Search PubMed⌕ Search

Biomedical subjects

S Ishiwata

Publications and source records attributed to S Ishiwata.

At least 127 records · Page 7Linked to original sources
PubMed1987

Beta-actinin is not distinguishable from an actin barbed-end capping protein in chicken breast muscle.

beta-Actinin is an actin-pointed end capping protein in skeletal muscle. Casella et al. have reported that a protein isolated from muscle acetone powder by procedures similar to those used for beta-actinin purification caps the barbed end of an actin filament (J. Biol. Chem. 261, 10915-10921 (1986)). We have confirmed the above results. However, it turned out that the two proteins were identical as to subunit sizes, peptide maps, and cross-reactivities with anti-beta-actinin IgG. The binding of the two proteins to opposite ends of an actin filament remains unexplained.

Actin Depolymerizing Factors↗
PubMed1986

Dynamic light-scattering study on polymerization process of muscle actin.

Globular actin (G-actin) polymerizes into a fibrous form (F-actin) under physiological salt conditions. The polymerization process of muscle actin was studied by a dynamic light-scattering method. The intensity correlation functions G2(tau) of scattered light from a G-actin solution containing 2 mM Tris-HCl (pH 8.0) and 0.1 mM ATP were analyzed by a cumulant expansion method, and the translational diffusion coefficient was determined to be D = (8.07 +/- 0.10) X 10(-7) cm2/s at 20 degrees C. This D value gave a diameter of 5.3 nm for spherical G-actin including a hydration layer. Polymerization of 1-3 mg/ml G-actin in a solution containing 10 mM Tris-HCl (pH 8.0), 0.2 mM ATP and 60 mM KCl was followed by successive measurements of G2(tau) for a data accumulation period of 60-300 s/run. The time evolution of G2(tau) was analyzed by a least-squares fitting to the field correlation function of a multiexponential form g1(tau) = sigma iAi exp(-gamma i tau) with gamma 1 greater than gamma 2 greater than 3 greater than ..., and the static scattering intensity I(t) = mean value of I as a function of time t after initiation of polymerization was decomposed as I(t) = mean value of I sigma iAi. At the early stage of polymerization, a two-exponential fit gave results indicating that component 1 came from G-actin and component 2 from F-actin growing linearly with t. At the middle stage of polymerization, a three-exponential fit gave the results that component 1 came from G-actin and possibly its small oligomers, component 2 from polymers with a number-average length Ln of about 900 nm which was independent of t, and component 3 from 'ghosts' in dynamic light scattering in a semidilute regime. Component 3 was concluded to arise from restricted motions of polymers with lengths much longer than Ln in cages formed by polymers giving component 2, and a fragmentation-elongation process of F-actin was suggested to start at the middle stage of polymerization, resulting in the size redistribution of F-actin.

Actins↗
PubMed1986

Accumulated strain mechanism for length determination of thick filaments in skeletal muscle. I. Experimental bases.

The kinetics of dissociation of myosin from both ends of thick filaments in glycerinated skeletal muscle fibres and myofibrils was studied in the presence of MgATP by use of an optical diffraction method and phase-contrast microscopy. The dissociation velocity, v (identical to -dL/dt where L is the length of thick filaments at time t), increased with increasing KCl concentration (0.225 to 0.5 M), or increasing pH (6.5 to 8.0) but hardly changed with temperature (5 and 25 degrees C), micromolar concentrations of Ca2+ or sarcomere length (2.4 and 2.75 micron). Over a wide range of filament length, the dissociation velocity could be expressed by v0exp(alpha L), where v0 and alpha are positive constants depending upon the dissociation condition. When the effects of crossbridge formation are minimized it was thus shown that the structural stability of thick filaments in a muscle fibre and a myofibril gradually decreases from the central part to the tips of the filaments. On the basis of these results we propose that the length of thick filaments is largely regulated by an accumulated strain mechanism in which the free energy of association of myosin molecules increases with filament length.

Animals↗
PubMed1986

Saturation transfer electron paramagnetic resonance study of the mobility of myosin heads in myofibrils under conditions of partial dissociation.

