Gomez-Cambronero J, Mato J M, Vivanco F, Sanchez-Crespo M
Laboratorio de Nefrologia, Instituto de Investigaciones Medicas de la Fundacion Jimenez Diaz, Madrid, Spain.
Biochem J. 1987 Aug 1;245(3):893-7. doi: 10.1042/bj2450893.
A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.
本文描述了一种从大鼠脾脏中纯化1-O-烷基-2-溶血-sn-甘油-3-磷酸胆碱：乙酰辅酶A乙酰转移酶（EC 2.3.1.67）的新的改良方法。在MgATP存在下，环磷酸腺苷依赖性蛋白激酶的催化亚基使这种部分纯化的酶活性提高了约3倍。当在测定混合物中加入[γ-32P]ATP时，通过SDS/聚丙烯酰胺凝胶电泳和放射自显影对磷蛋白产物进行分析，结果显示[32P]磷酸盐掺入了一条约30 kDa的单一蛋白带中。对磷酸化氨基酸的分析表明，磷酸盐掺入了一个丝氨酸残基中。蛋白激酶对乙酰化反应的激活是可逆的。激活的逆转与掺入30 kDa蛋白带中的[32P]磷酸盐的丢失同时发生，这表明乙酰转移酶受依赖于环磷酸腺苷的磷酸化-去磷酸化机制调节。
