Tadakazu Maeda

Flexural rigidity of singlet microtubules estimated from statistical analysis of their contour lengths and end-to-end distances.

Microtubules in solutions, observed under a dark-field microscope, show incessant Brownian movement such as translational, rotational and flexing motion. A large number of microtubules, spontaneously stuck to the under surface of a coverslip, were photographed and the contour lengths and end-to-end distances of their images were measured. From the statistical analysis of the contour lengths and end-to-end distances, a value for the parameter lambda representing the flexibility of singlet microtubules was estimated to be lambda = (6.8 +/- 0.8) . 10(-3) micrometers-1. From the value of lambda, the elastic modulus for bending, epsilon, and Youngs modulus, Y, of singlet microtubules were computed to be epsilon = approximately 10(-16) and Y = approximately 10(9) dyne . cm-2, respectively. The microscopic elastic constant, k, of bonding between two tubulin monomers neighboring along the singlet microtubule was computed to be k = congruent to 10(2) dyne . cm-1. A singlet microtubule is an order of magnitude as strong against bending and as weak against stretching as an F-actin filament.

Quasielastic Light Scattering under Optical Microscope

A system for quasielastic light scattering under an optical microscope is proposed. This system makes it possible to obtain some information about dynamic properties of macromolecules in a limited part (100×100 μ2) of a living cell.

F-actin-heavy meromyosin complex studied by optical homodyne and heterodyne methods.

Abstract Quasi-elastic scattering of laser light is now becoming a powerful tool for the study of dynamic properties of both biological and non-biological macromolecules. In solutions of biological macromolecules, aggregation-disaggregation of molecules under study usually depends on solvent conditions. Therefore, special attention must be paid in light-beating spectroscopy. By use of both homodyne and heterodyne methods, the spectral densities of solutions of F-actin and of a complex of F-actin and heavy meromyosin were measured. The half-width of the heterodyne spectrum was much wider than that of the homodyne spectrum. This was probably due to polydispersity of F-actin. The results showed that laser light scattering gave information about the dynamics of free filaments, although low frequency rheometry has suggested the rubber-like elasticity of the solution of F-actin and heavy meromyosin. The interaction between F-actin and heavy meromyosin was studied in the presence of pyrophosphate (PPi). At PPi concentrations between 10 and 100 μM, the spontaneous bending motion of F-actin occurred as if heavy meromyosin were not present, although turbidity of the solution indicated that heavy meromyosin was, in fact, bound to F-actin. This meant that there were two types of binding state of the F-actin-heavy meromyosin complex. A plausible model is that, in the absence of PPi, the two heads of one heavy meromyosin molecule simultaneously interact with two neighbouring monomers in F-actin, whereas a single head binding occurs in the presence of PPi. Above 100 μM PPi, heavy meromyosin seemed to dissociate from F-actin. However, it was not clear whether or not this was due to a direct effect of PPi on heavy meromyosin alone, because the dynamic properties of F-actin also changed. Assuming that actin filaments are cross-linked by myosin, a simple model is proposed to explain qualitatively the rubber-like elasticity of the solution of an F-actin-heavy meromyosin complex.

Quasi-Elastic Light Scattering from Muscle F-Actin

Photon counting autocorrelation functions of light scattered from solutions of muscle F-actin were measured by use of a 128 channel digital correlator. Measured correlation functions showed highly non exponential decay, which came mainly from the existence of very slow decay components, much slower than 1 s -1 . These slow decay components were observed not to produce the occupation number fluctuation. The average decay rate, (bar{varGamma}), dispersion, (mu_{2}/bar{varGamma}^{2}), and skewness, (mu_{3}/bar{varGamma}^{3}), obtained by the cumulant expansion method were strongly dependent on the channel width of the correlator, and on the position in, and preparation of the solution. These results were discussed in relation to the existence of aggregates and exponential length distribution of actin filaments. It was shown that, in highly polydisperse samples, correlation functions should be measured at various channel widths, and that apparent non reproducibility of the results could be avoid...

Light-beating study of the effect of β-actinin on the interaction between F-actin and heavy meromyosin

Abstract The effects of β-actinin on the interaction between F-actin and heavy meromyosin were studied by quasi-elastic scattering of laser light. The main findings are as follows: 1. 1. Molecular flexing of F-actin in solution has been known to be promoted by bound heavy meromyosin. However, this effect of heavy meromyosin disappeared on the addition of β-actinin (10% by wt to F-actin) in the absence of ATP. 2. 2. When β-actinin was added in the presence of suitable amounts of ATP, the above mentioned effect of β-actinin could not be observed after the ATP was hydrolyzed. However, the effect of β-actinin was observable after application of sheer force to the solution. Subsequent addition of ATP did not eliminate the effect of β-actinin again. 3. 3. Even when β-actinin was added in the presence of ATP, if the concentration of ATP was higher than about 4 mM, the effect of β-actinin was observed. Experiments suggested that the system was not sensitive to the final concentration of ADP, but to the initial concentration of Mg-ATP.