The rotational motion of rigidly spin-labeled myosin heads of glycerinated myofibrils as reflected in saturation-transfer EPR spectra behaves to a first approximation as though the heads consist of two populations with different rotational motions. An immobilized fraction has a correlation time (tau 2) of approximately 0.5 ms, comparable to that of spin-labeled subfragment-1 (S1) bound to thin filaments, while a mobile fraction has a tau 2 of 10 microseconds, comparable to that of the heads of purified myosin filaments. The effects of nonhydrolyzable ATP analogues, potassium pyrophosphate (PPi), or adenylyl imidodiphosphate, Ca2+, temperature, or ionic strength on the spectra can be analyzed in terms of the fraction of myosin heads immobilized by attachment to thin filaments, without requiring changes in the motion of either attached or detached heads.

Adenylyl Imidodiphosphate↗
PubMed1986

The state of the art on the chemical characterization of diesel particulates in Japan.

In this paper, the state of the art of the chemical characterization of diesel particulates in Japan is presented. However, some of these results are only preliminary. The principal contents may be summarized as follows: Characterization of Diesel Particulates; Examples of the application of the chemical characterization to various studies are described. Measurements for elemental composition and distributions of molecular weight and boiling point of diesel particulates and related samples were carried out to gain a better understanding of the main components of diesel particulates. Determinations of carbonaceous components, elemental carbon and organic carbon, in diesel particulates were made by a thermal carbon analyzer, and these results were applied to the study of the relations between such components and the driving conditions of diesel vehicles and to the estimate of emission rates of carbonaceous components from on-road diesel vehicles in a highway tunnel. It seems that the thermal carbon analyzer can be used to advantage in the determination of such carbonaceous components. Artifacts of PAH and 1-NP during Collection of Diesel Particulates; In order to minimize the artifacts of PAH during collection of diesel particulates, three methods were investigated for the artifacts of BkF, BaP and BghiP; two methods incorporated the removal of reactive gaseous components such as NO2, with an ACF method and an impinger method, and the third was a high dilution sampling method. These results suggest that the high dilution sampling method is very promising. From the results of various studies including model experiments, it is clear that the artifact formation of 1-NP is of first order dependence on total amounts of nitrate on carbon black and diesel particulates, not on gaseous nitric acid, although second order dependence of the artifact formation on NO2 concentration is ambiguous. Aside from the above studies, the relation between 1-NP concentrations in diesel exhaust and NO2 concentrations in the dilution tunnel with changing the dilution ratio at a fixed driving condition was utilized for an estimate of the artifact formation of 1-NP, and the estimated results are discussed in connection with the reaction order of the artifact formation on NO2 concentration. Concentrations of Various Components in Diesel Exhaust and Ambient Air; Analytical results are presented for the PAH, mononitro-PAH and dinitro-PAH in particulate samples collected from the exhaust of both IDI and DI diesel vehicles and the ambient air in several areas in Japan and China.(ABSTRACT TRUNCATED AT 400 WORDS)

Air↗
PubMed1985

Disassembly from both ends of thick filaments in rabbit skeletal muscle fibers. An optical diffraction study.

We show in this paper that the change of the internal structure of a sarcomere in a rabbit glycerinated psoas muscle fiber can be examined by analyzing the intensity change of the first- and the second-order optical diffraction lines. A unit-cell (sarcomere)-structure model has been applied to the estimation of the length of thick filaments in a muscle fiber while they undergo dissociation. The optical factors, except for the unit-cell-structure factor, hardly changed during the dissociation of the filaments. Our results show that thick filaments dissociate from both ends on increasing the KCl concentration in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed to some extent the dissociation of thick filaments. The disassembly of thick filaments occurred at higher KCl concentrations in the absence of pyrophosphate. There was a correlation between the stability of the thick filament structure and cross-bridge formation, which was induced either by the addition of micromolar concentrations of Ca2+ in the presence of Mg-pyrophosphate or by removal of Mg-pyrophosphate.

Animals↗
PubMed1985

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.

Animals↗
PubMed1985

Does actin bind to the ends of thin filaments in skeletal muscle?

