Disappearance Of M r 25,000 Protein, a New Protein Kinase Substrate, in Parallel with a kind of Serpin During Embryogenesis

Abstract

M(r) 25,000 protein (pp25) is a protein kinase substrate detected recently in Xenopus laevis oocytes [Hashimoto, E. et al. (1995) J. Biochem. 118, 453-460], but the physiological role of this protein remains to be determined. In order to elucidate some characteristics of pp25, a polyclonal antibody was raised against it and the distribution and quantitative changes of this protein were examined using various tissues and biological systems. In Western blot analysis, pp25 was detected only in Xenopus oocytes and not in other frog tissues when the heat-stable cytosolic fraction from each tissue was examined. Although the amount of pp25 apparently did not change during oocyte maturation induced by progesterone, pp25 disappeared in embryos around Nieuwkoop/Faber stages 45-48 in parallel with the change of pNiXa (a kind of serpin). These results suggest that pp25 plays some specific role(s) in Xenopus oocytes and that the level of pp25 changes dramatically during embryonic development.

Mr 25 000 protein, a substrate for protein serine/threonine kinases, is identified as a part of Xenopus laevis vitellogenin B1

“…However, it is not known whether they function only as nutrient sources. In a previous report (Funami et al 2002), we showed that the level of pp25 did not change markedly until stage 40 of early embryogenesis, and that pp25 disappeared completely after stage 48. If pp25 exists in the cytosol only as a nutrient source in a non-crystallized form, it seems somewhat surprising that pp25 disappeared suddenly during embryonic development.…”

“…In parallel with these experiments, we also examined the expression pattern of putative vitellogenin B1 protein in female tissues using pp25-specific antibody. In a previous study, we detected pp25 in the ovary but not in other tissues (Funami et al 2002). However, a larger protein with Mr of approximately 220 000, probably corresponding to vitellogenin B1, was detected in all of the organs examined (Fig.…”

“…ovary (Funami et al 2002). To investigate whether the predicted sequence included pp25 cDNA, we obtained cDNA of IMAGE clones 4056112 and 4058379 and sequenced them.…”

“…A portion of each tissue extract (corresponding to 20-40 µg protein) was subjected to SDS-PAGE followed by western blotting. The blot was probed with an antibody against the amino-terminal region of pp25 and detected by using enhanced chemiluminescence (ECL) reagents essentially as described previously by Funami et al (2002). A recent report from our laboratory (in Yonago) described the method for the preparation of this antibody .…”

“…Various tissues were also obtained from female Xenopus adults and processed as described previously by Funami et al (2002). A portion of each tissue extract (corresponding to 20-40 µg protein) was subjected to SDS-PAGE followed by western blotting.…”