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1.  Identification of α-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle 
BMC Biochemistry  2004;5:18.
The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus oocyte maturation.
We identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus, then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase).
Six of α-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently.
PMCID: PMC544557  PMID: 15603592
2.  Regulated interaction between polypeptide chain elongation factor-1 complex with the 26S proteasome during Xenopus oocyte maturation 
BMC Biochemistry  2003;4:6.
During Xenopus oocyte maturation, the amount of a 48 kDa protein detected in the 26S proteasome fraction (p48) decreased markedly during oocyte maturation to the low levels seen in unfertilized eggs. The results indicate that the interaction of at least one protein with the 26S proteasome changes during oocyte maturation and early development. An alteration in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycle, in the early embryo. In this study, we identified p48.
p48 was purified by conventional column chromatography. The resulting purified fraction contained two other proteins with molecular masses of 30 (p30) and 37 (p37) kDa. cDNAs encode elongation factor-1γ and δ were obtained by an immuno-screening method using polyclonal antibodies against purified p48 complex, which recognized p48 and p37. N-terminal amino acid sequence analysis of p30 revealed that it was identical to EF-1β. To identify the p48 complex bound to the 26S proteasome as EF-1βγδ, antibodies were raised against the components of purified p48 complex. Recombinant EF-1 β,γ and δ were expressed in Escherichia coli, and an antibody was raised against purified recombinant EF-1γ. Cross-reactivity of the antibodies toward the p48 complex and recombinant proteins showed it to be specific for each component. These results indicate that the p48 complex bound to the 26S proteasome is the EF-1 complex. MPF phosphorylated EF-1γ was shown to bind to the 26S proteasome. When EF-1γ is phosphorylated by MPF, the association is stabilized.
p48 bound to the 26S proteasome is identified as the EF-1γ. EF-1 complex is associated with the 26S proteasome in Xenopus oocytes and the interaction is stabilized by MPF-mediated phosphorylation.
PMCID: PMC179889  PMID: 12864926

Results 1-2 (2)