Room Temperature Phosphorescence Study of Refolding of Disulfide Reduced RNase T1.

Refolding kinetics of disulfide reduced ribonuclease T 1 (RNase T 1 ) was studied by the recovery of room temperature phosphorescence emitted from a tryptophan residue (Trp 59) of the protein. When unfolded in a deoxygenated aqueous solution, the enzyme does not emit phosphorescence at room temperature, but as soon as the protein was transferred into an anoxic refolding buffer solution, it began emitting phosphorescence within the mixing dead time (∼30 s) of our apparatus. The initial quick recovery of RNase T 1 phosphorescence was followed by a gradual increase in the phosphorescence lifetime lasting over several tens of minutes and then slight decrease to a final value comparable to that of native RNase T 1 . The initial quick recovery of phosphorescence suggests that the tryptophan residue, being exposed to surrounding water molecules when the protein is unfolded, is quickly isolated from the external water when the enzyme starts refolding, and the gradual change in the phosphorescence lifetime implies...

Conformation of antithrombin iii with defective biological functions derived from a thrombophilic patient

Antithrombin III (AT III) with defective biological functions and altered antigenicity has been found in a thrombophilic patient. Circular dichroism (CD) spectra of antithrombin III purified from the patients plasma were measured in near and far-ultraviolet (UV) wavelength regions. The far-UV CD spectrum of the patients AT III was similar at room temperature to that of normal AT III derived from healthy adult males. Mean residue ellipticity at 221 nm of the patients AT III, however, decreased its magnitude by 6% gradually as temperature increased up to 74 degrees C, whereas that of the normal AT III reduced its magnitude sharply at around 63-64 degrees C and by 18%. The near-UV CD spectrum of patients AT III was different from that of normal AT III. These results indicate that the secondary structure of patients AT III is similar to that of normal AT III at room temperature, while local conformation of aromatic amino acid residues in the abnormal protein is different from that in the normal one and that the secondary structure of the patients AT III is heat-stable, while that of the normal one is heat-labile.

Dynamic light-scattering study of monodisperse collagen in the less-dilute regime

Physicochemical properties of monodisperse type I collagen were studied using the dynamic light scattering and analytical ultra-centrifugation techniques. The measured osmotic second virial coefficient A2 is much smaller than the excluded volume limit calculated from the length L, diameter b and molecular weight M of collagen. The strength kD of the concentration dependence of the mutual diffusivity *DG of collagen is negative in the sign and large in the magnitude. The relationship kD equals 2A2M - v - k(zetz ) does not hold for collagen, while it holds for fd virus and poly((gamma) - benzyl L-glutamate), where v is the partial specific volume, and k(zetz ) the strength of the concentration dependence of the friction coefficient (zetz) . We conclude from these three lines of evidence that water is a poor solvent of collagen and that there must be very weak attractive interactions among collagen filaments which can easily be destroyed by external perturbations.

Dynamical structure factor of a solution of charged rod-like polymers in the isotropic phase

A theory of the static and dynamical structure factors of a charged rod suspension was developed. The theory treats the isotropic and depolarized scattering in a unified way and reduces to our previous theory for uncharged rods in the limit of zero polymer charge or zero Debye length. The evaluation of the structure factors and their initial decay rates is quite easy and straightforward. The following are the major conclusions. (i) It is well to approximate a charged rod with an uncharged one having an effective diameter beff the value of which is smaller for the depolarized scattering than for the polarized one. (ii) For strongly charged rods our theory is valid when the solvent ionic strength is higher than 1 mM. (iii) The theory does not give the correlation function a slow tail which has been observed experimentally. 1.

Dynamic Light Scattering Study of Muscle F-Actin in Solution

G-actin is globular in shape (Fig. 1a). Its molecular weight is about 42k daltons. G-actin polymerizes into F-actin under physiological salt concentrations (Fig. 1b). Based on observations by electron microscopy, a “pearl-and-necklace” model is proposed for the ultrastructure of F-actin. F-actin is a two-stranded helical polymer. The half pitch of the helix is 35 nm and within this length, there are 13 G-actins. The total length of F-actin varies according to polymerization conditions and, roughly speaking, is longer than 1 pm. As might be supposed from its structure, F-actin is rather stiff. Electron micrographs show the images of gradually curved F-actin. Tropomyosin is a rodlike protein (Fig. lc). When tropomyosin molecules are added to the solution of F-actin, they bind to F-actin and settle in the grooves of F-actin helix forming tropomyosin strands (Fig. ld). Myosin has two heads called subfragment-1 (S-1) and binds to F-actin in the absence of ATP. Partial digestion by some kind of proteases produces heavy meromyosin (HMM) and also S-1 (Fig. le).