Biomedical subjects

K Tokuraku

Publications and source records attributed to K Tokuraku.

7 recordsLinked to original sources

PubMed2001

Phalloidin affects the myosin-dependent sliding velocities of actin filaments in a bound-divalent cation dependent manner.

We examined sliding velocities in vitro of four types of actin filaments, that is, filaments with Ca2+ or Mg2+ bound at the high affinity metal binding site, each with rhodamine phalloidin bound with a high or low stoichiometry. When surfaces coated with a high density of heavy meromyosin (HMM) were used, high stoichiometric concentrations of rhodamine phalloidin reduced sliding velocities of only Ca2+-actin filaments, by 40%. As the HMM density on surfaces was reduced, continuous movement of actin filaments became dependent on the presence of methylcellulose and sliding velocities of all four types became progressively slower. Interestingly, Ca2+-actin filaments with a high stoichiometric concentration of rhodamine phalloidin were the fastest among the four types of filaments on sparse HMM surfaces. In contrast, phalloidin did not affect steady state ATPase activities of HMM in the presence of Ca2+- or Mg2+-actin filaments. We speculate that the reversal of the order of sliding velocities among the four types of actin filaments between high and low densities of HMM relates with different axial elasticity of the actin filaments, so that stiffer filaments move slower on dense HMM surfaces, but faster on sparse surfaces, than elastic ones.

Actin Cytoskeleton↗

PubMed2001

The actin-depolymerizing factor destrin has an actin-stabilizing domain.

Destrin is a 19 kDa actin-depolymerizing protein of the ADF-cofilin family. Destrin was digested with trypsin to a structurally stable 9.2 kDa fragment that contains the actin-binding sequence. The purified 9.2 kDa fragment has an actin filament stabilizing activity, rather than an actin filament depolymerizing activity. The deleted region is probably essential for the actin filament depolymerizing activity of intact destrin. Surprisingly, the 9.2 kDa fragment also has an assembly-promoting activity in the absence of ATP.

Actin Depolymerizing Factors↗

PubMed2000

Purification and characterization of a new, ubiquitously distributed class of microtubule-associated protein with molecular mass 250 kDa.

A heat-stable microtubule-associated protein (MAP) with relative molecular mass 250 000, termed 250-kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250-kDa MAP was able to bind to taxol-stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western-blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250-kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250-kDa MAP colocalized with free tubulin. Under electron microscopy, 250-kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.

Animals↗

PubMed1999

A new model for microtubule-associated protein (MAP)-induced microtubule assembly. The Pro-rich region of MAP4 promotes nucleation of microtubule assembly in vitro.

The microtubule-binding domains of microtubule-associated protein (MAP) 2, tau, and MAP4 are divided into three distinctive regions: the Pro-rich region, the AP sequence region and the tail region (Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Electron microscopic observation showed that the taxol-stabilized microtubules alone and those mixed with the A4T fragment (containing the AP sequence region and the tail region) had a long, wavy appearance, while those mixed with the PA4T fragment (containing the Pro-rich region, the AP sequence region, and the tail region) or the PA4 fragment (containing the Pro-rich region and the AP sequence region) were shorter and straighter. Stoichiometries of the binding between the fragments and the tubulin dimers were approximately between 1 and 2, suggesting that not all of the AP sequences in the AP sequence region bound to tubulin. Binding affinity of the PA4T fragment is only four times higher than that of the A4T fragment, while the microtubule nucleating activity of the PA4T fragment is far greater. Based on these results, we propose that the nucleation of microtubule assembly is promoted by the bridging activity of the Pro-rich region in the MAPs.

Binding Sites↗

PubMed1999

Microtubule-binding property of microtubule-associated protein 2 differs from that of microtubule-associated protein 4 and tau.

The microtubule-binding domains of microtubule-associated protein (MAP) 2, MAP4, and tau are structurally similar [Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855]. To compare the microtubule-binding mechanisms of the three MAPs, we performed a quantitative competition analysis using the three MAPs and the microtubule-binding domain fragment of MAP4 (PA4T fragment). The two-cycled microtubule protein fraction from bovine brain contains MAP1, MAP2, MAP4, and tau. When an excess of the PA4T fragment was added to the microtubule protein fraction, MAP4 and tau were completely released from the microtubules, while MAP1 remained bound. MAP2 was only partially released from the microtubules. The competition between MAP2 and MAP4 was further analyzed using purified MAP2, the PA4T fragment, and tubulin. About half of the MAP2 was still bound to the microtubules, even in the presence of an excess amount of the PA4T fragment. The microtubule-binding mechanisms of MAP2 and MAP4 seem to be different, in spite of their similar primary structures.

Animals↗

PubMed1998

Human natural resistance-associated macrophage protein is a new type of microtubule-associated protein.

Natural resistance-associated macrophage protein 1 (NRAMP1) is a putative membrane protein that dominates natural resistance to infection. An NRAMP1-glutathione S-transferase fusion protein was used to test the ability of the NRAMP1 NH2-terminal domain to bind to taxol-stabilized microtubules. Co-sedimentation analysis showed that the fusion protein binds to microtubules. Although the NH2-terminal domain of the NRAMP1 molecule has structural homology with the Pro-rich region of microtubule-associated protein 4 (MAP4), the presence of the MAP4 microtubule-binding domain fragment had little effect on the binding of the fusion protein to microtubules.

Animals↗

PubMed1997

The 'assembly-promoting sequence region' of microtubule-associated protein 4 failed to promote microtubule assembly.

In order to study the function of the bovine MAP4 microtubule-binding domain (the assembly-promoting (AP) sequence region), a fragment corresponding to the AP sequence region was prepared using an Escherichia coli expression system. When the fragment was mixed with purified tubulin at 37 degrees C, the fragment caused a time- and dose-dependent turbidity increase, and the fragment bound to tubulin. However, the products were cold-stable, and amorphous aggregates were observed by electron microscopy. Using axonemes as the seeds for microtubule assembly, the microtubule-elongating activity of the fragment was examined. A dose-dependent turbidity increase of the sample was observed, and electron microscopic observation revealed that microtubules were dose-dependently elongated from the axonemes. Consequently, the AP sequence region does not nucleate microtubules, but elongates them.

Adrenal Cortex↗