Effect of C-protein on actomyosin ATPase

The effect of C-protein on the actin-activated ATPase of column-purified skeletal muscle myosin has been investigated at varied ionic strength. At ionic strengths below about 0.1, C-protein is a potent inhibitor. The inhibition is not reversed by increasing the actin concentration, showing that it is caused by C-protein bound to the myosin filaments. When the ionic strength is raised above about 0.12, on the other hand, the inhibition vanishes and C-protein becomes a mild activator of the actomyosin ATPase. Both effects appear rapidly upon addition of C-protein to pre-formed myosin filaments, so C-protein probably acts by binding to the surface of the filaments.     

Sensitivity of cultured and skinned chick myotube to calcium, strontium, and barium ions examined by recording isometric contractions.

Sensitivity of cultured chick myotubes to alkaline earth metal ions was investigated by recording contractile isometric tension through a semiconductor transducer. The myotubes were obtained by culturing myoblasts of chick embryo breast muscles, and skinned chemically before physiological experiments. Contractions developed in response to Ca2+ in a bathing medium higher than 3 x 10(-7) M and reached maximum at 1 x 10(-5) M. Sr2+ was less effective than Ca2+; the threshold concentration was 1 x 10(-5) M and the tension reached maximum at 1 x 10(-3) M. Ba2+ was the least effective among the three alkaline earth metal ions; only one fifth of the Ca(2+)-induced maximum tension was attained at 1 x 10(-3) M. The sensitivity was similar to that of the mature pectoral muscle fiber, a fast twitch muscle fiber, rather than that of the anterior latissimus dorsi, a slow tonic muscle fiber. The sensitivity was shown to be dependent on its troponin C by replacing it with troponin C from the mature pectoral or cardiac muscle. This indicates that TnC of a fast-muscle type is expressed in the cultured chick myotube as in the mature pectoral muscle. The contractile apparatus was thus shown to be well developed in the cultured myotube with characteristics similar to the mature fast twitch muscle fiber.     

Purification of chicken gizzard myosin light-chain kinase, and its calcium and strontium sensitivities as compared with those of superprecipitation and ATPase activities of actomyosin

1. A purified preparation of myosin light-chain kinase (MLCK) was obtained from chicken gizzard, and it was shown to consist of two subunits; 130,000 (130 K)-dalton subunit and 17,000 (17 K)-dalton subunit. In amino acid composition the 130 K and 17 K subunits were identical with the 105 K and 17 K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17 K subunit of our MLCK preparation responded to Ca2+ ions in the same way as bovine calmodulin, and differently from skeletal troponin C. There appeared to be one minor difference between 17 K subunit and calmodulin in the primary structure of the C-terminal region. 2. The Ca2+ and Sr2+ concentrations required for the three activities (ATPase and superprecipitation activities and MLCK activity) were measured. Two types of "reconstituted" myosin B were used; one contained 17 K subunit of gizzard MLCK and the other contained bovine brain calmodulin. The two types of "reconstituted" myosin B were practically identical with "natural" myosin B in the Ca2+ and Sr2+ requirements for the three activities measured above. 3. Both the extent and the activity of superprecipitation, and both the limited and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring 32 P incorporation from [gamma-32P]ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.     

Inhibition of muscle actomyosin ATPase activity by mitochondrial ATPase inhibitor.

Ascites hepatoma cell line AH-130 was tested for the ability to transport various amino acids and glutathione before and after γ-glutamyl transpeptidase of the cells was affinity-labeled and inactivated by 6-diazo-5-oxo-L-norleucine, a glutamine analog. The rate of uptake of alanine, glycine, leucine and glutamine by the cells remained unchanged after γ-glutamyl transpeptidase was inactivated by this affinity label. This indicated that γ-glutamyl transpeptidase of the cell was not involved in the transport process of these amino acids tested. The uptake of glutathione was also tested before and after affinity labeling the enzyme. The total amount of the radioactivity incorporated into the cells was not significantly affected by the enzyme inactivation. However, the relative amount of incorporated intact glutathione was found to be slightly but significantly increased after membraneous γ-glutamyl transpeptidase was inactivated by the affinity label, while that of component amino acid, glycine, was found to decrease. This indicated that glutathione was taken up by the cell in its intact form as well as in degraded forms into its component amino acids, and γ-glutamyl transpeptidase in the ascites tumor cell AH-130 seemed to be involved in the metabolic process via the latter system.     

Activation of skinned muscle fibers by calcium and strontium ions

Intact and mechanically skinned skeletal muscle fibers of the crab Carcinus maenas have been used. The aim of the experiments was to determine the origin of the mechanical activity recorded in intact crab muscle fibers exhibiting an inward strontium current in strontium solution without calcium. To do so, the effect of strontium ions in inducing activation of contractile proteins and calcium release from the sarcoplasmic reticulum has been studied. The properties of the sarcoplasmic reticulum membrane towards strontium ions, i.e., the efficiency of the calcium ATPase towards strontium ions and the capability to release strontium ions have been investigated. Results show that the contractile proteins have a lower affinity for strontium than for calcium ions. However, the maximum bound strontium is identical to the maximum bound calcium. As for the sarcoplasmic reticulum, strontium ions can induce a calcium release and also can be taken up by the calcium ATPase and be released. We concluded that the mechanical activity in intact fibers bathed in a strontium medium has two origins: first, a direct and partial activation of the contractile proteins by strontium ions flowing through the calcium channel; second, a contractile proteins activation of calcium ions released by the sarcoplasmic reticulum by a "strontium-induced calcium release" mechanism.     

