A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized actomyosin.
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ABSTRACT:
SUBMITTER: Hartshorne DJ
PROVIDER: S-EPMC1271232 | biostudies-other | 1967 Sep
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. Tropomyosin preparations of the Bailey type, and those prepared in the presence of dithiothreitol to prevent oxidation of protein thiol groups, inhibit the Ca(2+)-activated adenosine triphosphatase (ATPase) of desensitized actomyosin by up to 60%. 2. The inhibitory activity of myofibrillar extracts and tropomyosin survives various agents known to denature proteins but to the action of which tropomyosin is unusually stable, namely heating at 100 degrees and mild tryptic digestion. It is destroyed by prolonged treatment with trypsin. 3. The ethylenedioxybis-(ethyleneamino)tetra-acetic acid (EGTA)-sensitizing factor present in extracts of natural actomyosin and myofibrils could be selectively destroyed, leaving unchanged the inhibitory effect on the Ca(2+)-activated ATPase. There was no correlation between the EGTA-sensitizing and the Ca(2+)-activated inhibitory activities of tropomyosin prepared under different conditions. 4. Optimum inhibition was achieved when tropomyosin and the myosin of desensitized actomyosin were present in approximately equimolar proportions. Tropomyosin had no effect on the Ca(2+)-activated ATPase of myosin measured under similar conditions. 5. Evidence is presented showing that the tropomyosin binds to desensitized actomyosin under the conditions in which the ATPase is inhibited.
| S-EPMC1198446 | biostudies-other
Project description:1. A simple procedure involving repeated washings of actomyosin, extracted as the complex from myofibrils (natural actomyosin) at ionic strength less than 0.002, is described for the preparation of a desensitized actomyosin. 2. The Mg(2+)-activated adenosine triphosphatase of natural actomyosin was markedly inhibited by ethylenedioxybis(ethyleneamino)tetra-acetic acid, whereas that of the desensitized actomyosin was unaffected. 3. The activity of the Ca(2+)-activated adenosine triphosphatase of natural actomyosin was generally lower than that of the Mg(2+)-activated adenosine triphosphatase, whereas in the desensitized actomyosin the difference between the activities was considerably less. In both natural and desensitized actomyosin the adenosine-triphosphatase activities in the presence of Mg(2+) were similar. 4. The conversion of the natural into the desensitized actomyosin was accompanied by the removal of a protein fraction containing the factors responsible for the sensitivity to ethylenedioxybis(ethyleneamino)tetra-acetic acid and for modifying the Ca(2+)-activated adenosine triphosphatase. When added to a desensitized actomyosin this fraction effected a reversal to the natural form. The recombination was facilitated by increasing the ionic strength of the medium. The two factors showed different stabilities to heat and tryptic digestion.
| S-EPMC1270571 | biostudies-other
Project description:The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.
| S-EPMC1168088 | biostudies-other
Project description:1. The effects of Ca(2+) and Mg(2+) on the enzymic activity of myosin were studied with myosin preparations treated by the ion-exchange resin Chelex-100. A reaction mixture containing 0.05m-potassium chloride was chosen in which the effects of univalent ions such as K(+), Na(+) and Cl(-) do not change significantly with small variations in their concentrations. 2. The relationship between the rate of hydrolysis of ATP or ITP and the concentration of Ca(2+) suggests that a relatively weak binding of Ca(2+) either to myosin or to the substrate nucleotide is responsible for the activation of the enzymic activity. According to the experiments with an ultrafiltration technique, the binding of Ca(2+) to myosin proceeds in at least two steps, the first occurring at one site on every 500000 atomic mass units of myosin with an apparent association constant, K(app.), 1.3x10(6)m(-1), and the second seeming to be so weak that its binding parameters cannot be determined by the method used. The first type of Ca(2+) binding is not observable with N-ethylmaleimide-modified myosin, yet this modified myosin shows activation by Ca(2+) of its adenosine triphosphatase and inosine triphosphatase. 3. The inhibition by Mg(2+) can be related to a binding reaction of Mg(2+) with myosin having K(app.) approximately 10(6)m(-1). Mg(2+) replaces the Ca(2+) bound tightly to myosin. The K(app.) for Mg(2+)-myosin binding calculated by assuming a competition between Ca(2+) and Mg(2+) for the same site is 2.1x10(5)-3.0x10(5)m(-1). When myosin is modified with a thiol reagent (p-mercuribenzoate) at a certain ratio to myosin, the inhibition by Mg(2+) becomes unobservable. 4. The behaviour of the hydrolytic activity of myosin on ATP or ITP in the presence of both Ca(2+) and Mg(2+) is consistent with the explanation that the inhibition by Mg(2+) is due to the tight binding of Mg(2+) to myosin, whereas the activation by Ca(2+) is caused either by a weak binding of Ca(2+) to myosin or by CaATP(2-) or by both.
