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Biomedical subjects

M A Geeves

Publications and source records attributed to M A Geeves.

At least 73 records · Page 4Linked to original sources
PubMed1993

Kinetic characterization of a cytoplasmic myosin motor domain expressed in Dictyostelium discoideum.

A detailed kinetic study of the interaction of a recombinant myosin head fragment (MHF) of Dictyostelium discoideum with actin and adenine nucleotides has been made by using a combination of rapid-reaction, equilibrium, and fluorescence methods. MHF is equivalent in size to a proteolytic fragment of skeletal muscle myosin, subfragment 1 (S1), the simplest unit of myosin to retain enzymatic and functional activity. The results show that qualitatively the interactions of MHF with nucleotides and actin are the same as those of S1. Both bind to rabbit actin with the same affinity, although differences in the rate constants of their interactions with nucleotides in the presence and absence of actin occur. The rate of ATP binding to MHF and the subsequent cleavage step are significantly slower than the corresponding rates with S1. The dissociation of a fluorescent analog of ADP from MHF was 5-fold faster than from S1, while its rate of binding MHF was 3-fold slower, resulting in a weaker association equilibrium constant. The ATP-induced isomerization of the actoMHF complex was 10-fold slower than for actoS1, but the binding affinities of ADP for actoMHF and actoS1 were indistinguishable. The results suggest a different degree of coupling between the nucleotide and actin binding sites of MHF and S1 which may be a common feature of nonmuscle myosins. They also provide the basis for a study of specifically modified myosins with which one can probe the sites of interaction with nucleotides or actin, as well as functional motility.

Actins↗
PubMed1993

Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament.

Equilibrium titrations and kinetic experiments were used to define the cooperative binding of myosin subfragment 1 (S1) to actin-troponin-tropomyosin. Both types of experiment require an equilibrium between two states of the thin filament in which one state (the off state) binds S1 less readily than the other. Equilibrium titrations are compatible with > 95% of the actin7.Tn.Tm units being in the off state in the absence of calcium and 80% in the off state in the presence of calcium. Kinetic binding data suggest that the presence of calcium switches the thin filament from 70% in the off state to < 5%. The two experiments, therefore, define quite different populations of the off states. We propose a three-state model of the thin filament. A "blocked state" which is unable to bind S1, a "closed state" which can only bind S1 relatively weakly and an "open state" in which the S1 can both bind and undergo an isomerization to a more strongly bound rigor-like conformation. The equilibrium between the three states is calcium-dependent; KB = [closed]/[blocked] = 0.3 and > or = 16 and KT = [open]/[closed] = 0.09 and 0.25 in the absence and presence of calcium, respectively. This model can account for both types of experimental data.

Actins↗
PubMed1993

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.

Animals↗
PubMed1992

The actomyosin ATPase: a two-state system.

Studies of the interaction between actin and myosin subfragment 1 (S1) in solution have shown that the association reaction takes place in at least two steps. Initially the association is relatively weak to form a complex called the A state which can then isomerize to the R state. The rate and equilibrium constants for the isomerization have been measured and are shown to depend upon the nucleotide bound to the S1 ATPase site; with ATP bound the A state is preferred but as ATP is hydrolysed and the products are sequentially released then the complex gradually shifts to the A state. An extensive series of experiments have characterized the A-to-R isomerization both in solution and in contracting muscle fibres and have shown it to be closely associated with the key events in the ATP-driven contraction cycle: the conformational change from the A to the R state can be monitored by fluorescent probes on either actin or the nucleotide; the isomerization can be perturbed by increases in hydrostatic pressure; the actin-induced acceleration of the rate of product release from myosin is coupled to the A-to-R isomerization; tropomyosin may control actin and myosin interaction by controlling the isomerization step and finally pressure perturbations of contracting muscle fibres shows there to be a close coupling between the isomerization of acto.S1 and the force generating event of muscle contraction.

Animals↗
PubMed1991

Regulation of the acto.myosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two acto.myosin subfragment 1 states.

The co-operative binding of myosin subfragment 1 (S1) to reconstituted skeletal-muscle thin filaments has been examined by monitoring the fluorescence of a pyrene probe on Cys-374 of actin. The degree of co-operativity differs when phosphate, sulphate or ADP are bound to the S1 active site. Binding isotherms have been analysed according to the Geeves & Halsall [(1987) Biophys. J. 52, 215-220] model, which proposed that troponin and tropomyosin effected regulation of the actomyosin interaction by controlling an isomerization of the actomyosin complex. The data support the proposal that seven actin monomers associated with a single tropomyosin molecule act as a co-operative unit that can be in one of two states. In the 'closed' state myosin can bind to actin, but the subsequent isomerization is prevented. The isomerization is only allowed after the seven-actin unit is in the 'open' form. Ca2+ controls the proportion of actin filaments in the 'closed' and 'open' forms in the absence of myosin heads. The ratio of 'closed' to 'open' forms is approx. 50:1 in the absence of Ca2+ and 5:1 in its presence.

