Komori K, Robinson K A, Block N E, Roberts R C, Buse M G
Department of Medicine and Biochemistry/Molecular Biology, Medical University of South Carolina, Charleston 29425.
Endocrinology. 1992 Sep;131(3):1288-96. doi: 10.1210/endo.131.3.1380438.
alpha 1-Inhibitor-III (alpha 1I3), a broad range proteinase inhibitor, member of the alpha-macroglobulin family, is abundant in normal rat plasma. Insulin-dependent tyrosine phosphorylation of a monomeric 195K glycoprotein (pp195) was observed in wheatgerm agglutinin (WGA)-Sepharose-purified insulin receptor preparations from rat liver and muscle. Phosphorylation of pp195 in vitro required a basic poly-amino acid, i.e. poly-L-lysine. We present evidence identifying pp195 as alpha 1I3. In situ perfusion with saline essentially removed pp195 from rat livers. Addition of normal rat plasma to liver homogenates or to WGA eluates restored insulin-stimulated phosphorylation of pp195; plasma from streptozotocin-diabetic rats was much less effective. Liver-derived pp195 copurified with an abundant plasma protein, with the characteristics of alpha 1I3, on size exclusion and ion-exchange chromatography. An approximately 195K protein, comigrating with alpha 1I3, was markedly diminished in plasma from diabetic rats, and alpha 1I3 concentration was decreased by approximately 70% upon immunoblot analysis. Highly purified alpha 1I3 was phosphorylated by muscle- or liver-derived insulin receptors in the presence of 1 microM poly-L-lysine and comigrated with pp195 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 1I3 phosphorylation was half-maximal at approximately 70 nM and was stimulated by insulin 7-fold. Hindlimb perfusion removed more than 90% plasma albumin but only approximately 20% pp195 from muscles. alpha 1I3 messenger RNA was identified in liver but not in muscle. A specific antibody against alpha 1I3 immunoprecipitated phosphorylated pp195 in WGA-purified insulin receptor preparations from nonperfused liver and from saline perfused and nonperfused muscle. alpha 1I3 is bound and internalized by alpha-macroglobulin receptors; whether it is phosphorylated in vivo is unknown. Hepatic alpha 1I3 synthesis may diminish in diabetic rats.
α1-抑制剂-III（α1I3）是一种广泛作用的蛋白酶抑制剂，属于α-巨球蛋白家族成员，在正常大鼠血浆中含量丰富。在从小麦胚芽凝集素（WGA）-琼脂糖纯化的大鼠肝脏和肌肉胰岛素受体制剂中，观察到一种单体195K糖蛋白（pp195）的胰岛素依赖性酪氨酸磷酸化。体外pp195的磷酸化需要一种碱性多聚氨基酸，即聚-L-赖氨酸。我们提供证据表明pp195就是α1I3。用生理盐水原位灌注基本上从大鼠肝脏中去除了pp195。向肝脏匀浆或WGA洗脱液中加入正常大鼠血浆可恢复胰岛素刺激的pp195磷酸化；链脲佐菌素诱导的糖尿病大鼠的血浆效果则差得多。肝脏来源的pp195与一种丰富的血浆蛋白在尺寸排阻色谱和离子交换色谱上共纯化，该血浆蛋白具有α1I3的特征。在糖尿病大鼠的血浆中，一种与α1I3共迁移的约195K蛋白明显减少，免疫印迹分析显示α1I3浓度降低了约70%。在存在1μM聚-L-赖氨酸的情况下，高度纯化的α1I3被肌肉或肝脏来源的胰岛素受体磷酸化，并在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上与pp195共迁移。α1I3磷酸化在约70 nM时达到半最大反应，并被胰岛素刺激7倍。后肢灌注从肌肉中去除了超过90%的血浆白蛋白，但仅去除了约20%的pp195。在肝脏中鉴定出α1I3信使RNA，但在肌肉中未鉴定出。一种针对α1I3的特异性抗体在未灌注肝脏以及生理盐水灌注和未灌注肌肉的WGA纯化胰岛素受体制剂中免疫沉淀磷酸化的pp195。α1I3被α-巨球蛋白受体结合并内化；其在体内是否被磷酸化尚不清楚。糖尿病大鼠肝脏中α1I3的合成可能会减少。
