Casein kinase G (CKG) with more than 2500-fold enrichment was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit ...Casein kinase G (CKG) with more than 2500-fold enrichment was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major target for the enzyme auto-phosphorylation. Each full-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0.38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induced maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine. The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an substrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphorylation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesterone-induced oocyte maturation, 55 kD protein was dephospho-rylated. Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental results indicated strongly that CKG may be the physiological target of spermine.展开更多
Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation. The ID50 for spermine inhibition via intra - oocyte mi-croinjection on maturation induced by progestero...Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation. The ID50 for spermine inhibition via intra - oocyte mi-croinjection on maturation induced by progesterone was 6. 8 mM(100 nl). Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation. Spermine selectively promoted the level of phosphorylation of ?this protein in both progesterone - stimulated and hormone - untreated oocytes. The extent of its dephosphorylation was fairly correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the period of 0. 40 GVBD50 and 0. 60 GVBD50, at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone - stimulated protein synthesis in almost the same dose dependent manner as its inhibitory effect on the hormone - induced maturation. The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF. It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein (mRNP) particles.展开更多
文摘Casein kinase G (CKG) with more than 2500-fold enrichment was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major target for the enzyme auto-phosphorylation. Each full-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0.38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induced maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine. The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an substrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphorylation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesterone-induced oocyte maturation, 55 kD protein was dephospho-rylated. Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental results indicated strongly that CKG may be the physiological target of spermine.
文摘Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation. The ID50 for spermine inhibition via intra - oocyte mi-croinjection on maturation induced by progesterone was 6. 8 mM(100 nl). Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation. Spermine selectively promoted the level of phosphorylation of ?this protein in both progesterone - stimulated and hormone - untreated oocytes. The extent of its dephosphorylation was fairly correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the period of 0. 40 GVBD50 and 0. 60 GVBD50, at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone - stimulated protein synthesis in almost the same dose dependent manner as its inhibitory effect on the hormone - induced maturation. The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF. It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein (mRNP) particles.
