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Query: UNIPROT:Q8N5D0 (ADP)
 37,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor. 145 20

The structure of myosin subfragment 1 (S1) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/50-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes.
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PMID:Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state. 148 70

AMP deaminase catalyzes the deamination of AMP to inosine 5'-monophosphate (IMP) and ammonia. Factors controlling the enzyme in muscle can rapidly promote high rates of IMP formation when ATP utilization exceeds supply. We evaluated whether binding of AMP deaminase to myosin, which occurs during intense contraction conditions, alters the kinetic behavior of the enzyme. Reaction kinetics of myosin-bound and free AMP deaminase were evaluated. Reaction kinetics of the free enzyme yielded a near-linear double-reciprocal plot with an expected Km of approximately 1 mM AMP concentration (AMP). In contrast, reaction kinetics of AMP deaminase became bimodal when bound to myosin. At [AMP] less than 0.15 mM, a high-affinity Km (0.05-0.10 mM) with maximal velocity approximately 20% that of free enzyme was evident. At [AMP] greater than 0.15 mM, the Km and maximal velocity values were similar to that of the free enzyme. The 10- to 20-fold higher affinity Km would allow for a higher rate of AMP deamination at the low [AMP] found physiologically. AMP deaminase binding to myosin also induced a marked resistance to orthophosphate inhibition (10 mM) in the presence of 50 microM ADP. Results were similar for purified preparations of AMP deaminase bound to myosin subfragment 2 and crude extracts obtained from contracting muscle. Our results add further support to the hypothesis that AMP deaminase binding to myosin serves an important role in control of enzyme activity in contracting muscle.
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PMID:Altered kinetics of AMP deaminase by myosin binding. 151 76

In muscle fibres labelled with iodoacetamidotetramethylrhodamine at Cys707 of the myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power stroke, we monitored tension and linear dichroism of the probes in single glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by laser pulse photolysis of caged ATP and caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of ATP from caged ATP in the presence of calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-ATP) upon release of ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an ATP-containing relaxing solution to a rigor medium lacking ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of ATP. Neither adding nor removing ADP produced substantial changes in force. These results demonstrate that orientation of the rhodamine probes on the myosin head reflects mainly structural changes linked to nucleotide binding and release, rather than rotation of the cross-bridge during force generation.
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PMID:Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres. 153 Sep 78

Inhibition of the myosin subfragment 1 (S-1) ATPase activity by beryllium fluoride was studied directly in the presence of MgATP and following preincubation of samples with MgADP. In both cases, the rates of inhibition were very slow, with kapp = 0.5 and 58 M-1 s-1, respectively, in analogy to the rates of inhibition of myosin ATPase by vanadate [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2620-2624]. The very different rates of inhibition in the presence of MgATP and on preincubation with MgADP suggested that beryllium fluoride binds to the M.ADP state of myosin. The slow inhibition rates and the nonlinear dependence of the observed rates on beryllium fluoride concentration were consistent with a two-step inhibition process involving a rapid binding equilibrium to yield a collisional complex, M.ADP.BeF3-, and its slow isomerization into M++.ADP.BeF3-. A third, much slower, step was required to account for the conversion of the stable M++.ADP.BeF3- to a virtually irreversibly inhibited complex. Kinetic description of the inhibition pathway was derived from the observed rates of inhibition of myosin ATPase, information on the binding of beryllium fluoride to M.ADP, and measurements of epsilon ADP chase from M++.epsilon ADP.BeF3-. The isomerization rate and equilibrium constants were 1.4 x 10(-2) s-1 and 50, respectively, and the overall binding constant of beryllium fluoride to M.ADP was 5 x 10(5) M-1. The inhibitory complex showed a 16% enhancement to tryptophan fluorescence of S-1 and a reduced quenching of epsilon ADP by acrylamide. It is concluded that M++.ADP.BeF3- is analogous to the M++.ADP.Vi and M**.ADP.Pi states of myosin.
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PMID:Inhibition of myosin ATPase by beryllium fluoride. 153 58

It is well established that caldesmon binds to actin (Kb = 10(7) - 10(-8) M-1) and to tropomyosin (Kb = 10(6) M-1) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 10(6) M-1 in low salt buffer, but is weakened by increasing [KCl] reaching 10(5) M-1 in 100 mM KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg2+ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen 'frayed' from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous 'whiskers' are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3 mM MgATP shows there is a high affinity binding (Kb = 10(6) M-1) which is independent of [Ca2+] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments. 153 66

