Tadashi Ishimoda-Takagi

Evidence for the involvement of muscle tropomyosin in the contractile elements of the coelom-esophagus complex in sea urchin embryos

The sea urchin morphogenesis, especially formation of the coelom-esophagus complex, was observed correlating the distribution of tropomyosin-specific immunofluorescence. Coelomic cells arranged at both sides of the esophagus extended their pseudopods toward the esophagus to form the contractile bands, which surrounded the esophagus and brought about the contraction of the esophagus. The earliest stage at which the tropomyosin-specific immunofluorescence was recognized coincided with the appearance of the coelomic pseudopods. The tropomyosin-specific immunofluorescence located at the contractile bands and the cell bodies from which they derived, when the ectoderm-disrupted embryos were used to investigate the detailed distribution of tropomyosin. The tropomyosin-specific immunofluorescence remained in the same regions when the embryos were stained with the antiserum absorbed with egg tropomyosin, which detected only muscle tropomyosin. From these observations, the coelomic pseudopod-forming cells were conclusively shown to be muscle cells.

Molecular heterogeneity and tissue specificity of tropomyosin obtained from various bivalves

Abstract 1. 1. Molecular heterogeneity and tissue specificity of tropomyosin from various bivalves were investigated by electrophoresis and limited proteolysis with trypsin or Staphylococcus aureus V8 protease. 2. 2. In scallops, a single striated muscle-specific tropomyosin isoform was detected in adductor-striated muscle, two tropomyosin isoforms in adductor-smooth muscle, one specific to smooth muscle and the other identical with the cardiac muscle isoform and a single tropomyosin isoform in cardiac muscle. 3. 3. In eulamellibranchs, the tropomyosin isoform which existed through translucent and opaque portions of the adductor muscle and cardiac muscle was detected in addition to the opaque portion-specific tropomyosin isoform.

Polymorphism and tissue specificity of scallop tropomyosin

Abstract 1. 1. Molecular polymorphism of tropomyosin from various muscle sources of the scallop, Patinopecten yessoensis, was investigated by electrophoretic and immunochemical methods. 2. 2. Treatment of the muscle sources with trichloroacetic acid (TCA) prior to tropomyosin preparation was found useful to prevent proteolytic degradation of this protein. 3. 3. Electrophoretic and immunochemical analysis revealed that at least six kinds of tropomyosin isoforms may exist in scallop muscle tissues. 4. 4. The tropomyosin isoforms showed tissue-specific distribution in amounts and molecular species among the various muscle sources.

Distribution of tropomyosin isoforms in spiny lobster muscles

Heterogeneity and tissue specificity of tropomyosin isoforms included in various muscles of the spiny lobster, Panulirus japonicus, were investigated using two-dimensional urea-shift gel electrophoresis, two-dimensional gel electrophoresis with first dimension isoelectric focusing and second dimension sodium dodecyl sulfate gels, and immunoblotting. Four kinds of tropomyosin isoforms were recognized as major components in spiny lobster muscles, and distribution of these isoforms was considerably tissue-specific. Three kinds of tropomyosin isoforms designated as TMa, TMb, and TMc in the present study were detected in abdominal and cephalothoracic muscles. TMa was detected predominantly in fast muscles, TMb predominantly in slow muscles and it was the only isoform in leg muscles, and TMc was found preferentially in the dorsal-side fast muscles. Six distribution patterns of tropomyosin isoforms, two fast-type, two slow-type, thoracic-type and leg-type, were detected in the skeletal muscle tissues. A unique tropomyosin constituent designated as TMh was observed as the main component in the heart muscle. The antisera against TMa and TMb showed that these isoforms had the epitopes specific to the respective isoforms as well as those common to both isoforms. The antisera specific to TMa and TMb were prepared by absorption of the antibodies common to both isoforms from the respective antisera. From immunoblot analysis of TMc and TMh with the absorbed antisera, it was shown that these isoforms had rather TMa-type antigenicities. J. Exp. Zool. 277:87–98, 1997.

Serine/threonine phosphorylation associated with hamster sperm hyperactivation

Background and AimsMammalian sperm activation and hyperactivation is regulated by protein phosphorylation. Although tyrosine phosphoiylation is considered very important, several studies have investigated whether serine and threonine phosphoiylation are also associated with sperm activation and hyperactivation, and that was also the aim of the present study.MethodsProtein phosphorylation of hamster spermatozoa was detected by Western blotting using antiphospho-amino acid monoclonal antibodies after tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences were analyzed using a peptide sequencer.ResultsFour proteins were phosphorylated at serine residues during hyperactivation via activation and their approximate molecular weights were 90, 38, 32 and 10 kDa, respectively. Five proteins were phosphorylated or dephosphorylated at threonine residues and their approximate molecular weights were 90, 70, 65, 35 and 10 kDa, respectively. The 10-kDa protein corresponded to a previously reported 10-kDa tyrosine phosphoprotein. N-terminal sequences of the 10-kDa protein were similar to carcinustatin, which is a neuropeptide.ConclusionsDuring hyperactivation, four serine phosphorylation and five threonine phospho- or dephosphorylations occurred, which suggested that the 10-kDa protein was phosphorylated at tyrosine residues when spermatozoa were activated and then dual-phosphorylated at the serine and threonine residues during hyperactivation.

