in the absence of calcium, increases from 0.2 at 5 degrees C to 0.6 at 40 degrees C.

Actins↗

PubMed1995

Pressure-induced changes in the isometric contractions of single intact frog muscle fibres at low temperatures.

Effects of increased hydrostatic pressure (range 0.1-10 MPa) on isometric twitch and tetanic contractions of single intact muscle fibres, isolated from frog tibialis anterior muscle, were examined at 4-12 degrees C. The tension changes produced on exposure to steady high pressures are compared with those produced on exposure to low concentrations of caffeine (0.5 mM, subthreshold for contracture) and when pressure is rapidly released during a contraction. The peak twitch tension was potentiated by pressure accompanied by increased rate of tension rise and increased duration; the pressure sensitivity of twitch tension was approximately 8% MPa-1. The correlation between the rate of tension rise and peak tension in caffeine-induced twitch tension potentiation was quantitatively similar to that in pressure-induced twitch potentiation. Experiments involving the rapid release of pressure (approximately 2 ms) during twitch contractions demonstrate that high pressure need only be maintained for a brief period during the early part of tension development to elicit full twitch potentiation. The tetanic tension was depressed by pressure (approximately 1% MPa-1). Results demonstrate that the major effect of increased hydrostatic pressure on intact muscle fibres, which results in tension potentiation, is complete very early during contraction and is similar to that of caffeine.

Animals↗

PubMed1995

Strain-dependent cross-bridge cycle for muscle.

The cross-bridge cycle for actin, S1 myosin, and nucleotides in solution is applied to the sliding filament model for fully activated striated muscle. The cycle has attached and rotated isomers of each actomyosin state. It is assumed that these forms have different zero-strain conformations with respect to the filament and that strain-free rate constants are the nominal solution values. Only one S1 unit of heavy meromyosin is considered. Transition-state theory is used to predict the strain dependences of S1 binding to actin, the force-generating transition to rotated states, and the release/binding of nucleotide and phosphate. We propose that ADP release and ATP binding are blocked by positive strain and phosphate release by negative strain. At large strains, rapid dissociation of S1 nucleotide from actin is expected when the compliant element of the cross-bridge is strained in either direction beyond its elastic limits. The dynamical behavior of this model of muscle contraction is discussed in general terms. Its computed steady-state properties are presented in an accompanying paper.

Actins↗

PubMed1995

Strain-dependent cross-bridge cycle for muscle. II. Steady-state behavior.

Quantitative predictions of steady-state muscle properties from the strain-dependent cross-bridge for muscle are presented. With a stiffness of 5.4 x 10(-4) N/m per head, a throw distance of 11 nm, and three allowed actin sites/head, isometric properties and their dependence on phosphate and nucleotide levels are well described if the tension-generating step occurs before phosphate release. At very low ATP levels, rigorlike states with negative strain are predicted. The rate-limiting step for cycling and ATP consumption is strain-blocked ADP release for isometric and slowly shortening muscle. Under rapid shortening, ATP hydrolysis on detached heads is the rate-limiting step, and the ratio of bound ATP to bound ADP.Pi increases by a factor of 7. At large positive strains, bound heads must be forcibly detached from actin to account for tension in rapid extension, but forced detachment in shortening has no effect without destroying isometric attached states. Strain-blocked phosphate release as proposed produces modest inhibition of the ATPase rate under rapid shortening, sufficient to give a maximum for one actin site per helix turn. Alternative cross-bridge models are discussed in the light of these predictions.

Actins↗

PubMed1995

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.

Actins↗

PubMed1995

The role of three-state docking of myosin S1 with actin in force generation.

It has been shown that in solution myosin subfragment 1 binds to actin in three principal steps: [formula: see text] The nucleotide bound to myosin has a major influence on the equilibrium constant of the third of these steps but little effect on the other two. The third step is thought to be coupled to the force-generating event. Three-step binding and structure: The formation of the collision complex is strongly ionic strength dependent but independent of temperature. The isomerization to the A state is not strongly dependent on ionic strength but is affected by organic solvent and temperature. In contrast the isomerization to the R state-is affected by both ionic strength and organic solvent but little affected by temperature. The recent docking of the three-dimensional structures of actin and S1 suggest possible structural correlates of these events. These studies lead to predictions for the docking process, which may be tested using site-directed mutagenesis or peptide inhibitors. Three-step binding and head-head interactions: Studies of HMM binding to actin compared with S1 binding show that binding of two heads in the A state are unlikely presumably because of strain effects. However, binding of two heads as one A and one R state shows little evidence of strain while the isomerization of the second head to give two R states is fivefold weaker than for an isolated S1 head. These results suggest that in a rapidly shortening muscle only one head is likely to be attached at a time. Under isometric conditions, although it is possible for both heads to bind to adjacent actins, it is unlikely that both will be in the force holding R state simultaneously. Three-step binding and regulation by tropomyosin-troponin:Our recent solution studies have established that the thin filament can exist in three calcium-dependent states which we termed blocked, closed and open. A blocked state cannot form the A state with S1 and a closed state cannot form the force holding R state nor accelerate product release from S1. Thus control operates at two distinct points in the docking process. The docking process itself is coupled to hydrolysis of ATP (the A-to-R isomerization is inhibited by the presence of the gamma Pi on ATP), and therefore all of these events are interrelated.The coming together of these different strands provides a biochemical framework that should allow the dynamic properties of the crossbridge in muscle to be understood.

