Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reaction of trypsin on the heavy chain of gizzard myosin and chymotryptic HMM was investigated under restricted fragmentation conditions. The three fragments of the head part with 29 kDa, 50 kDa and 26 kDa were isolated and identified. The 66 K heavy chain segment containing the S1-S2 junction was slowly but extensively degraded liberating a S1-like entity which lacked an intact COOH-terminal 26 kDa region; this isolated species displayed full intrinsic ATPase activities but little actin-binding ability. Tryptic HMM was also formed bearing a fragmented heavy chain and lacking the 20 kDa light chain. Its actin-activated ATPase was derepressed upon cleavage of the 66 kDa segment by papain. We propose that the integral 66 kDa heavy chain component is directly involved in the regulation of the gizzard actomyosin ATPase.
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PMID:Structural and actin-binding properties of the trypsin-produced HMM and S1 from gizzard smooth muscle myosin. 634 18

Sedimentation in a preparative ultracentrifuge was used to determine the affinity of heavy meromyosin, HMM, for regulated actin, F-actin plus troponin-tropomyosin, in the presence of MgATP at pH 7.0, 20 degrees C, and mu = 18 mM. HMM was prepared from vertebrate striated muscle myosin by a mild chymotryptic digestion. This HMM contained 85-90% intact 19 000-dalton light chains, LC2. In the presence of calcium, 90% of the HMM bound to regulated actin with an association constant of (2-4) X 10(4) M-1. In the absence of calcium, while one-third of the HMM bound with an affinity similar to that observed in the presence of calcium, the rest bound much more weakly. It was not possible to accurately determine the association constant for this weakly binding HMM, but a 20-fold reduction in affinity is consistent with the binding data. The binding of single-headed heavy meromyosin to regulated actin was similarly sensitive to the calcium concentration. Since removal of calcium inhibits the regulated actin-activated ATPase of HMM greater than 20-fold, troponin-tropomyosin must be capable of inhibiting both the binding of HMM to regulated actin and a step which occurs after binding but prior to product release. Removal of LC2 increased the fraction of HMM with calcium-insensitive binding, and adding LC2 back to this depleted HMM restored most of the calcium sensitivity. Chymotryptic cleavage of LC2 to a 17 000-dalton fragment destroyed the calcium-sensitive binding of HMM to regulated actin. Phosphorylation of LC2, however, had no detectable effect on this binding. Thus, the calcium-sensitive binding of HMM to regulated actin requires that both the head-tail junction and the N-terminal part of LC2 be intact. Binding studies with cross-linked regulated actins and kinetic measurements of the rates of change in turbidity demonstrate that this calcium sensitivity is due to calcium binding to troponin and not to LC2.
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PMID:Calcium-sensitive binding of heavy meromyosin to regulated actin requires light chain 2 and the head-tail junction. 640

We have established a method to estimate the values of various kinetic parameters of acto-heavy meromyosin (acto-HMM) ATPase, using a fluorescent ATP analog, beta-naphthyl triphosphate (beta-NapP3); from the fluorescence intensity change accompanying beta-NapP3 hydrolysis, the various kinetic parameters of beta-NapP3 hydrolysis, including its product inhibition, were obtained. beta-NapPd3 hydrolysis is inhibited competitively by ATP, resulting in different time courses of fluorescence intensity change in the presence and absence of ATP. From this difference, the values of kinetic parameters of ATP hydrolysis, including its product inhibition, can be estimated. By extending this method to the acto-HMM system, seventeen parameters in a reaction scheme for the concurrent hydrolysis of ATP and beta-NapP3, including association constants between F-actin and substrate-free or substrate-bound HMM, were obtained. The kinetic-parameters estimated for ATP hjydrolsis were in good agreement with those in the literature.
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PMID:Fluorometric method for estimating the kinetic parameters of beta-naphthyl triphosphate and ATP hydrolysis by acto-heavy meromyosin. 644 47

ATPase activity and ATP-Pi exchange of unregulated (without tropomyosin-troponin) and regulated (with tropomyosin-troponin) acto-HMM were measured in media containing 0.2 mg/ml actin, HMM, and (when present) tropomyosin-troponin, 2 mM MgCl2, 10 mM KCl, 2 mM NaN3, 10 mM Pi (pH 7.0), 3 MM ATP. The following mean values for ATPase activity and for the rate of incorporation of Pi into ATP (each per mg HMM and per min) were obtained: unregulated acto-HMM 0.33 mumol Pi and 0.33 nmol Pi, regulated acto-HMM 0.54 mumol Pi and 1.06 nmol Pi. The ratio of Pi incorporation rate to ATPase activity was 1.01 x 10(-3) for unregulated and 2.02 x 10(-3) for regulated acto-HMM. From these ratios and from the overall free energy change of ATP hydrolysis it was calculated that under the prevailing experimental conditions in unregulated acto-HMM 62% and in regulated acto-HMM 66% of the free energy change of ATP hydrolysis occurs after the release of phosphate from actomyosin. It is probably this part of the free energy change that is used by the muscle for the performance of work.
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PMID:Tropomyosin-troponin-induced changes in the partitioning of free energy release of actomyosin-catalyzed ATP hydrolysis as measured by ATP-phosphate exchange. 644 20

