Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6.
Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6.
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DOI：
10.1016/s0021-9258(17)42478-1
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发表时间：
1986-01
期刊：
影响因子：
--
通讯作者：
E. Erikson;J. Maller
中科院分区：
文献类型：
--
作者：
E. Erikson;J. Maller
在非洲爪蟾卵母细胞中，核糖体蛋白S6在丝氨酸残基上发生磷酸化，这是对激素或生长因子的反应，并在劳斯肉瘤病毒或阿贝尔森小鼠白血病病毒相关酪氨酸特异性蛋白激酶的微注射后发生的。为了开始表征该系统中负责S6磷酸化的酶，我们从未受精的爪蟾卵中纯化了S6蛋白激酶。粗提物deae - sepacel层析显示两个S6激酶活性峰，选择160 mM NaCl洗脱峰进行进一步纯化。在Mono S、Sephacryl S-200、Mono Q和肝素- sepharose上进行连续层析，将酶纯化为单个蛋白，在聚丙烯酰胺凝胶上以Mr = 92,000迁移。最终产物从DEAE-Sephacel峰纯化约500倍，回收率为10%。该酶对ATP和40s亚基的表观Km值分别为28和5微米，对330微米ATP和5.6微米40s亚基的比活性为300 nmol/min/mg。该酶被-甘油磷酸、氟化钠、磷酸钾、ADP、肝素、槲皮素和精胺抑制。纯化的S6蛋白激酶的可用性将有助于阐明生长刺激过程中S6磷酸化的分子机制。
In Xenopus oocytes ribosomal protein S6 becomes phosphorylated on serine residues in response to hormones or growth factors and following microinjection of the tyrosine-specific protein kinases associated with Rous sarcoma virus or Abelson murine leukemia virus. To begin characterization of the enzymes responsible for S6 phosphorylation in this system, we have undertaken the purification of S6 protein kinases from unfertilized Xenopus eggs. DEAE-Sephacel chromatography of crude extracts revealed two peaks of S6 kinase activity, and the peak eluting at 160 mM NaCl was chosen for further purification. Successive chromatography on Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose resulted in purification of the enzyme to a single protein migrating at Mr = 92,000 on polyacrylamide gels. The final preparation was purified about 500-fold from the DEAE-Sephacel peak with a recovery of 10%. Apparent Km values of the enzyme for ATP and 40 S subunits were 28 and 5 microM, respectively, and the specific activity with 330 microM ATP and 5.6 microM 40 S subunits was 300 nmol/min/mg. The enzyme was inhibited by beta-glycerophosphate, sodium fluoride, potassium phosphate, ADP, heparin, quercetin, and spermine. The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation.