Aggarwal S R, Palmer T N
Department of Biochemistry, University of Western Australia, Nedlands.
Biochim Biophys Acta. 1994 Nov 10;1224(2):311-22. doi: 10.1016/0167-4889(94)90205-4.
One-dimensional SDS-PAGE of cytosolic phosphopeptides confirms that glucagon promotes the phosphorylation of 11 phosphopeptides in isolated rat hepatocytes pre-equilibrated with 32PO4(3-). Nine of these phosphopeptides are tentatively identified, whereas two phosphopeptides (48 kDa and 46 kDa) remain unidentified. Transfer of the glucagon-challenged hepatocytes to medium free of 32PO4(3-) and glucagon led to the rapid net dephosphorylation of the phosphopeptides and to a rapid decline in the specific radioactivity of the [32P]ATP pool. There were profound differences between the post-glucagon rates of net dephosphorylation of the different hepatic phosphopeptides, consistent with net dephosphorylation being asynchronous during the recovery phase from acute glucagon challenge. On the basis of descending rates of dephosphorylation, four major groups of phosphopeptides were delineated. Okadaic acid, a potent inhibitor of protein phosphatase 2A and to a lesser extent protein phosphatase 1, inhibited the dephosphorylation of all of the phosphopeptides. A role for protein phosphatase 2A in protein dephosphorylation may be indicated by the observation that spermine, a specific activator of protein phosphatase 2A, stimulates the dephosphorylation of some, but not all, of the glucagon-stimulated phosphopeptides. Although phosphorylation during the recovery phase from glucagon challenge may be a complicating factor, the results suggest that post-glucagon dephosphorylation is a complex asynchronous process. The physiological consequences of this asynchrony may be that the suppression of glycogenolysis and gluconeogenesis and the activation of glycolysis are early events in the recovery process.
对胞质磷酸肽进行的一维SDS-PAGE证实，胰高血糖素可促进在含32PO4(3-)的缓冲液中预平衡的离体大鼠肝细胞中11种磷酸肽的磷酸化。其中9种磷酸肽已初步鉴定，而另外两种磷酸肽（48 kDa和46 kDa）仍未鉴定出来。将受胰高血糖素刺激的肝细胞转移至不含32PO4(3-)和胰高血糖素的培养基中，导致磷酸肽迅速发生净去磷酸化，并使[32P]ATP池的比放射性迅速下降。不同肝磷酸肽在胰高血糖素作用后的净去磷酸化速率存在显著差异，这与急性胰高血糖素刺激后的恢复阶段净去磷酸化不同步一致。根据去磷酸化速率的降序排列，划分出了四大类磷酸肽。冈田酸是蛋白磷酸酶2A的强效抑制剂，对蛋白磷酸酶1的抑制作用较弱，它可抑制所有磷酸肽的去磷酸化。蛋白磷酸酶2A的特异性激活剂精胺可刺激部分但并非全部受胰高血糖素刺激的磷酸肽的去磷酸化，这一现象表明蛋白磷酸酶2A在蛋白质去磷酸化过程中发挥了作用。尽管胰高血糖素刺激后的恢复阶段的磷酸化可能是一个复杂因素，但结果表明胰高血糖素作用后的去磷酸化是一个复杂的异步过程。这种异步性的生理后果可能是糖原分解和糖异生的抑制以及糖酵解的激活是恢复过程中的早期事件。
