Reversible changes in the ATPase activity and in the regulatory light chain content upon heat (30 degrees C)-treatment of "Akazara" striated adductor myosin

Myosin from striated adductor muscle of "Akazara" scallop was incubated at 30 degrees C for 5 min in a medium containing 2 mM MgCl2 and various concentrations of Ca2+ ions. It was observed that the 30 degrees C-treatment resulted in a decrease in the Ca2+-sensitivity of myosin-ATPase as well as in the release of the regulatory light chain (EDTA-LC) of myosin. The 30 degrees C-treated myosin was then subjected to a cooling treatment, being kept for 18 h at 0 degrees C. It was found that EDTA-LC recombined with myosin and that Ca2+-sensitivity of myosin-ATPase was restored. It was also found that Ca2+ alone was about 70 times more effective than Mg2+ alone in preventing the heat-induced release of EDTA-LC from occurring and also in recombination of EDTA-LC with the heat-treated myosin.     

A binary complex of troponin-I and troponin-T from Akazara scallop striated adductor muscle.

A binary complex consisting of Mr 19,000 and Mr 40,000 components was co-purified with troponin from a crude troponin fraction of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle. This complex is incapable of conferring Ca(2+)-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, rather strongly inhibiting it, but became capable on further complexing with Akazara scallop troponin-C. To examine the effects of the Mr 19,000 and Mr 40,000 components on the ATPase activity, they were separated from each other by CM-Toyopearl column chromatography. The Mr 19,000 component strongly inhibited the Mg-ATPase activity of actomyosin-tropomyosin and the inhibition was reversed by further addition of the Akazara scallop troponin-C. On the other hand, the Mr 40,000 component slightly increased it. On hybridization with the Akazara scallop troponin subunits, the Mr 19,000 and Mr 40,000 components were shown to be able to substitute for troponin-I and troponin-T, respectively. The amino acid compositions of the Mr 40,000 component and troponin-T were almost identical, and those of the Mr 19,000 component and Mr 17,000 C-terminal fragment of the troponin-I resembled each other fairly well. From these results, it may be concluded that the Mr 19,000-40,000 binary complex is the troponin-I-troponin-T complex.     

Akazara scallop troponin C: Ca(2+)-induced conformational change and interaction with rabbit troponin subunits.

The number of specific Ca2+ bound to Akazara scallop troponin C was estimated to be 0.7 with an apparent binding constant of 5 x 10(5) M-1 (T. Ojima and K. Nishita, 1986, J. Biol. Chem. 261, 16749-16754). In the present paper, we report on the Ca(2+)-induced conformational changes in the troponin C and the interaction of the troponin C with rabbit troponin subunits. The Ca2+ binding to the troponin C caused a marked change in difference uv absorption spectra and a retardation of elution on Sephacryl S-200 gel filtration. However, its circular dichroism spectrum was hardly changed by the Ca2+ binding. These results suggest that the Ca2+ binding to the troponin C induced changes predominantly in tertiary structure rather than in secondary structure. Akazara scallop troponin C was shown to be able to bind to rabbit troponin I-Cellulofine affinity column, but the affinity was not greatly increased by Ca2+ unlike the case of rabbit troponin C. On hybridizing with rabbit troponin T and I, Akazara scallop troponin C was shown to be incapable of substituting rabbit troponin C; i.e., the hybrid troponin strongly inhibited the Mg-ATPase activity of rabbit actomyosin-tropomyosin irrespective of the presence or absence of Ca2+, thus recovering no Ca2+ sensitivity.     