We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ionic strength, marked binding of actin to the free ends, i.e., the pointed ends, of thin filaments was observed when actin was added at an early phase of polymerization. As the polymerization of actin proceeded, the binding efficiency decreased. The critical actin concentration for this binding was higher than that for polymerization in solution. The binding of G-actin was not observed at low ionic strength. On the basis of these results, we suggest that a particular structure suppressing the binding of actin is present at the free ends of thin filaments in intact myofibrils and that a part of the end structure population is eliminated or modified at high ionic strength so that further binding of actin becomes possible. The myofibril and I-Z-I brush appear to be useful systems for studies aimed at elucidating the organizational mechanisms of actin filaments in vivo.

Actins↗
PubMed1985

Characterization of beta-actinin: a suppressor of the elongation at the pointed end of thin filaments in skeletal muscle.

We examined the physico-chemical properties and the functions of beta-actinin by using a beta-actinin preparation having the same properties as those reported by Maruyama et al. (J. Biochem. 81, 215-232, 1977). beta-Actinin was composed of two components with molecular weights (estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis) of 35,000 and 31,000 daltons. Their isoelectric points in 8 M urea were, respectively, 5.9 and 5.4, clearly distinguishable from those of tropomyosin, troponin T and some enzymes having similar molecular weights. beta-Actinin suppressed the polymerization of actin onto the free end, i.e., the pointed end, of thin filaments in an I-Z-I brush prepared by dissolving thick filaments of a myofibril at high ionic strength. Further, beta-actinin suppressed the association of actin to the whole region of an I-Z-I brush. The present study indicates that beta-actinin is composed of two components and functions as a suppressor of elongation at the pointed end of thin filaments, supporting the conclusions of Maruyama et al. (J. Biochem. 81, 215-232, 1977).

Actinin↗
PubMed1984

Dynamic light scattering study of muscle F-actin.

By use of digital autocorrelation and fast Fourier methods, dynamic light-scattering studies of in vitro reconstituted muscle F-actin were made over a wide range of concentrations, 0.01-2 mg/ml F-actin. Measurements of correlation function [g1(t)]2 showed that a transition from a dilute to a semidilute regime for the Brownian motions of filaments occurred at around 0.3 mg/ml F-actin. Beyond this concentration, profiles of successively measured [g1(t)]2 showed very poor reproducibility. This resulted from the existence of very slow components, which could not be measured with a high statistical accuracy even for a measuring time of 3600 s/run. On the other hand, subtraction of these components automatically by an electronic circuit, [g-1(t)]2, or by computer processing, [g1(t)]2, resulted in a fairly good reproducibility of the profiles. The decay characteristics of [g1(t)]2 (and [g-1(t)]2) were very similar to those of [g1(t)]2 for dilute solutions. A theoretical model will be discussed which could account for the above situation. The time sequence [n(t,T)] of photoelectron counts at a sampling time T of light scattered from semidilute solutions of F-actin was stored on magnetic tapes, and both power spectra S(f) and correlation functions [g-1(t)]2 were computed by taking the ensemble average over many short records with duration 1024T. Since both S(f) and [g-1(t)]2 lacked frequency components lower than 1/(2048T) Hz, their profiles were highly reproducible. An analysis of S(f) confirmed our earlier results which had shown an apparent contradiction to later results by a correlation method. A comparison of S(f) and [g-1(t)]2 based on the same [n(t,T)] clarified the reasons why the bandwidth gamma of S(f) largely differed from the bandwidth gamma of [g1(t)]2 and [g-1(t)]2. The temperature dependence of gamma suggested that F-actin would be flexible and that the flexibility parameter would change with temperature.

Actins↗
PubMed1980

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.

Actins↗
PubMed1978

Studies on the F-actin.tropomyosin.troponin complex. II. Partial reconstitution of thin filament by F-actin, tropomyosin and the tropomyosin binding component of troponin (TN-T).

It was found that thin filaments are reconstituted from F-actin, tropomyosin and the tropomyosin binding component of troponin (TN-T) in vitro according to a self-assembly mechanism. Although a gel structure is formed when the three proteins are mixed in the order F-actin, TN-T and tropomyosin, it is in a metastable state and spontaneously transforms to dispersed filaments in an equilibrium state. The rate of the transformation is largely dependent on temperature. When the mixing order of TN-T and tropomyosin is reversed, large amounts of the complex appear immediately in the dispersed filament form. Dependence on the order of mixing is observed only when the molar ratio of TN-T to tropomyosin is about one, i.e. at the physiological ratio. When the molar ratio of TN-T to tropomyosin is either above or below one, a stable gel form or a stable dispersed filament form, was respectively, obtained independently of the mixing order of the three proteins.