Actins↗
PubMed1991

The effect of hydrostatic pressure on the interaction of actomyosin subfragment 1 with nucleotides.

Increased hydrostatic pressure has previously been shown to reduce the tension of isometrically contracting skinned muscle fibres. An isomerization of the actomyosin complex is known to be pressure sensitive, but the pressure sensitivity of other steps in the ATPase pathway has not been characterised. We report here the effect of pressure on the ATP hydrolysis step of the myosin subfragment 1 ATPase, ADP binding to actomyosin subfragment 1 and the rate of ATP induced dissociation of actomyosin subfragment 1. We discuss the relationship of these changes to the observed effect of pressure on skinned muscle fibres.

Actomyosin↗
PubMed1991

Tension responses to rapid pressure release in glycerinated rabbit muscle fibers.

We have previously shown that the isometric tension of a fully calcium-activated skinned rabbit psoas muscle fiber is reversibly depressed by increased hydrostatic pressure. We report here the characterization of tension transients induced by a rapid (less than 1-ms) release of increased pressure at 12 degrees C. The tension transient consists of three clear phases, an initial further decrease of tension in phase with pressure change followed by two phases of tension increase back to the level recorded at ambient pressure. The mean reciprocal relaxation time for phase 2 (1/tau 2) was approximately 17 s-1 and that for phase 3 (1/tau 3) was 3 s-1. The presence of 20 mM inorganic phosphate markedly increased 1/tau 2 to approximately 52 s-1 and decreased 1/tau 3 to approximately 1.7 s-1. These observations are interpreted in terms of a pressure-sensitive transition between two attached crossbridge states of low (or zero) and higher force. This is compatible with the pressure-sensitive isomerization of actomyosin previously observed in solution. The results presented allow us to propose a coupling between a specific pressure-sensitive isomerization of purified actomyosin, the phosphate release step of the ATPase pathway, and the force-generating event of the cross-bridge cycle.

Animals↗
PubMed1991

Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes.

We have used a fluorescent analogue of ATP, mantATP [2'(3')-O-(N-methylanthraniloyl)-adenosine 5'-triphosphate; Hiratsuka T. (1983) Biochim. Biophys. Acta 742, 496-508], and made a detailed kinetic study of the interaction of mantATP and mantADP with S1 and acto-S1. We have shown that these analogues behave like ATP and ADP, respectively. In addition, we have demonstrated that this analogue can distinguish between two acto-S1 complexes, the A-M.N (attached) and A.M.N (rigor-like) states [Geeves, M. A., Good, R. S., & Gutfreund, H. (1984) J. Muscle Res. Cell Motil. 5, 351-361]. Previously, these two states were observed with a pyrene label on Cys 374 of actin. This isomerization can now be monitored at two spatially distinct sites on the ternary complex, indicative of a major conformational change in the ternary complex. Also, we have measured the rate of ADP dissociation from both A-M.N and A.M.N directly and shown these to differ by a factor of 1000. Thus the results presented here support the model of Geeves et al. and are consistent with the A-M.N to A.M.N transition being coupled to the force-generating event of the crossbridge cycle.

Actomyosin↗
PubMed1991

A nucleation--elongation mechanism for the self-assembly of side polar sheets of smooth muscle myosin.

Self-assembled filaments of smooth muscle myosin were observed by low dose electron microscopy to be flat side-polar sheets, in which the component molecules appeared straight and close-packed. Fraying experiments released small oligomers, in which molecules were staggered in parallel by about +/- 14 nm relative to two immediate neighbours, and were bound also to an antiparallel partner via a approximately 14 nm overlap at the very tip of the tail. We suggest a filament model which preserves these packing relationships. Adding stoichiometric amounts of MgATP to the filaments caused them to disassemble completely by progressive loss of material from their ends, at a limiting rate equivalent to about 2 monomers per second per end in physiological saline. The rate of the competing association reaction varied linearly with the monomer concentration, as determined in pressure-jump experiments. This suggests that myosin monomers, rather than dimers or higher oligomers, are the building blocks of these filaments. Shearing and annealing of assembled filaments appeared negligible on a time scale of a few hours. In consequence, filament number and filament length were dependent on the rate at which monomers were supplied to the assembly reaction, and on the number of filaments already present at the start of the assembly reaction.

Actin Cytoskeleton↗
PubMed1991

A "slow" temperature jump apparatus built from a stopped-flow machine.

A simple modification to a standard thermostated stopped-flow machine is described which allows it to be used as a temperature jump machine. Temperature jumps larger than 10 degrees C can be achieved in less than 150 ms which makes it useful for the range of times where conventional rapid temperature jumps are not applicable. The apparatus has a sample size of 300 microliters and can produce temperature jumps both above and below the initial temperature.

