Search PubMed⌕ Search

Biomedical subjects

M A Geeves

Publications and source records attributed to M A Geeves.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transient kinetic analysis of the 130-kDa myosin I (MYR-1 gene product) from rat liver. A myosin I designed for maintenance of tension?

The 130-kDa myosin I (MI(130)), product of the myr-1 gene, is one member of the mammalian class I myosins, a group of small, calmodulin-binding mechanochemical molecules of the myosin superfamily that translocate actin filaments. Roles for MI(130) are unknown. Our hypothesis is that, as with all myosins, MI(130) is designed for a particular function and hence possesses specific biochemical attributes. To test this hypothesis we have characterized the enzymatic properties of MI(130) using steady-state and stopped-flow kinetic analyses. Our results indicate that: (i) the Mg(2+)-ATPase activity is activated in proportion to actin concentration in the absence of Ca(2+); (ii) the ATP-induced dissociation of actin-MI(130) is much slower for MI(130) than has been observed for other myosins (-Ca(2+), second order rate constant of ATP binding, 1.7 x 10(4) M(-1) s(-1); maximal rate constant, 32 s(-1)); (iii) ADP binds to actin-MI(130) with an affinity of approximately 10 microM and competes with ATP-induced dissociation of actin-MI(130); the rate constant of ADP release from actin-MI(130) is 2 s(-1); (iv) the rates of the ATP-induced dissociation of actin-MI and ADP release are 2-3 times greater in the presence of CaCl(2), indicating a sensitivity of motor activity to Ca(2+); and (v) the affinity of MI(130) for actin (15 nM) is typical of that observed for other myosins. Together, these results indicate that although MI(130) shares some characteristics with other myosins, it is well adapted for maintenance of cortical tension.

Actins↗
PubMed1999

Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin.

Motifs N2 and N3, also referred to as switch-1 and switch-2, form part of the active site of molecular motors such as myosins and kinesins. In the case of myosin, N3 is thought to act as a gamma-phosphate sensor and moves almost 6 A relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the gamma-phosphate. The closed form seems to be necessary for hydrolysis and is stabilised by the formation of a salt-bridge between an arginine residue in N2 and a glutamate residue in N3. We examined the role of this salt-bridge in Dictyostelium discoideum myosin. Myosin motor domains with mutations E459R or R238E, that block salt-bridge formation, show defects in nucleotide-binding, reduced rates of ATP hydrolysis and a tenfold reduction in actin affinity. Inversion of the salt-bridge in double-mutant M765-IS eliminates most of the defects observed for the single mutants. With the exception of a 2,500-fold higher KMvalue for ATP, the double-mutant displayed enzymatic and functional properties very similar to those of the wild-type protein. Our results reveal that, independent of its orientation, the salt-bridge is required to support efficient ATP hydrolysis, normal communication between different functional regions of the myosin head, and motor function.

Adenosine Diphosphate↗
PubMed1999

Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.

Adenosine Triphosphate↗
PubMed1999

Kinetic analysis of Dictyostelium discoideum myosin motor domains with glycine-to-alanine mutations in the reactive thiol region.

Three conserved glycine residues in the reactive thiol region of Dictyostelium discoideummyosin II were replaced by alanine residues. The resulting mutants G680A, G684A, and G691A were expressed in the soluble myosin head fragment M761-2R [Anson, M., Geeves, M. A., Kurzawa, S. E., and Manstein, D. J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A showed no major alterations except for a marked increase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitated by this mutation, and the addition of actin to G691A stimulated ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A and G684A showed a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A were small ( approximately 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding were 20-30-fold slower and the affinity for mantADP was approximately 10-fold increased due to a 200-fold reduction in the dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.680A complex was normal, but the communication between ADP and actin binding was altered such that the two sites are thermodynamically uncoupled but kinetically actin still accelerates ADP release.

Adenosine Triphosphatases↗
PubMed1999

Microsecond folding of the cold shock protein measured by a pressure-jump technique.

A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding. In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 microseconds and then held constant. Thus, unfolding and refolding reactions in the time range from 70 microseconds to 70 s can be followed with this technique. Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions. We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region. For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains. The Phe27Ala mutation affected the rate of unfolding only; the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding. Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.

Alanine↗
PubMed1999

Cooperativity and switching within the three-state model of muscle regulation.

