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K Mihashi

Publications and source records attributed to K Mihashi.

At least 37 records · Page 2Linked to original sources
PubMed1990

Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate.

In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.

Adenosine Diphosphate↗
PubMed1989

Subunit flow in F-actin under steady-state conditions. Application of a novel method to determination of the rate of subunit exchange of F-actin at the terminals.

We developed a novel method to determine the subunit exchange rates of F-actin at its terminals under quasi-steady-state conditions by using a powerful fluorescent probe, N-(1-pyrenyl)iodoacetamide. The applicability of the method was checked with regard to both theoretical and experimental aspects. We determined the rates of subunit exchange of F-actin and F-actin-tropomyosin complex under various ionic conditions. We found that: (i) KCl accelerated both on and off rates at each end, and lowered the critical concentration of the P-end while the critical concentration of the B-end was not affected; (ii) binding of tropomyosin drastically reduced the subunit flow in F-actin by suppressing the off rate principally of the P-end. It is therefore believed that tropomyosin exerts an anisotropic constraint on F-actin and regulates its dynamic polarity.

Actins↗
PubMed1989

Ca2+-dependent regulation of the dynamic polarity of F-actin under the influence of tropomyosin and troponin.

A novel method that we have developed in the preceding paper to study the subunit exchange rates of F-actin (N. Suzuki and K. Mihashi, Biophys. Chem. 33 (1989) 177) was applied to regulated F-actin (a complex of F-actin, tropomyosin and troponin). We found that the dynamic polarity of regulated F-actin is modulated in a Ca2+-dependent manner, giving rise to strong suppression of the on/off rates of subunit exchange at the P-end. We interpreted this characteristic suppression as follows. Removal of Ca2+ from troponin C in regulated F-actin produces strong constraints on fluctuations in potential energy of an intermediate conformation of the terminal structure (P-end) which would be formed in the course of association and dissociation of the actin subunit.

Actins↗
PubMed1986

The excimer fluorescence of N-(1-pyrenyl)iodoacetamide labeled to myosin and its subfragment 1.

Myosin and its subfragment 1 were labeled with the fluorescent probe N-(1-pyrenyl)iodoacetamide. Both of the labeled complexes exhibited the excimer band at 480 nm (pH 8.0, 25 degrees C). SH1 and SH2 are labeled with this probe as judged by Ca2+-ATPase of the labeled complex. Excimers arise both from the interaction of PIAAs in the two different heads within a single myosin molecule and also from the interaction of PIAAs in the same head. ATP affects these excimers depending on the concentration of Ca2+.

Adenosine Triphosphate↗
PubMed1984

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range.

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.

Actins↗
PubMed1984

Structure determination of polysaccharides in Aloe saponaria (Hill.) Haw. (Liliaceae).

Neutral polysaccharides that inhibit carrageenin-induced edema in rats were isolated from the nondialysate of the pulp of Aloe saponaria by gel filtration. These were shown to be a linear polymer of a 1,4-linked beta-D-mannopyranose (mol. wt. 15,000) containing 18% acetyl groups (As mannan 1), and a 1,4-linked alpha-D-mannopyranose polymer containing a single branch on the principal chain consisting of D-glucose residues linked at C-2 and C-4 (mol. wt. 66,000), with 10% acetyl groups (As mannan 2). As mannan 1 inhibited carrageenin-induced hind paw edema at 50 mg/kg ip in rats; As mannan 2 was not tested for pharmacological activity. A crude preparation of both As mannans was effective when given intraperitoneally, but was ineffective when given orally.

Aloe↗
PubMed1984

Ca-dependent regulation of the subunit exchange of F-actin under the influence of tropomyosin and troponin.

A quasi-steady state rate of actin subunit incorporation into a complex of F-actin, tropomyosin, and troponin was studied in a solution containing MgCl2 0.5 mM, ATP 0.2 mM, Tris-HCl 5 mM (pH 8.0), and either CaCl2 64 muM or EGTA 644 muM at 33 degrees C by adding a small amount (less than 1 muM) of fluorescence-labeled actin monomer. Incorporation of the labeled actin monomer was remarkably much slower in the presence of EGTA, but the rate recovered instantaneously upon addition of an excess amount of CaCl2. The rate was very much reduced by the presence of cytochalasin D (1 muM) and again showed Ca-dependence.

Actins↗
PubMed1983

Conservation, restoration and rehabilitation of voice in treating patients with carcinoma of the larynx. A 10-year review of 244 patients.

We reviewed clinical records of 244 patients with carcinoma of the larynx treated during the 10 years from 1971 to 1980 in the Department of Otolaryngology, Kurume University Hospital. Of the 244 patients, 111 patients (45.5%) were treated with their voice conserved and 133 underwent total laryngectomy. In the latter, information on the modality of speech communication was obtained from 73 patients. Forty-nine (67%) of these 73 patients was able to speak by some means but the remaining 24 (33%) had no means to speak.

Adult↗
PubMed1983

Internal motion of F-actin in 10(-6)-10(-3) s time range studied by transient absorption anisotropy: detection of torsional motion.

By means of laser flash photolysis, the transient absorption anisotropy (TAA) of the triplet probe, 5-iodoacetamide-Eosin, labeling rabbit skeletal F-actin was measured in the 10(-6)-10(-3) s time range. The TAA curve at 20 degrees C showed a relatively slow decay phase covering several hundred microseconds and a large residual anisotropy (approximately 0.1 at 2 ms). After analysis with Barkley & Zimm's formula, it was concluded that the TAA of Eosin-F-actin can be approximated by the anisotropy decay due to torsional motion of F-actin.

Actins↗
PubMed1982

The binding of myosin subfragment-1 to F-actin in the absence of nucleotide. Evidence for dependence on F-actin concentration.

The binding of myosin subfragment-1 (S-1) to F-actin in the absence of nucleotide was examined by the sedimentation method using 1,5-IAEDANS-labeled S-1. We found that the binding affinity of F-actin to S-1 was dependent on the concentration of F-actin, and the binding was weaker at higher concentrations of F-actin. The apparent association constant determined from a linearly extrapolated Scatchard plot was 6.5 x 10(6) M-1 at 8.1 microns F-actin, and 1.7 x 10(7) M-1 at 2.0 microns F-actin in 120 mM KC1, 2 mM MgCl2, 0.1 mM CaCl2, and 20 mM Tris-acetate (pH 7.6) at 20 degrees C. Furthermore, the Scatchard plot revealed the existence of cooperativity in the binding of S-1 to F-actin. In order to obtain higher precision we developed a new method for the chromatographic determination of free S-1 in acto-S-1 solution. By this method we could determine free S-1 concentrations of the order of 10(-9) M easily and accurately. The above conclusion obtained by the sedimentation method was confirmed by this chromatographic method, and these effects can be well explained by considering the length distribution of F-actin. We propose an allosteric model in which both the length distribution and the polarity of F-actin are taken into consideration.

Actins↗
PubMed1981

Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin.

A fluorescent reagent, N-(1-pyrenyl)iodoacetamide, was conjugated to rabbit skeletal muscle actin at the site of the most reactive sulfhydryl group, and fluorescence characteristics (excitation and emission spectra, quantum yields, lifetimes) of the conjugate were investigated. Associated with polymerization of labelled G-actin, the fluorescence intensity at 407 nm, after excitation at 365 nm, was enhanced by a factor of about 25. It was reduced to about 25% on the binding of heavy meromyosin (or subfragment 1). The results suggest that binding of heavy meromyosin to the protomer of F-actin alters the local structure of the protomer towards a G-actin-like one.

Actins↗