Actins↗

PubMed1994

Contractile activation and force generation in skinned rabbit muscle fibres: effects of hydrostatic pressure.

1. Effects of hydrostatic pressure (range 0.1-10 MPa) on the isometric tension of skinned (rabbit psoas) muscle fibres were examined at 12 degrees C and at different levels of Ca2+ activation (pCa range 4-7); the effects on both the steady tension and the tension transients induced by rapid pressure release (< 1 ms) are described. 2. The steady tension was depressed by increased pressure (approximately 1% MPa-1) at a high level of Ca2+ activation (pCa approximately 4) whereas it was potentiated at lower Ca2+ levels (pCa > 6); the effects were reversible. 3. At maximal Ca2+ activation, the tension recovery following pressure release (10 MPa to atmospheric) consisted of a fast (approximately 30 s-1) and a slow (2-3 s-1) phase; the rate and the normalized amplitude (normalized to the steady tension at atmospheric pressure for a particular pCa) of the fast phase were invariant with changes in Ca2+ level. 4. The effects of changing Ca2+ level on the slow phase were complex; its positive amplitude at high Ca2+ levels changed to negative and the rate decreased to approximately 1 s-1 at low Ca2+ levels (pCa > 6.0). 5. Results are discussed in relation to previous studies on the effect of pressure on intact muscle fibres and the actin-myosin interaction. This work supports calcium regulation of cross-bridge recruitment rather than calcium regulation of the rate of a specific step in the cross-bridge cycle.

Animals↗

PubMed1994

The influence of 2,3-butanedione 2-monoxime (BDM) on the interaction between actin and myosin in solution and in skinned muscle fibres.

2,3-butanedione 2-monoxime (BDM) inhibits muscle contraction and actomyosin ATPase both in fibres and in solution. It is potentially useful as a tool for exploring weak interactions between actin and myosin. We have examined the effect of BDM on several key steps of the myosin subfragment-1 and actomyosin subfragment-1 ATPase in solution. These studies show that BDM shifts the equilibrium between two actomyosin states towards a more weakly bound form when the acto.myosin complex has ADP alone or ADP and phosphate bound. We also confirm the findings of Herrmann and colleagues (1993, Biochemistry, 31, 12227-32) that the main effect of BDM on the myosin subfragment-1 ATPase is to slow the release of phosphate following ATP hydrolysis. Skinned fibre studies show that the effects of BDM and phosphate on the steady isometric tension of the fibres are additive. This is consistent with the interpretation that BDM is reducing fibre tension either by increasing phosphate binding or by a direct effect on the crossbridge. Tension transients induced by rapid pressure release were examined in single muscle fibres; they showed that BDM reduces the rate of tension generation following pressure release. This result suggest that BDM directly affects the force generating event in the crossbridge.

Actins↗

PubMed1994

Dynamics of the muscle thin filament regulatory switch: the size of the cooperative unit.

Actin thin filaments containing bound tropomyosin (Tm) or tropomyosin troponin (Tm.Tn) exist in two states ("off" and "on") with different affinities for myosin heads (S1), which results in the cooperative binding of S1. The rate of S1 binding to, and dissociating from, actin, Tm.actin, and Tm.Tn.actin, monitored by light scattering (LS), was compared with the rate of change in state, monitored by the excimer fluorescence (Fl) of a pyrene label attached to Tm. The ATP-induced S1 dissociation showed similar exponential decreases in LS for actin.S1, Tm.actin.S1, and Tm.Tn.actin.S1 +/- Ca2+. The Fl change, however, showed a delay that was greater for Tm.Tn.actin than Tm.actin, independent of Ca2+. The S1 binding kinetics gave observed rate constants for the S1-induced change in state that were 5-6 times the observed rate constants of S1 binding to Tm.actin, which were increased to 10-12 for Tm.Tn.actin, independent of Ca2+. The rate of the Fl signals showed that the on/off states were in rapid equilibrium. These data indicate that the apparent cooperative unit for Tm.actin is 5-6 actin subunits rather than the minimum structural unit size of 7, and is increased to 10-12 subunits for Tm.Tn.actin, independent of the presence of Ca2+. Thus, Tm appears semi-flexible, and Tn increases communication between neighboring structural units. A general model for the dynamic transitions involved in muscle regulation is presented.

Actins↗