Alexander M C, Palmer J L, Pointer R H, Kowaloff E M, Koumjian L L, Avruch J
J Biol Chem. 1982 Feb 25;257(4):2049-55.
We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.
我们通过两种方法估算了胰岛素刺激的ATP-柠檬酸裂解酶的磷酸化情况。分离的肝细胞在1小时内将细胞外32P掺入[γ-32P]ATP中，并将免疫沉淀的ATP-柠檬酸裂解酶达到稳态水平。稳态时肝细胞ATP-柠檬酸裂解酶中酸稳定的32P含量为0.33±0.038摩尔磷/摩尔（四聚体）全酶。胰岛素（1毫单位/毫升）在10分钟内使免疫沉淀的裂解酶的32P含量增加2至3倍。从对照细胞和胰岛素处理的细胞中分离的酶中，裂解酶上超过90%的酸稳定32P是32P-丝氨酸。从肝细胞中分离的ATP-柠檬酸裂解酶每摩尔全酶含有0.95±0.1摩尔碱不稳定磷酸盐。用胰岛素处理肝细胞（1毫单位/毫升，处理10分钟）可使碱不稳定磷含量增加45%。有证据表明，胰岛素刺激的磷酸化并非由催化磷酸酶中间体的分子内迁移引起。这些观察结果支持这样的结论：胰岛素刺激的ATP-柠檬酸裂解酶磷酸化是由胰岛素诱导的裂解酶激酶活性增加和/或裂解酶磷酸酶活性降低介导的。ATP-柠檬酸裂解酶亚化学计量磷酸化的功能作用仍然未知。