| S-EPMC1184772 | biostudies-other
Project description:1. After removal of tropomyosin and troponin from the ;natural' actomyosin complex, the adenosine triphosphatase activity of the resulting ;desensitized' actomyosin is stimulated to the same extent by various bivalent cations with an ionic radius in the range 0.65-0.99å when tested at optimum concentration of the metal ion in the presence of 2.5mm-ATP at low ionic strength and pH7.6. Under identical conditions the adenosine triphosphatase activity of myosin alone is stimulated to an appreciable extent only by Ca(2+) (ionic radius 0.99å). 2. Tropomyosin narrows the range of size of the stimulatory cations by inhibiting specifically the adenosine triphosphatase activity of ;desensitized' actomyosin when stimulated by Ca(2+) or the slightly smaller Cd(2+) (ionic radius 0.97å). Tropomyosin has no effect on the adenosine triphosphatase activity of ;desensitized' actomyosin when stimulated by the smaller cations, nor on the Ca(2+)-activated adenosine triphosphatase activity of myosin alone. 3. The adenosine triphosphatase activity of the ;natural' actomyosin system (containing tropomyosin and troponin) stimulated by the smallest cation, Mg(2+) (ionic radius 0.65å), is low when the system is deprived of Ca(2+) but high in the presence of small amounts of Ca(2+). This sensitivity to Ca(2+) seems to be a unique feature of the Mg(2+)-stimulated system. 4. The changes in specificity of the myosin adenosine triphosphatase activity in its requirement for bivalent cations caused by interaction with actin, tropomyosin and troponin primarily concern the size of the metal ions. The effects on enzymic properties of myofibrils due to tropomyosin and troponin can be demonstrated at low and at physiological ionic strength.
| S-EPMC1187607 | biostudies-other
Project description:The kinetics of the Mg2+-dependent ATPase (adenosine triphosphatase) activity of bovine cardiac myosin and its papain subfragment-1 were studied by using steady-state and pre-steady-state techniques, and results were compared with published values for the corresponding processes in the ATPase mechanism of rabbit skeletal-muscle myosin subfragment-1. The catalytic-centreactivity for cardiac subfragment-1 is 0.019s-1, which is less than one-third of that determined for the rabbit protein. The ATP-induced isomerization process, measured from enhancement of protein fluorescence on substrate binding, is similarly decreased in rate, as is also the isomerization process associated with ADP release. However, the equilibrium constant for ATP cleavage, measured by quenched-flow by using [gamma-32P]ATP, shows little difference in the two species. Other experiments were carried out to investigate the rate of association of actin with subfragment-1 by light-scattering changes and also the rate of dissociation of the complex by ATP. The dissociation rate increases with increasing substrate concentration, to a maximum at high ATP concentrations, with a rate constant of about 2000s-1. It appears that isomerization processes which may involve conformational changes have substantially lower rate constants for the cardiac proteins, whereas equilibrium constants for substrate binding and cleavage are not significantly different. These differences may be related to the functional properties of these myosins in their different muscle types. Kinetic heterogeneity has been detected in both steady-state and transient processes, and this is discussed in relation to the apparent chemical homogeneity of cardiac myosin.