Thin filament regulation is mediated by the presence of tropomyosin (Tm) and troponin (Tn) on the actin filament. Binding of Tm alone induces two states, closed and open (with the equilibrium between them defined by KT), which differ in their affinity for myosin subfragment 1 (S1). Cooperative switching between the states results in characteristic sigmoidal myosin S1 binding curves. In the presence of Tn and absence of Ca2+, a third state, blocked, has previously been kinetically shown to be present, leading to the three state model of McKillop and Geeves [(1993) Biophys. J. 65, 693-701]. We have measured equilibrium binding of S1 to phalloidin-stabilized pyrene-actin filaments by monitoring the pyrene fluorescence at 50 nM, a concentration 10-fold lower than previously possible. In combination with kinetic studies, we show that the data can be fitted to a modified version of the three-state model with an additional term allowing for a varying apparent cooperative unit size (n). Our results show that the apparent cooperative unit size (n) is dependent upon both the presence of Tn and of Ca2+. Also in the absence of Ca2+, the occupancy of the blocked state (defined by KB) is accompanied by a 2-3-fold reduction in KT. These results are discussed in comparison to the Hill model [(1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] and a flexible model of thin filament regulation based upon that of Lehrer et al. [(1997) Biochemistry 36, 13449-13455].

Actins↗
PubMed1999

Force generation upon hydrostatic pressure release in tetanized intact frog muscle fibres.

Single intact muscle fibres isolated from the tibialis anterior muscle of the frog were exposed to hydrostatic pressures of 1-10 MPa, at 2-4 degrees C and sarcomere length of 2.1-2.2 microm. The pressure was rapidly released (ca. 1 ms) to atmospheric level (0.1 MPa) during the plateau of a tetanic contraction (Po) and the resultant tension (= force) transient examined. The pressure release induced tension transient consisted of an initial tension drop coincident with pressure release (ca. 4% Po per MPa, Phase 1), followed by a rapid recovery (Phase 2a) and a slower rise of tension (Phase 2b). Phase 1 was partly due to a length release at fibre ends (ca. 0.1 nm per half-sarcomere per MPa) induced by pressure-release effects on the steel chamber and fibre attachments, and partly due to 'expansion' upon pressure release within muscle fibre (ca. 0.2 nm per half-sarcomere per MPa), probably in the myofilaments and cross-bridges. The rate of tension recovery during phase 2a (ca. 600/s) was comparable to that of the quick tension recovery (T1-T2 transition) reported from moderately fast small length releases; the time course of Phase 2b (rate ca. 40/s) was similar to the late phase of tension rise in a tetanus, and hence compared with Phase 4 (T4) of a length release tension transient. Results are compared with the previously reported findings from analogous experiments on Ca2+ -activated skinned (rabbit) muscle fibres.

Animals↗
PubMed1999

Structural mechanism of muscle contraction.

X-ray crystallography shows the myosin cross-bridge to exist in two conformations, the beginning and end of the "power stroke." A long lever-arm undergoes a 60 degrees to 70 degrees rotation between the two states. This rotation is coupled with changes in the active site (OPEN to CLOSED) and phosphate release. Actin binding mediates the transition from CLOSED to OPEN. Kinetics shows that the binding of myosin to actin is a two-step process which affects ATP and ADP affinity. The structural basis of these effects is not explained by the presently known conformers of myosin. Therefore, other states of the myosin cross-bridge must exist. Moreover, cryoelectronmicroscopy has revealed other angles of the cross-bridge lever arm induced by ADP binding. These structural states are presently being characterized by site-directed mutagenesis coupled with kinetic analysis.

Actins↗
PubMed1998

Kinetic characterization of myosin head fragments with long-lived myosin.ATP states.

We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mantATP [2'(3')-O-(N-methylanthraniloyl)-ATP] and mantADP binding, the most dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by 4 (E476K) and 6 (E459V) orders of magnitude. Thus, both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin.ATP states. The E459V mutation allowed for a direct measurement of the ATP off rate constant from myosin, which was found to be 2 x 10(-)5 s-1. Actin accelerated ATP release from this E459V construct by at least 100-fold. Additionally, we found that the affinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with actin normally. Their functional properties and the fact that they can be produced and purified in large amounts make the E476K and E459V constructs ideal tools to elucidate key structural features of the myosin ATPase cycle. These constructs should allow us to address important questions, including how binding of ATP to myosin heads results in a >3 order of magnitude reduction in actin affinity.

Actins↗
PubMed1998

Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms.