In a previous paper (Kimura, I., Arai, K., & Watanabe, S. (1979) J. Biochem. 86, 1629-1638), we reported that when myosin was subjected to heat treatment, inactivation of actomyosin ATPase occurred in two steps; an early fast inactivation, followed by a slow inactivation. A further study was conducted on the fast inactivation in the early stage of heat treatment of myosin, and the following results were obtained: 1. As the weight ratio of actin to heat-treated myosin increased, the rate of early inactivation of actomyosin ATPase increased. 2. The superprecipitation activity of actomyosin was also decreased in two steps by heat treatment of myosin: an early fast inactivation and a subsequent slow inactivation were distinguishable. 3. The turbidity of myosin suspensions in 0.05 M KCl increased during heat treatment of myosin. The increase also proceeded in two distinct steps, and the rate constants for the two steps of increase were comparable to those for inactivation of actomyosin ATPase. 4. In an electron microscopic study, a new change in the morphology of "thick filaments" of myosin was detectable in the early period of heat treatment, when the fast inactivation of actomyosin ATPase was found to occur; myosin filaments were shorter and thinner than the "regular thick filaments" of untreated myosin, and their size was much more heterogeneous. Heterogeneity of myosin aggregates was also detectable in the sedimentation patterns. 5. The actin-binding ability of myosin or HMM remained unaffected during heat treatment of myosin. The binding ability was estimated in three different ways; by measuring the ATP-induced changed in the turbidity of actomyosin solution, the actin-inhibition of EDTA-ATPase of myosin, and the actin-activation of Mg-ATPase of HMM. 6. In a sedimentation study, formation of myosin dimers was not detectable in the early period of heat treatment of myosin, and was detectable only in the later period. Likewise, increase in the light scattering intensity of myosin solutions occurred only in the later period of heat treatment of myosin. 7. Heat treatment caused a decrease in the ATP-induced enhancement of the HMM fluorescence. The decrease was fitted by a single exponential (first-order reaction), and not by two exponentials. 8. Heat-induced change was not detectable either in the light scattering intensity of LMM solutions in 0.5 M KCl or in the turbidity of LMM suspensions in 0.05 M KCl. Based on these results, it is strongly suggested that the earliest event in heat denaturation of myosin is loss of the ability of myosin to form "regular thick filaments."
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PMID:Earliest event in the heat denaturation of myosin. 645 Jul 57

Actin modulating (AM-) protein from Physarum binds as monomer and as a stable heterodimer with one actin molecule to actin and induces the formation of oligomeric actin complexes and short-chained filaments with no definite stoichiometry. While inhibiting the extent of actin polymerization by reducing the size of the aggregates formed, AM-protein increases the velocity of polymerization. The AM-protein monomer depolymerizes F-actin very rapidly by breaking the filaments into small pieces and oligomeric complexes. When a heterodimer of actin an AM-protein is reconstituted the polymerization inhibitor activity is identical to that of the monomer, but depolymerization of actin is almost completely abolished. The action of the monomeric AM-protein on actin is highly Ca++-dependent as it requires micromolar amounts of Ca++ for full activation. The inhibitory activity of both the natural and the reconstituted heterodimer has only little Ca++-sensitivity: A prolonged exposure to Ca++-chelating agents is necessary to obtain a partial inactivation of the heterodimers. The depolymerizing effect on actin of the AM-protein monomer is inhibited by tropomyosin and also by heavy meromyosin. Addition of phalloidin to the actin reduces only the velocity of depolymerization by AM-protein. In the presence of AM-protein the actomyosin ATPase or Acto-HMM ATPase is strongly inhibited. The supposed function of the AM-protein in Physarum is that of a powerful regulator of the polymer state of actin. Such a regulation system is necessary for the dynamic actin transformation processes during ectoplasm-endoplasm transitions and the assembly-disassembly of contractile and cytoskeletal structures. The Ca++-sensitivity of the AM-protein indicates that these processes are controlled by calcium.
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PMID:An actin-modulating protein from Physarum polycephalum. II. Ca++-dependence and other properties. 645 27

