Pelech S L, Meijer L, Krebs E G
Howard Hughes Medical Institute, University of Washington, Seattle 98195.
Biochemistry. 1987 Dec 1;26(24):7960-8. doi: 10.1021/bi00398a062.
DEAE-Sephacel chromatography of cytosolic extracts from sea star oocytes resolved at least two distinct peaks of maturation-activated protein kinase activity, each of which catalyzed the phosphorylation of histone H1, ribosomal protein S6, and Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), a synthetic peptide based on the sequence of a phosphorylation site in the latter protein. The first peak (elution conductivity approximately equal to 6 mmho) contained the major activated kinase with respect to the phosphorylation of histone H1, and the second peak (elution conductivity approximately equal to 10.5 mmho) contained the major activated kinase with respect to the phosphorylation of S6 and RRLSSLRA. These kinase activities were barely detectable in extracts from immature oocytes. The major stimulated histone H1 kinase exhibited an apparent Mr of approximately 90 000 on Sephacryl S-300 but eluted from TSK-400 with an apparent Mr of approximately 10 000. After DEAE-Sephacel fractionation, this kinase was shown to utilize both ATP (apparent Km approximately equal to 45 microM) and GTP (apparent Km approximately equal to 10 microM), although the Vmax was 8-fold higher with ATP than with GTP. The enzyme phosphorylated histone H1 with an apparent Km approximately equal to 50 micrograms/mL. Its properties resembled those of the growth-associated histone kinase. The major stimulated RRLSSLRA kinase had an apparent Mr of approximately 84 000 on Sephacryl S-300 and approximately 40 000 on TSK-400. After DEAE-Sephacel chromatography, this kinase selectively utilized ATP (apparent Km approximately equal to 25 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
对海星卵母细胞的胞质提取物进行二乙氨基乙基-葡聚糖凝胶（DEAE-Sephacel）层析，分离出至少两个不同的成熟激活蛋白激酶活性峰，每个峰都能催化组蛋白H1、核糖体蛋白S6以及基于后者蛋白磷酸化位点序列合成的肽段精氨酸-精氨酸-亮氨酸-丝氨酸-丝氨酸-亮氨酸-精氨酸-丙氨酸（RRLSSLRA）的磷酸化。第一个峰（洗脱电导率约为6毫姆欧）含有相对于组蛋白H1磷酸化的主要激活激酶，第二个峰（洗脱电导率约为10.5毫姆欧）含有相对于S6和RRLSSLRA磷酸化的主要激活激酶。这些激酶活性在未成熟卵母细胞的提取物中几乎检测不到。主要受刺激的组蛋白H1激酶在Sephacryl S-300上的表观分子量约为90000，但在TSK-400上洗脱时表观分子量约为10000。经过DEAE-Sephacel分级分离后，该激酶显示既能利用ATP（表观米氏常数约为45微摩尔）也能利用GTP（表观米氏常数约为10微摩尔），尽管以ATP时的最大反应速度比以GTP时高8倍。该酶磷酸化组蛋白H1的表观米氏常数约为50微克/毫升。其特性类似于生长相关组蛋白激酶。主要受刺激的RRLSSLRA激酶在Sephacryl S-300上的表观分子量约为84000，在TSK-400上约为40000。经过DEAE-Sephacel层析后，该激酶选择性地利用ATP（表观米氏常数约为25微摩尔）。（摘要截短于250字）