The striated muscle myosin of Placopecten moves actin faster in in vitro motility assays and has a higher actin-activated ATPase turnover rate than the myosin of the catch muscle. The heavy chain sequences of the two PlacoS1s are almost identical except at the surface loop 1 near the nucleotide binding pocket, where the two sequences vary significantly. Argopecten striated muscle myosin is 96% identical to Placopecten striated myosin, and both move actin with a similar velocity. To identify the individual kinetic steps which differ between these myosins, we completed a transient kinetic characterization of the three myosin S1s. The two striated S1s have similar rates of nucleotide binding to S1 and to acto.S1. The largest differences between the two are in the rate of ADP dissociation from S1 and affinity of ADP to S1, which differ by a factor of 2. The rates of nucleotide binding, nucleotide dissociation and affinity to nucleotides of the two Placopecten S1s are similar and agree within a factor of 2. In contrast, the affinity of acto.S1 for ADP is nine times weaker for the striated acto.S1 than for the catch acto.S1, compatible with the differences in motility of the Placopectenmyosins. Thus the differences in ADP affinity to acto.S1 and in the in vitro motility can be attributed to the differences in surface loop 1.

Actins↗
PubMed1998

Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain.

The effects of mutations in an actin-binding surface loop of myosin (loop 2) are described. Part of loop 2, the segment between myosin residues 618 and 622, was replaced with sequences enlarged by the introduction of positively charged GKK or neutral GNN motifs. Constructs with loops carrying up to 20 additional amino acids and charge variations from -1 to +12 were produced. Steady-state and transient kinetics were used to characterize the enzymatic behavior of the mutant motor domains. Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with moderate charge changes (-1 to +2) displayed wild-type-like behavior. Introduction of more than one GKK motif led to stronger coupling between the actin- and nucleotide-binding sites of myosin and an up to 1000-fold increased affinity for actin in the absence of ATP and at zero ionic strength. In comparison to the wild-type construct M765, constructs with 4-12 extra charges displayed an increased dependence on ionic strength in their interaction with actin, a 2-3-fold increase in kcat, a more than 10-fold reduction in Kapp for actin, and a 34-70-fold increase in catalytic efficiency.

Actins↗
PubMed1998

The muscle thin filament as a classical cooperative/allosteric regulatory system.

It is generally accepted that the regulation of muscle contraction involves cooperative and allosteric interactions among the protein components, actin, myosin, tropomyosin and troponin. But, as yet, the individual role of each component has not been clearly identified. Here we compare the properties of the components of the muscle regulatory system with the corresponding components of two systems, hemoglobin and aspartate transcarbamylase, that are well described by the classical Monod, Wyman and Changeux (MWC) model. The analogy indicates that actin is the catalytic subunit, tropomyosin is the regulatory subunit and troponin in the absence and presence of Ca2+ is the allosteric inhibitor and activator, respectively. The analogy additionally indicates that the substrate is myosin-ATP (or myosin-ADP-Pi) rather than ATP. Also, in contrast to other MWC systems, the activating ligand for actin-tropomyosin is a myosin-nucleotide intermediate or product that binds tightly to actin, rather than the substrate which binds weakly. This tightly bound intermediate switches the system from the off-state to the on-state (T to R-state in MWC nomenclature) in a concerted transition, affecting n actin subunits, allowing force to be developed.

Actins↗
PubMed1998

Substrate specificity of human nucleoside-diphosphate kinase revealed by transient kinetic analysis.

Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of gamma-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 x 10(6) M-1 s-1. Dephosphorylation by NDPs was 2-3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3-4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5'-O-(thiotriphosphate) (ATPgammaS) and guanosine 5'-O-(thiotriphosphate) (GTPgammaS) to NDP, but at (1)/(1000) of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATPgammaS and GTPgammaS occurred with kmax of 2.8 and 1.35 s-1 and Kd of 145 and 36 muM respectively. For dethiophosphorylation by a range of NDPs, kmax was in the range of 5-30 s-1, whereas Kd varied between 0.16 and 3.3 mM. Guanine had the lowest Kd values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2'-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3'-ribose.

Adenosine Triphosphate↗
PubMed1998

Interaction of actin and ADP with the head domain of smooth muscle myosin: implications for strain-dependent ADP release in smooth muscle.

