Heterogeneity and tissue specificity of tropomyosin isoforms from four species of bivalves.

Heterogeneity and tissue specificity of tropomyosin isoforms obtained from four species of bivalves (Scapharca broughtonii (ark shell), Mytilus galloprovincialis (mussel), Atrina pectinata (surf clam) and Crassostrea gigas (Pacific oyster)), were examined. Tropomyosins were extracted from translucent and opaque portions of posterior adductor muscle, respectively, and cardiac muscle of each bivalve. There were two tropomyosin isoforms in the ark shell, the surf clam and the Pacific oyster. They were designated as TMa and TMb. In the ark shell, TMa was the common isoform and TMb was specific for the opaque portion of the adductor muscle. In the surf clam, TMb was the common isoform present in all tissues. TMa was found only in the translucent portion of muscle. In the Pacific oyster, TMb was the major component in both portions of adductor muscle and TMa was the major component in cardiac muscle, although both tropomyosins were included in all tissues. The mussel had only one tropomyosin.

Identification of 36 kDa phosphoprotein in fibrous sheath of hamster spermatozoa.

In our previous studies (Fujinoki et al., 2001, 2003), we reported that two types of 36 kDa proteins, designated 36K-A protein and 36K-B protein, obtained from hamster sperm flagella, are associated with motility activation and phosphorylated in a cAMP-dependent manner at serine residues. In the present experiments, we focused on the hamster (Mesocricetus auratus) 36K-A protein, which was analyzed by peptide mass finger printing and amino acid sequencing. The results suggest that 36K-A protein is a pyruvate dehydrogenase E1 component beta subunit lacking the N-terminal 30 amino acids. Moreover, our results suggest that 36 K-A protein is localized in the fibrous sheath of the principal piece of hamster spermatazoa.

Serine/threonine phosphorylation associated with hamster sperm hyperactivation.

Background and Aims: Mammalian sperm activation and hyperactivation is regulated by protein phosphorylation. Although tyrosine phosphorylation is considered very important, several studies have investigated whether serine and threonine phosphorylation are also associated with sperm activation and hyperactivation, and that was also the aim of the present study. Methods: Protein 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. Results: Four 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. Conclusions: During 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. (Reprod Med Biol 2004; 3: 223-230).

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

Background and Aims: Sperm motility is regulated by protein phosphorylation. 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. Methods: The 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. Results: The 66 kDa protein was tubulin ß chain. Conclusion: The 66 kDa protein is one of the tubulin ß chain isoforms and phosphorylated in relation to the motility initiation. (Reprod Med Biol 2004; 3: 133-139).

Identification of 36-kDa flagellar phosphoproteins associated with hamster sperm motility.

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that two types of 36-kDa protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with the regulation of motility activation. In the present experiments, it was suggested that these two types of 36-kDa protein were phosphorylated in a cAMP-dependent manner associated with motility activation of hamster spermatozoa. Because the 36K-B protein was the most intensely phosphorylated in a cAMP-dependent manner, attempts were made to further characterize it. The 36K-B protein was assumed to be localized in the middle piece. The localization of the 36K-B protein was the same as that of the 36-kDa protein reported in our previous paper [Y. Si et al. (1999) Mol. Reprod. Dev. 52, 328-334]. In order to identify the 36K-B protein, it was analyzed by peptide mass finger printing and amino acid sequencing. The results suggested that the 36K-B protein was a pyruvate dehydrogenase E1 component beta subunit and a component of the mitochondrial sheath of the middle piece.

Tropomyosin isoforms present in the sea anemone, Anthopleura japonica (Anthozoa, Cnidaria).

Five isoforms of tropomyosin, designated as TMa, TMb, TMc, TMd, and TMe, were detected in the sea anemone, Anthopleura japonica. The apparent molecular weights of these isoforms were estimated to be approximately 30 kD to 37.5 kD, and their pI values were approximately 4.55 (TMa and TMb) and 4.65 (TMc, TMd, and TMe). Although sea anemone tropomyosin isoforms have the ability to bind to rabbit skeletal muscle actin, they preferably bind to actin at higher concentrations of Mg(2+) (10-20 mM) and slightly lower pH (6.2-7.2) than those used in conventional conditions. Antigenic properties of sea anemone tropomyosin seemed to be considerably specific to each isoform. Distribution of tropomyosin isoforms in the sea anemone body was somewhat portion-specific. TMa, TMb, and TMe were detected similarly in the extracts from tentacle, oral disc, column, mouth, and pedal disc. Although TMc and TMd were detected abundantly in the tentacle extract and moderately in the column and mouth extracts, these components were not contained in the pedal disc extract and detected only faintly in the oral disc extract.

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.