Showing papers by "Toshinobu Tokumoto published in 2000"
TL;DR: The results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interactions between regulators.
Abstract: To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. At least two protein bands, at 30 kDa (detected by GC3beta antibody) and 62 kDa (detected by 1-4D5 antibody), differed between 26S proteasomes. A monoclonal antibody, GC3beta cross-reacted with two bands in the 26S proteasome from immature oocytes, however, the upper band was absent in the 26S proteasome from mature oocytes. The 62-kDa protein band detected by 1-4D5 antibody was not detected in the immature oocyte 26S proteasome; however, a band was detected in mature oocyte 26S proteasome. The cDNAs encoding these proteins were isolated by an immunoscreening method using the monoclonal antibodies. The 30-kDa protein was an alpha4 subunit, which is one of the alpha-subunit group of the 20S proteasome, and the 62-kDa protein was a homologue of CCTepsilon, one of the components of eukaryotic molecular chaperones. Phosphatase treatment of the 26S proteasome revealed that a part of the alpha4 subunit of goldfish 20S proteasome, alpha4_ca, is phosphorylated in G2-phase and dephosphorylated in M-phase. A binding assay using a recombinant goldfish CCTepsilon revealed that unmodified CCTepsilon interacts with the 26S proteasome. Fertilization triggers a transition from meiotic metaphase to mitotic interphase. During fertilization, a GC3beta cross-reacting upper band reappeared. The 62-kDa band dissociated from the 26S proteasome. As a result, the 26S proteasome changed to an immature type from a mature type during fertilization. These results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interactions between regulators.
32 citations
TL;DR: The cloning, sequencing and expression analysis of goldfish, Carassius auratus, E1 from goldfish ovary shows that Recombinant goldfish E1 could transfer ubiquitin to cyclin-selective ubiquit in-conjugating enzyme.
9 citations
TL;DR: Results suggest that the β6 subunit of the goldfish 20S proteasome, β6_ca, is responsible for anchoring proteasomes in the nucleus, which is highly homologous to those of β 6 subunits of other vertebrates.
7 citations
TL;DR: It is concluded that the metallopeptidase purified from Xenopus oocytes is a member of the TOP family, and one major species of Xenopus-TOP mRNA of 3.0 kb was expressed relatively strongly from early stage (III) of xenopus oogenesis, its level decreasing in later stages (V and VI).
Abstract: We have isolated a cDNA clone for a zinc-requiring metallopeptidase in Xenopus oocytes from Xenopus ovary library using oligonucleotides synthesized on the basis of the partial amino acid sequence. The full-length 2,055 bp cDNA encodes a protein of 685 amino acid residues with a predicted molecular mass of 78,136 Da. The deduced amino acid sequence of this protein exhibits high similarity to that of human (74.1%), pig (75.3%) and rat (74.1%) thimet oligopeptidase (TOP) [EC 3.4.24.15]. Expression of the cDNA in bacterial cells resulted in the production of an active metalloenzyme. Thus, we concluded that the metallopeptidase purified from Xenopus oocytes is a member of the TOP family. In Northern blot analyses, one major species of Xenopus-TOP (X-TOP) mRNA of 3.0 kb was expressed relatively strongly from early stage (III) of Xenopus oogenesis, its level decreasing in later stages (V and VI). This result suggests that the expression of X-TOP mRNA is regulated during Xenopus oogenesis.
2 citations
1 citations
TL;DR: The Xenopus homologue to the ubiquitin-activating enzyme (El) from a Xenopus ovary is described and the deduced amino acid sequence is highly homologous to El from other species.
Abstract: The Xenopus homologue to the ubiquitin-activating enzyme (El) from a Xenopus ovary is described. The deduced amino acid sequence is highly homologous to El from other species.