Transient kinetic methods were used to study interactions between actin, MgADP, and smooth muscle (chicken gizzard) myosin subfragment 1 (smS1). The equilibrium dissociation constant (Kd) of actin for smS1 was 3.5 nM, tighter than that of skeletal S1 (skS1). Actin binding to smS1 was weakened 5-fold by saturation with ADP compared to 30-60-fold for skS1. The Kd of ADP for smS1 was increased from 1.2 to 5 microM by actin, whereas for skS1 values increased from 2 to 100 microM. Thus, coupling between ADP and actin binding is weaker for smS1. Previous studies show that release of ADP from actin.smS1.ADP produces a tilt of the regulatory domain [Whittaker, M., Wilson-Kubalek, E. M., Smith, J. E., Faust, L., Milligan, R. A., and Sweeney, H. L. (1995) Nature 378, 748-751]. This result was confirmed by independent structural methods; tilting was absent for skS1, and the Kd for ADP was in agreement with the values measured here [Gollub, J., Cremo, C. R., and Cooke, R. (1996) Nat. Struct. Biol. 3, 796-802; Poole, K. I. V., Lorenz, M., Ellison, P., Evans, G., Rosenbaum, G., Boesecke, P., Holmes, K. C., and Cremo, C. R. (1997) J. Muscle Res. Cell Motility 18, 264]. We discuss tilting upon ADP release with respect to our measurements, previous measurements with skS1, and nucleotide concentrations in smooth muscle. We propose that these data suggest a strain-dependent ADP release mechanism that may be accentuated in smooth muscles.

Actins↗
PubMed1998

Differential scanning calorimetric study of the thermal unfolding of the motor domain fragments of Dictyostelium discoideum myosin II.

The thermal unfolding of two recombinant fragments of the head of Dictyostelium discoideum myosin II was studied by differential scanning calorimetry. These fragments M754 and M761 correspond to the globular motor portion of the myosin head that contains ATP- and actin-binding sites but lacks the light chain binding domain. Our results show that M754 is less thermostable than M761: the maximum of the thermal transition occurred at 41.7 degrees C for M754 and at 45.6 degrees C for M761, and the calorimetric enthalpy value determined for M754 (677 kJ/mol) was about half of that for M761 (1417 kJ/mol). This indicates that the region containing residues 755-761 plays a very important role in the structural stabilization of the entire globular motor part of the myosin head. ADP binding induces structural changes in both myosin fragments which are reflected in a 2-3.5 degrees C shift of the thermal transitions to higher temperature. The formation of stable ternary complexes of these myosin fragments with ADP and phosphate analogues such as orthovanadate, beryllium fluoride or aluminium fluoride causes additional structural changes which are reflected in a pronounced increase of thermal stability. The effect of beryllium fluoride was less distinct than that of aluminium fluoride or orthovanadate. In general, the changes caused by various phosphate analogues were similar to those observed with skeletal myosin subfragment 1. Thus, structural changes revealed by differential scanning calorimetry in the myosin head, that are due to the formation of stable ternary complexes with ADP and Pi analogues, occur mainly in the globular motor portion of the head.

Actins↗
PubMed1998

The use of fast kinetic systems for the study of ng-microgram quantities of purified muscle proteins.

Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However these methods traditionally require relatively large amounts of protein (several mg) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g. Dictyostelium). We have been developing methods which would allow fast transients to be studied on the much smaller quantities of protein available from individual drosophila muscles, single mammalian muscle fibres, human biopsies and non-muscle myosins. The use of fluorescent labels on actin, myosin or ATP can be used in modern stopped-flow equipment at concentrations as low as 10 nM. These labels can report on the interactions between the three molecules using a few microgram of protein. However the stopped flow systems requires large volumes of sample to give sufficient acceleration of the sample to achieve good mixing. We have therefore been using a similar optical set up but initiating the reaction using caged ATP. In this device the same measurements can be made using 10-20 fold less material. This is the quantity of myosin which can be purified from a few mm of a single mammalian muscle fibre. Using these methods the following measurements can be made: rate of ATP induced dissociation of actomyosin, the affinity of ADP for actomyosin, the rate of ADP release from actomyosin and the affinity of actin for myosin and myosin ADP.

Animals↗
PubMed1998

Cooperativity between the two heads of rabbit skeletal muscle heavy meromyosin in binding to actin.

An extensive series of experiments in this laboratory has shown that the binding of actin to rabbit skeletal muscle myosin subfragment-1 (a single-headed subfragment) can be described by a two-step model, with formation of a weakly bound complex, the A-state, followed by an isomerization to a more tightly bound complex, the R-state. In this paper, we report on additional experiments comparing the subfragment-1 with heavy meromyosin (a two-headed subfragment). Using a modeling approach, we have quantitated the two-step binding for each of the two heads. This indicates that the binding is cooperative and leads to a more complex view of the acto-myosin interaction than has previously been acknowledged. Implications for the dynamic behavior of the two heads during muscle contraction are discussed.

Actins↗