Sparks J D, Sparks C E, Roncone A M, Amatruda J M
Department of Pathology, University of Rochester School of Medicine and Dentistry, New York 14642.
J Biol Chem. 1988 Apr 15;263(11):5001-4.
Apolipoprotein B (apo B) phosphorylation was examined in primary cultures of hepatocytes from control and nonketotic streptozotocin diabetic rats. Following a 5-h incubation with ortho[32P]phosphate, media lipoproteins (d less than 1.063 g/ml) were isolated, and delipidated apolipoproteins were separated by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGGE), and gels were heat fixed. Autoradiographic bands corresponding to high (apo BH) and low molecular weight apo B (apo BL) were observed in media lipoproteins isolated from control rats, and these bands were more prominent in media lipoproteins isolated from diabetic rats. Apo B-specific activity was estimated from aqueous alcohol-precipitated radioactivity and apo B monoclonal immunoassay of isolated media lipoproteins. In lipoproteins secreted by hepatocytes of diabetic rats, the calculated apo B specific activity was between 18- and 31-fold greater than that secreted by hepatocytes of control rats, consistent with the SDS-PAGGE gel data. The increase in secretory 32P-labeled apo B from hepatocytes of diabetic rats was due, at least in part, to an increase in labeled phospho-tyrosine as determined by phosphoamino acid analysis. These data demonstrate that apo BH may be secreted as a phosphorylated protein and that apo B phosphorylation occurs on tyrosine as well as serine residues.
在对照大鼠和非酮症链脲佐菌素糖尿病大鼠的原代肝细胞培养物中检测了载脂蛋白B（apo B）的磷酸化情况。用正[32P]磷酸盐孵育5小时后，分离出培养基脂蛋白（d小于1.063 g/ml），脱脂载脂蛋白通过十二烷基硫酸钠-聚丙烯酰胺梯度凝胶电泳（SDS-PAGGE）进行分离，然后对凝胶进行热固定。在从对照大鼠分离的培养基脂蛋白中观察到对应于高分子量（apo BH）和低分子量apo B（apo BL）的放射自显影条带，并且这些条带在从糖尿病大鼠分离的培养基脂蛋白中更明显。通过水醇沉淀的放射性和对分离的培养基脂蛋白进行apo B单克隆免疫测定来估计apo B的比活性。在糖尿病大鼠肝细胞分泌的脂蛋白中，计算出的apo B比活性比对照大鼠肝细胞分泌的比活性高18至31倍，这与SDS-PAGGE凝胶数据一致。糖尿病大鼠肝细胞分泌的32P标记的apo B增加至少部分是由于通过磷酸氨基酸分析确定的标记磷酸酪氨酸增加。这些数据表明，apo BH可能以磷酸化蛋白的形式分泌，并且apo B的磷酸化发生在酪氨酸以及丝氨酸残基上。