Histones bind to contractile proteins and inhibit actomyosin ATPase

Nuclear histones bind to and precipitate the major contractile proteins, actin and myosin. The binding of histone to actin seems to reach saturation at 2:1 ratio, the interaction may serve some regulatory function(s) in intranuclear events. The binding of histone to myosin is not saturable, and, although it inhibits the actin-activated Mg2+-dependent myosin ATPase activity, does therefore not seem of physiological importance.     

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.     

Regulation of molluscan actomyosin ATPase activity

The interaction of myosin and actin in many invertebrate muscles is mediated by the direct binding of Ca2+ to myosin, in contrast to modes of regulation in vertebrate skeletal and smooth muscles. Earlier work showed that the binding of skeletal muscle myosin subfragment 1 to the actin-troponin-tropomyosin complex in the presence of ATP is weakened by less than a factor of 2 by removal of Ca2+ although the maximum rate of ATP hydrolysis decreases by 96%. We have now studied the invertebrate type of regulation using heavy meromyosin (HMM) prepared from both the scallop Aequipecten irradians and the squid Loligo pealii. Binding of these HMMs to rabbit skeletal actin was determined by measuring the ATPase activity present in the supernatant after sedimenting acto-HMM in an ultracentrifuge. The HMM of both species bound to actin in the presence of ATP, even in the absence of Ca2+, although the binding constant in the absence of Ca2+ (4.3 X 10(3) M-1) was about 20% of that in the presence of Ca+ (2.2 X 10(4) M-1). Studies of the steady state ATPase activity of these HMMs as a function of actin concentration revealed that the major effect of removing Ca2+ was to decrease the maximum velocity, extrapolated to infinite actin concentration, by 80-85%. Furthermore, at high actin concentrations where most of the HMM was bound to actin, the rate of ATP hydrolysis remained inhibited in the absence of Ca+. Therefore, inhibition of the ATPase rate in the absence of Ca2+ cannot be due simply to an inhibition of the binding of HMM to actin; rather, Ca2+ must also directly alter the kinetics of ATP hydrolysis.     

Modeling of the actomyosin ATPase activity

In the rapid “quench” kientics of myosin, the “initial phosphate burst” is the excess inorganic phosphate that is produced during the early time-course of ATP hydrolysis by myosin subfragment-1 (S-1) or HMM. In general, the existence of a Pi burst implies a rapid (i.e., generally an order of magnitude faster than the steady-state hydrolysis rate) lysis of the phospho-anhydride bond within the ATP molecule, followed by one or more slower steps that are rate limiting for the process. Thus, the presence of a Pi burst can provide an important clue to the mechanism of the reaction. However, in the case of actomyosin, this clue as long been the subject of controversy and misunderstanding. To measure the (initial) Pi burst, myosin S-1 (or HMM) is rapidly mixed with ATP and then the mixture is acid quenched after a specific time period. The medium produced contains free Pi generated from hydrolysis of the ATP. The quantitative measure of the phosphate generated in this way has always been significantly greater than that expected by steady-state “release” of Pi alone, and it is that very difference between this measured Pi after the quench and that amount of Pi expected to be released by steady-state considerations in that same time period that has been referred to as the “initial Pi burst”. Recent investigations of the kinetics of Pi release have used an entirely new method that directly measures the release of Pi from the enzyme-product complex. These studies have made reference to the properties of the “initial Pi burst” in the presence of actin, as well as to a new kinetic entity: the “burst of Pi release”, and have been often vague concerning the true nature of the initial Pi burst, as well as the properties of Pi release as predicted by the current models of the actin activation of the myosin ATPase activity. The purpose of the current article is to correct this oversight, to discuss the “burst” in some detail, and to display the kinetics predicted by the current models for the actin activation of myosin. Furthermore, predictions for the kinetics of the new “burst of Pi release” are discussed in terms of its ability to discriminate between the two current competing models for actin activation of the myosin ATPase activity.     

Effect of nitric oxide on ATPase activity of smooth muscle actomyosin

The effects of nitric oxide donor sodium nitroprusside (SNP) on ATPase activities of smooth muscle actomyosin and myosin were investigated. The effect of SNP on actomyosin ATPase activity was biphasic: the low concentration of this reagent increased the actomyosin ATPase activity while the high concentration exerted opposite effect. These effects were similar to those induced by the specific thiol-alkylating agent N-ethylmaleimide. These data demonstrate that nitric oxide exert the direct effect on smooth muscle contractile proteins. Such effect may be involved in physiological action of NO on smooth muscle.     

The steady-state actomyosin ATPase: a further note.

The simplest kinetic scheme capable of describing the MgATPase of actomyosin was proposed by Lymn &; Taylor (1971) as

Previously (Lymn, 1974) I discussed some steady-state properties which could be expected from this scheme, in particular the character of plots of [myosin]/velocity versus 1/[actin]. This note discusses plots of [actin]/velocity versus 1/[myosin] and shows why the two types of plot will not in general give the same value for the extrapolated V_max of actomyosin.     

The effect of magnesium ions on the two-stage kinetics of superprecipitation and ATPase activity of natural actomyosin

The effect of magnesium ions on the two-stage kinetics of superprecipitation (SPP) and ATP activity of natural skeletal muscle actomyosin was studied. It was found that the changes in the ratios of two independent steps of SPP and ATPase activity are mainly induced by the Mg-ATP2- complex, but not by free Mg2+. These changes in the kinetics of SPP and ATPase are regarded as being due to the shift in the dynamic equilibrium between the two types of the actomyosin complexes in solution, each of which is characterized by different reaction mechanisms. The role of the Mg-ATP2(-)-induced alteration of at least two structural-and-functional states of actomyosin in muscle contractibility is discussed.