Sea urchin egg tropomyosin isoforms with muscle-type and nonmuscle-type antigenicities

Egg tropomyosins were prepared from four sea urchin species, Stronglyocentrotus intermedius, Anthocidaris crassispina, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, and their molecular heterogeneity was investigated by electrophoresis and immunoblotting. The molecular heterogeneity of egg tropomyosins was species-specific, and two to four kinds of tropomyosin isoforms were detected, the apparent molecular weights of which were 29,000-32,000. The egg tropomyosin isoforms could be classified into two groups with muscle- and nonmuscle-type antigenicities in each species. No obvious difference in their cytological localization was observed immunocytochemically in S. intermedius and H. pulcherrimus.

Identification of 66 kDa phosphoprotein associated with motility initiation of hamster spermatozoa

Background and AimsSperm motility is regulated by protein phospborylation. The 66 kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues associated with the motility initiation. In order to understand the regulatory mechanism of sperm motility, the 66 kDa protein was identified in the present study.MethodsThe 66 kDa protein was purified by 2-D gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry liquid chromatography-tandem mass spectrometry and peptide sequencer.ResultsThe 66 kDa protein was tubulin β chain.ConclusionThe 66 kDa protein is one of the tubulin β chain isoforms and phosphorylated in relation to the motility initiation.

Characterization of two distinct isoforms of tropomyosin present in the eggs of the sea urchin, Strongylocentrotus intermedius

Abstract 1. 1. Two distinct isoforms of tropomyosin, 32K and 30K components, were detected in a tropomyosin fraction prepared from eggs of the sea urchin. Strongylocentrotus intermedius, and some biochemical properties of these isoforms were investigated. 2. 2. The 32K component reacted strongly with the antisera raised to sea urchin muscle tropomyosin, whereas the 30K component showed only a very weak cross-reactivity with these antisera. 3. 3. The 32K component seemed to have a slightly higher actin-binding ability than the 30K component.

Comparative study of horseshoe crab tropomyosin

Abstract 1. 1. Immunological and biochemical properties of skeletal muscle tropomyosin were investigated to elucidate phylogenetic relationships among four extant species of horseshoe crabs, Tachypleus tridentatus, T. gigas, Carcinoscorpius rotundicauda , and Limulus polyphemus . 2. 2. Immunodiffusion tests using an antiserum against tropomyosin from T. tridentatus showed no difference of antigenicities among the four species. Peptide patterns of enzymatically or chemically cleaved tropomyosin also represented no significant difference. 3. 3. However, electrophoretic mobilities of the proteins were found to be significantly different between L. polyphemus and three other species when long distance electrophoresis was conducted. 4. 4. The mobility difference revealed a distinctive property of L. polyphemus tropomyosin and confirmed that the species is phylogenetically differentiated far from the other species.

Changes of tropomyosin isoforms during development of cross‐fertilized sea urchin embryos

Changes of tropomyosin isoforms during development of the sea urchin, Hemicentrotus pulcherrimus, were investigated using two‐dimensional urea‐shift gel electrophoresis. Tropomyosin isoforms included in the embryos were gradually increased after 2 cell stage and retained at a constant level after gastrula stage. To detect the tropomyosin isoforms derived from zygotic genomes, embryos cross‐fertilized between H. pulcherrimus and Pseudocentrotus depressus gametes were prepared. Since tropomyosin isoforms from H. pulcherrimus eggs and from P. depressus eggs could be distinguished from each other on a two‐dimensional electrophoretic gel, the paternal isoforms of tropomyosin in the cross‐fertilized embryos, which were not included endogenously in the egg, could be regarded as products derived from zygotic genomes. The paternal isoforms of tropomyosin were detected first at around the gastrula stage in embryos cross‐fertilized between H. pulcherrimus sperm and P. depressus eggs and also in the reverse combination of the gamete species. Muscle tropomyosins derived from H. pulcherrimus and P. depressus genomes were similarly detected in cross‐fertilized embryos at the pluteic stage when the muscle tropomyosin appeared in sea urchin embryos.