The structural properties of cardiac isomyosins from several species were compared using native gel electrophoresis, analysis of proteolytic digests, analysis of monoclonal antibody reactivity to specific proteolytic fragments on electroblots and S1 nuclease mapping with cDNA probes. The structure of specific regions of the myosin molecule was analyzed by reacting monoclonal antibodies with chymotryptic peptides of myosin separated by two-dimensional electrophoresis. The pattern of fragments reactive with antibody CCM-52 (epitope in LMM) was identical in all types of V3 isomyosin examined, and different in each type of V1 isomyosin. Peptides reactive with RCM-79 (epitope in HMM) were different from those reactive with CCM-52 and were also significantly different in each type of myosin examined. Thus, HC-alpha is structurally similar in the LMM portion of the molecule in all animals examined, while in the HMM region there are significant structural differences. HC-alpha differs from HC-beta, with structural differences in both LMM and HMM. We have also shown that atrial myosin HC and ventricular HC-alpha in the rabbit are indistinguishable both by RIA and peptide mapping analysis. The same conclusion was derived after analysis of the myosin HC mRNA expressed in rabbit atria and ventricles. Using cDNA probes specific for the alpha and beta myosin HC mRNA, we could not distinguish between the atrial myosin mRNA and ventricular HC alpha (V1 isomyosin) mRNA by S1 nuclease mapping experiments. Classification of different cardiac myosins is largely based on their mobility on native gel electrophoresis, immunological cross-reactivity, and ATPase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Classification and characterization of cardiac isomyosins. 653

Previously, several workers reported that at very low ionic strength and in the presence of ATP, the extent of binding of S-1 with the F-actin-tropomyosin-troponin complex or regulated actin (FA-TM-TN) is unaffected by removal of Ca2+. However, in this study we found that during the ATPase reaction at physiological ionic strength, the extent of binding of HMM with FA-TM-TN decreased markedly upon removal of CA2+. Therefore, the effects of Ca2+ were studied on the intermediate steps in the acto-HMM or acto-S-1 ATPase reaction. 1. The nucleotide-induced dissociation of acto-s-1 was studied using AMPPNP as substrate. The extent of binding of S-1 with regulated actin in the presence of Mg2+-AMPPNP increased in a sigmoidal manner as the S-1 concentration increased. When the molar ratio of actin monomer to S-1 was higher than 5-10, the removal of Ca2+ shifted the equilibrium of the dissociation reaction, FA-S-1-AMPPNP in equilibrium FA + S-1-AMPPNP, to the right. 2. The recombination rate of HMMPADP or S-1PADP with regulated actin in the absence of free Mg2+-ATP was estimated by measuring the time course of recovery in light-scattering intensity after addition of ATP. The rate decreased upon removal of Ca2+, when the molar ratio of actin monomer to S-1 was higher than 5-10. 3. The decomposition rate of HMMPADP was measured in the presence of Mg2+-ATP. In the absence of Ca2+, regulated actin did not affect this rate, whereas in its presence, regulated actin markedly accelerated the rate. These findings clearly indicated that at physiological ionic strength, removal of Ca2+ affects various elementary steps in the ATPase reaction to promote the dissociation of myosin heads from FA-TM-TN.
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PMID:Regulation of binding of myosin subfragments with regulated actin by calcium ions in the presence of magnesium ATP. 698 Feb 20

The recent communication by Bishop and Gray (J. Biochem. 90, (1981)) on the streaming in the circular slit of stream cells is probably based on observations under conditions where no active streaming can be produced because of a substantial lack of F-actin. According to our experience, these conditions are similar to those obtained when actins are partly denaturated. Hence, for the sake of their criticism, detailed comparisons should be added of the stability of streaming, the distribution of streaming velocities as well as their sizes, acto-HMM ATPase activities under various streaming velocities and the amount of proteins fixed to the Millipore filter.
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PMID:Comments on Bishop and Gray's criticism of the streaming driven by acto-heavy meromyosin. 703 Oct 47

A fluorescent ATP analog, 2'-(5-dimethylaminonaphthalene-1-sulfonyl)amino-2'-deoxy ATP (DNS-ATP), was synthesized. In water, the wavelengths of maximum excitation were 260 and 340 nm, and that of maximum emission was 554 nm. The fluorescence quantum yield with excitation at 340 nm was 0.052. In 80% dioxane-20% water solution, the wavelength of the maximum emission shifted to 527 nm and the quantum yield was about 5.4 times in water. When DNS-ATP was mixed with HMM in the presence of Mg2+ ions, the fluorescence intensity of DNS-ATP was enhanced by about 30%, and the wavelength of maximum emission shifted to 545 nm. The observed second-order rate constant for the change in fluorescence intensity after adding DNS-ATP to HMM was 1.6 x 10(-7) M-1 . s-1, while the observed first-order rate constant for its recovery was 0.17 s-1. When the HMM DNS-ATPase reaction was measured in terms of the TCA-Pi liberation, 1 mol of initial burst of Pi liberation per mol of myosin was observed. In 50 mM KCl and at 20 degrees C, the rate of the HMM DNS-ATPase reaction was increased by F-actin from 0.4 to 1.15 s-1 (in 3 mg/ml F-actin). The observed dissociation constant for the binding of DNS-ATP with HMM increased from 1.2 to 20 microM in the presence of 5 mg/ml F-actin. However, the extent of change in fluorescence intensity at infinite concentration of DNS-ATP was unaffected by the presence of F-actin.
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PMID:Preparation of a new fluorescent analog of ATP, 2'-(5-dimethylaminonaphthalene-1-sulfonyl)amino-2'-deoxy ATP, and its interactions with myosin and actomyosin. 703 Oct 48


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