Actins↗
PubMed1978

Studies on the F-actin.tropomyosin.troponin complex. III. Effects of troponin components and calcium ion on the binding affinity between tropomyosin and F-actin.

We studied the effects of the troponin components TN-T, TN-I and TN-C, and of Ca2+ concentration on the dissociation temperature of the F-actin.tropomyosin complex. Troponin components and their complexes increased the dissociation temperature in the following sequence of effectiveness: TN-C(+/- Ca2+) less than TN-I less than TN-I.TN-C(+/-Ca2+) less than TN-T.TN-C(+/- Ca2+) less than TN-T.TN-C(--Ca2+) approximately TN-T less than TN-T.TN-I.TN-C(+Ca2+) less than TN-T.TN-I.TN-C(--Ca2+) approximately TN-T.TN-I; TN-C has no effect. The effect of TN-I is small. TN-T is the most important component for increasing the dissociation temperature. The shifting by Ca2+ of the dissociation temperature to lower values is only seen when both TN-T and TN-C are present.

Actins↗
PubMed1977

Dynamic characteristics of F-actin and thin filaments in vivo and in vitro.

Measurements of birefringence, ultraviolet dichorism and quasielastic light scattering were carried out on F-actin in solution and on the thin filaments of glycerinated myofibrils. The birefringence of the I-bands of myofibrils was of the same order of magnitude as that of F-actin or the F-actin-tropomyosin-troponin complex oriented in vitro at the same concentration. The ultraviolet dichroism spectrum of the I-bands was very similar to that of F-actin or the F-actin complex in vitro, which is due to orientation of bound ADP and tryptophan residues in F-actin. Quasielastic light scattering measurements, electronmicroscopic observations and the analyses of the electro-optic effect of the I-bands suggested approximately the same flexibility for F-actin in vitro and for the thin filaments in vivo. These optical measurements which were made under various conditions provide evidence for a conformational change induced by calcium ions in F-actin both in vivo and in vitro. This conformational change was found to be amplified by the interaction of F-actin with myosin. This is a brief review of our investigation on the dynamics of F-actin and the thin filament in vivo and in vitro by optical methods.

Actins↗
PubMed1976

Uni-directional growth of F-actin.

In was shown that the growth of F-actin occurs uni-directionally in vitro; that is, actin monomers (G-ADP-actin) polymerize onto only one of the two ends of a pre-existing seed in the absence of ATP. Fragments of the F-actin-heavy meromyosin complex prepared by sonication were used as seeds. By studying a large number of electron micrographs, we concluded that growth occurs on the tail end of the arrow-head structure of the seed. This result suggests that the thin filament grows in vivo from the center of the sarcomere towards the Z-line. It was further shown that the fragments of F-actin combine with both ends of the seed with nearly equal probability. These results are well interpreted according to the theory developed by Oosawa (1,2).

Actins↗
PubMed1976

Freezing of actin. Reversible oxidation of a sulfhydryl group and structural change.

It was found that the essential change in actin (whether G- or F-actin) on freeze-thawing was the specific oxidation of one of five sulfhydryl (SH) groups, i.e. the SH group of Cys 373 in the amino acid sequence. Oxidized SH groups formed an inter-molecular disulfide (SS) bond to yield an actin dimer. F-actin, subjected to freeze-thawing (or F-actin obtained by the transformation of once frozen G-actin which is essentially a dimer), has anomalous physico-chemical properties and a different conformation from normal F-actin, as determined by optical and electron microscopic observations, as well as high steady ATP-splitting activity in the presence of Mg2+. However, it was found that those peculiarities disappeared and normal actin was reformed on reducing the oxidized SH group with dithiothreitol (DTT). It was also found that the normal characteristics of actin were preserved for more than four months on freezing in the presence of a sufficient amount of DTT.

Actins↗