Actins↗
PubMed1991

Changes produced by increased hydrostatic pressure in isometric contractions of rat fast muscle.

1. Muscle fibre bundles isolated from the extensor digitorum longus (a fast muscle) of the rat were exposed to different hydrostatic pressures (range 0.1-10 MPa), in order to determine the pressure dependence of their isometric contractions. 2. The pressure dependent changes in the contractions were reversible and linearly related to pressure. 3. The peak tension, the time to peak and the time to half-relaxation of a twitch contraction increased with pressure; the mean (+/- S.E.M.) percentage increases were 5.9 +/- 0.5% MPa-1, 2.7 +/- 0.2% MPa-1 and 2.7 +/- 0.4% MPa-1, respectively. 4. In a fused tetanus, the tension was typically depressed at high pressure (0.9 +/- 0.16% MPa-1); the half-time of tension rise was decreased (2.1 +/- 0.2% MPa-1) and the half-time of exponential relaxation was increased (2.4 +/- 0.3% MPa-1).

Animals↗
PubMed1991

The influence of pressure on actin and myosin interactions in solution and in single muscle fibres.

Studies of the molecular mechanism of motile activity require the capacity to examine the properties of individual, isolated molecular components and the properties of these same molecular components in the organised system. Pressure perturbation is one method which can be applied to motile systems at different levels of organisation. We show here that pressure perturbs a specific interaction between actin and myosin in solution and also perturbs the cycling crossbridge in a contracting muscle.

Actins↗
PubMed1990

Hydrostatic compression in glycerinated rabbit muscle fibers.

Glycerinated muscle fibers isolated from rabbit psoas muscle, and a number of other nonmuscle elastic fibers including glass, rubber, and collagen, were exposed to hydrostatic pressures of up to 10 MPa (100 Atm) to determine the pressure sensitivity of their isometric tension. The isometric tension of muscle fibers in the relaxed state (passive tension) was insensitive to increased pressure, whereas the muscle fiber tension in rigor state increased linearly with pressure. The tension of all other fiber types (except rubber) also increased with pressure; the rubber tension was pressure insensitive. The pressure sensitivity of rigor tension was 2.3 kN/m2/MPa and, in comparison with force/extension relation determined at atmospheric pressure, the hydrostatic compression in rigor muscle fibers was estimated to be 0.03% Lo/MPa. As reported previously, the active muscle fiber tension is depressed by increased pressure. The possible underlying basis of the different pressure-dependent tension behavior in relaxed, rigor, and active muscle is discussed.

Animals↗
PubMed1989

Dynamic interaction between actin and myosin subfragment 1 in the presence of ADP.

The equilibrium and dynamics of the interaction between actin, myosin subfragment 1 (S1), and ADP have been investigated by using actin which has been covalently labeled at Cys-374 with a pyrene group. The results are consistent with actin binding to S1.ADP (M.D) in a two-step reaction, A + M.D K1 equilibrium A-M.D K2 equilibrium A.M.D, in which the pyrene fluorescence only monitors the second step. In this model, K1 = 2.3 X 10(4) M-1 (k+1 = 4.6 X 10(4) M-1 s-1) and K2 = 10 (k+2 less than or equal to 4 s-1); i.e., both steps are relatively slow compared to the maximum turnover of the ATPase reaction. ADP dissociates from both M.D and A-M.D at 2 s-1 and from A.M.D at greater than or equal to 500 s-1; therefore, actin only accelerates the release of product from the A.M.D state. This model is consistent with the actomyosin ATPase model proposed by Geeves et al. [(1984) J. Muscle Res. Cell Motil. 5, 351]. The results suggest that A-M.D cannot break down at a rate greater than 4 s-1 by dissociation of ADP, by dissociation of actin, or by isomerizing to A.M.D. It is therefore unlikely to be significantly occupied in a rapidly contracting muscle, but it may have a role in a muscle contracting against a load where the ATPase rate is markedly inhibited. Under these conditions, this complex may have a role in maintaining tension with a low ATP turnover rate.

Actins↗
PubMed1989

Pressure sensitivity of active tension in glycerinated rabbit psoas muscle fibres: effects of ADP and phosphate.

The effect of changes in hydrostatic pressure (up to 10 MPa) on the maximally calcium-activated tension in glycerinated rabbit psoas fibres has been examined. The steady active tension was depressed by 0.8% per MPa pressure rise. This pressure sensitivity was enhanced by the pressure of millimolar phosphate and depressed by millimolar ADP. These results support the conclusions that increased pressure is perturbing a crossbridge event. The results are discussed in terms of a three state crossbridge model and are shown to be compatible with a pressure effect on the transition from an attached crossbridge state to a tension bearing state. This is compatible with the effects of pressure on the isolated proteins in solution.

Adenosine Diphosphate↗