Genetic diversity of MHC class II <Emphasis Type="Italic">DRB</Emphasis> alleles in the continental and Japanese populations of the sable <Emphasis Type="Italic">Martes zibellina</Emphasis> (Mustelidae,Carnivora, Mammalia)

The sable (Martes zibellina) is a medium-sized mustelid inhabiting forest environments in Siberia, northern China, the Korean Peninsula, and Hokkaido Island, Japan. To further understand the molecular evolution of the major histocompatibility complex (MHC), we sequenced part of exon 2 in MHC class II DRB genes, including codons encoding the antigen binding site, from 33 individuals from continental Eurasia and Japan. We identified 16 MHC class II DRB alleles (Mazi-DRBs), some of which were geographically restricted and others broadly distributed, and eight putative pseudogenes. A single-breakpoint recombination analysis detected a recombination site in the middle of exon 2. A mixed effects model of evolution analysis identified five amino acid sites presumably under positive selection. These sites were all located in the region 3′ to the recombination site, suggesting that positive selection and recombination could be committed to the diversity of the M. zibellina DRB gene. In a Bayesian phylogenetic tree, all Mazi-DRBs and the presumed pseudogenes grouped within a Mustelidae clade. The Mazi-DRBs showed trans-species polymorphism, with some alleles most closely related to alleles from other mustelid species. This result suggests that the sable DRBs have evolved under long-lasting balancing selection.     

Hybrid with rabbit skeletal DTNB-light chains of heavy meromyosin, myosin, and glycerinated fibers from Akazara scallop adductor

It was shown in our previous report (Ojima et al. (1983) J. Biochem. 94, 307-310) that hybridization of Akazara scallop "desensitized" myosin with rabbit skeletal DTNB-light chains led to inhibition of the Mg-ATPase activity of acto-desensitized myosin but to enhancement of its superprecipitation activity. The following are now found: Development of tension in desensitized glycerinated fibers of Akazara adductor is significantly improved when DTNB-light chains are added to the fiber bath. The actin-affinity of desensitized heavy meromyosin in the presence of ATP but in the absence of Ca2+ is decreased by hybridization with chicken gizzard 20K dalton-light chains but significantly increased by that with DTNB-light chains. It is therefore suggested that the increase in actin-binding may account for the enhancing effect of DTNB-light chains on the superprecipitation and on the tension development.     

Myosin from striated adductor muscle of Chlamys nipponensis akazara.

Myosin was isolated from striated adductor muscle of Akazara shell-fish, and purified on DEAE-Sephadex A50. The sedimentation constant (s 20,2 0 W) and the intrinsic viscosity, [eta] of Akazara myosin thus purified were estimated to be 6.6 S and 2.10 dl/g, respectively. In many respects, Akazara myosin was similar to scallop myosin. (1) Only one size of light-chain component (17,000 daltons) was detectable in SDS-gel electrophoresis of Akazara myosin, but two types of light-chain component were seen in urea-gel electrophoresis; these were equivalent to EDTA-light chain and SH-light chain of scallop myosin. The molar ratio of heavy chain (206,000 daltons), EDTA-light chain, and SH-light chain in Akazara myosin was estimated, from the staining densities of gel-electrophoretic bands, to be approximately 1 : 1 : 1. (2) EDTA-washing procedure removed EDTA-light chain only, causing desensitization of Akazara myosin. EDTA-light chain isolated from Akazara myofibrils was able to resensitize EDTA-washed Akazara myosin. Akazara myosin, however, was found to be different from scallop myosin in two important properties: (1) complete removal of EDTA-light chains was required to achieve a complete loss of calcium sensitivity, and full resensitization was attained on recombination of EDTA-light chains with desensitized myosin prepared essentially free from EDTA-light chains. (2) EDTA-light chains isolated from Akazara myofibrils show a calcium-induced UV absorption difference spectrum.     

Comparative immunochemical studies of carbonic anhydrase III in horses and other mammalian species

1. Carbonic anhydrase III (CA-III) from different mammalian species (horse, cow, dog, cat, rat and rabbit) has been analyzed by the immunodiffusion technique with anti-equine CA-III serum. 2. Immunodiffusion demonstrated the absence of cross-reactivity between isozyme CA-I, CA-II, and CA-III. 3. Cross-reactions were observed between the CA-III from all the species examined except the rabbit. 4. Molecular weights and isoelectric points of CA-III from different species were determined by Western blotting.     

Filopodia formation mediated by receptor tyrosine kinase Ror2 is required for Wnt5a-induced cell migration

The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt-JNK pathway and inhibiting the beta-catenin-TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.