| S-EPMC1164118 | biostudies-other
Project description:Magnesium deficiency increases the generation of pro-inflammatory cytokines, which is consistently accompanied by the sensitization of cells such as neutrophils, macrophages and endothelial cells. We investigated the potential of magnesium to regulate macrophage polarization and macrophage-induced inflammation with or without lipopolysaccharide (LPS) and interferon-? (IFN-?) activation and further elucidated whether these effects impact the inhibitory functions of activated macrophage-induced inflammation on cartilage regeneration. The results showed that magnesium inhibited the activation of macrophages, as indicated by a significant reduction in the percentage of CCR7-positive cells, while the percentage of CD206-positive cells decreased to a lesser degree. After activation, both pro-inflammatory and anti-inflammatory cytokines were down-regulated at the mRNA level and certain cytokines (IL-1?, IL-6 and IL-10) were decreased in the cell supernatant with the addition of magnesium. Moreover, magnesium decreased the nuclear translocation and phosphorylation of nuclear factor-?B (NF-?B) to impede its activation. A modified micromass culture system was applied to assess the effects of activated macrophage-conditioned medium with or without magnesium treatment on the chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Magnesium enhanced the chondrogenic differentiation of hBMSCs by reversing the adverse effects of activated macrophage-induced inflammation.
| S-EPMC5821731 | biostudies-literature
Project description:1. When rat spleen mitochondria are incubated with oxidizable substrates, added MgCl2 (greater than 150 muM free concentration) markedly stimulates state-4 respiration and lowers both the respiratory control and ADP/O ratios; this effect is reversible on addition of excess of EDTA. 2. With [gamma-32P]ATP as substrate, an Mg2+-stimulated ATPase (adenosine triphosphate) was identified in the atractyloside-insensitive and EDTA-accessible space of intact rat spleen mitochondria. 3. Oligomycin has no effect on the activity of the Mg2+-stimulated ATPase at a concentration (2.0mug/mg of protein) that completely inhibits the atractyloside-sensitive reaction. Of the two ATPase activities, only the atracytoloside sensitive reaction is stimulated (approx. 40%) by dinitrophenol. 4. On digitonin fractionation the atractyloside-insensitive Mg2+-stimulated ATPase co-purifies with the outer membrane-fraction of rat spleen mitochondria, whereas (as expected) the atractylosidesensitive activity co-purifies with the inner-membrane plus matrix fraction. 5. Stoicheiometric amounts of ADP and Pi are produced as the end products of ATP hydrolysis by purified outer-membrane fragments; no significant AMP production is detected during the time-course of the reaction. 6. The outer-membrane ATPase is present in rat kidney cortex and heart mitochondria as well as in spleen, but is absent from rat liver, thymus, brain, lung, diaphragm and skeletal muscle.
| S-EPMC1164245 | biostudies-other
Project description:The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.
| S-EPMC1184146 | biostudies-other
Project description:1. Two mutants of Escherichia coli K 12 were isolated which, although able to grow on glucose, are unable to grow with succinate or d-lactate as the sole source of carbon. 2. Genetic mapping of these mutants showed that they both contain a mutation in a gene (designated uncA) mapping at about minute 73.5 on the E. coli chromosome. 3. The uncA(-) alleles were transferred by bacteriophage-mediated transduction into another strain of E. coli and the transductants compared with the parent strain to determine the nature of the biochemical lesion in the mutants. 4. The mutants gave low aerobic growth yields when grown on limiting concentrations of glucose, but oxidase activities in membranes from both the mutants and the normal strain were similar. 5. Measurement of P/O ratios with d-lactate as substrate indicated that a mutation in the uncA gene causes uncoupling of phosphorylation associated with electron transport. 6. Determination of the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activities in the mutant and normal strains indicated that the uncA gene is probably the structural gene for Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 7. Mg(2+),Ca(2+)-stimulated adenosine triphosphatase therefore appears to be essential for oxidative phosphorylation in E. coli.
| S-EPMC1177115 | biostudies-other