The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (Vf) is analogous to the unloaded shortening velocity (Vu) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their Vf. Use of these techniques allowed comparison of Vf to literature values for Vu with regard to [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM), and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures greater than 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures less than 20 degrees C than Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths less than 50 mM, Vf is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect Vf. Thus, although Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures greater than 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.
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PMID:Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin. 155 Feb 12

Effects of [Ca2+] on isometric tension and unloaded shortening velocity were characterized in single chemically skinned myocytes from frog atrium and in mechanically disrupted myocardium from rat ventricle. The preparations were attached to a force transducer and piezoelectric translator and were viewed with an inverted microscope to allow continuous monitoring of sarcomere length during mechanical measurements. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of each preparation. Ca2+ sensitivity of isometric tension was assessed as pCa50, i.e., the Ca2+ concentration at which tension was 50% maximal, and was greater for frog atrial myocytes (pCa50 6.17) than for rat ventricular myocytes (pCa50 6.06). This difference in Ca2+ sensitivity may be due to variations in myofibrillar protein isoform composition in the two preparations. Inclusion of caffeine in the activating solutions substantially increased the Ca2+ sensitivity of tension, which may be a manifestation of a direct effect of caffeine on the myofibrillar proteins. Unloaded shortening velocity during maximal activation averaged 4.32 muscle lengths per second in frog atrial myocytes and 4.46 muscle lengths per second in rat ventricular myocytes. When [Ca2+] was reduced, unloaded shortening velocity decreased substantially in both preparations. Possible mechanisms for the effect of Ca2+ on shortening velocity in myocardium include Ca2+ dependence of the rate of ADP dissociation from actomyosin complexes or a shortening-dependent internal load involving structures such as C protein or long-lived myosin cross-bridges.
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PMID:Effects of calcium on shortening velocity in frog chemically skinned atrial myocytes and in mechanically disrupted ventricular myocardium from rat. 156 99

To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release.
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PMID:Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin. 176 36

A kinetic model of the cross-bridge cycle in skeletal muscle is presented with special reference to the rate limiting steps regulating the peak rate of force development (dP/dt), peak force (P0), and the maximal shortening speed (Vmax). Force production in skeletal muscle is dependent on the number of cross-bridges in the strongly bound, high-force state (AM'-ADP), and during a peak isometric contraction this state is the dominant cross-bridge form. The peak force and power output of a muscle depends upon numerous factors to include: (1) muscle and fiber size and length; (2) architecture, such as the angle and physical properties of the fiber-tendon attachment, and the fiber to muscle length ratio; (3) fiber type; (4) number of cross-bridges in parallel; (5) force per cross-bridge; (6) peak dP/dt; (7) force-velocity relationship; (8) fiber Vmax; (9) force-pCa2+ relationship: and (10) the force-frequency (action potential Hz) relationship. In this paper, we discuss these determinants of force and power output, and consider how they adapt to both muscle unloading (induced by hindlimb suspension) and programs of regular endurance exercise. Slow- and fast-twitch fibers have similar capacities to generate specific tension (kg cm-2). However, fast fibers show a considerably higher peak dP/dt, Vmax, and power output. The high Vmax of the fast-twitch fiber is likely due to the high myofibrillar ATPase activity of the fast myosin isozyme. Both hindlimb suspension and regular endurance exercise have been shown to induce fiber type specific changes in single fiber function. For example, fiber size and the peak tetanic tension of the slow oxidative (SO), fast oxidative glycolytic (FOG), and fast glycolytic (FG) fiber types were generally unaltered by endurance exercise-training. In contrast, hindlimb suspension produced cell atrophy in all fiber types and a reduced specific tension in the SO but not the FOG or FG fiber types. Both exercise-training and HS shifted the force-pCa curve to the right, and increased the Vmax of the SO fiber type. From the standpoint of work capacity or the ability to move a load, the important functional property is power output. Peak power is obtained at loads considerably below 50% of PO, and it is correlated with the percentage of fast-twitch fibers. Peak power can be increased by both dynamic and isometric programs of exercise-training.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The determinants of skeletal muscle force and power: their adaptability with changes in activity pattern. 179 Nov 72


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