Nishimura M, Fedorov S, Uyeda K
Department of Veterans Affairs Medical Center, Dallas, Texas 75216.
J Biol Chem. 1994 Oct 21;269(42):26100-6.
The effect of glucose on hepatic fructose (Fru) 2,6-P2 in starved rats was investigated. When livers were perfused with high glucose (40 mM), hexose-P in the liver increased immediately reaching the maximum within in 2 min, but Fru 2,6-P2 after a lag period of 4 min increased linearly. The activation of Fru 6-P,2-kinase and inactivation of Fru 2,6-Pase also showed a similar lag period. Determination of the phosphate contents of the bifunctional enzyme after 10 min of glucose perfusion revealed that 90% of the enzyme was in the dephospho form while only 10% of the control liver enzyme was dephosphorylated. Comparison of crude extracts of liver perfused with either high glucose or normal glucose (5.6 mM) showed that high glucose livers contained 50% higher protein phosphatase activity, which dephosphorylated the bifunctional enzyme. Subcellular fractionation of the extract showed that activation of the protein phosphatase occurred in the cytosol. Desalting of the cytosolic fraction resulted in a 50% loss of the protein phosphatase activity. The low molecular weight activator in the cytosol was isolated, and by various chemical and enzymatic methods it was identified as xylulose 5-P. The activation of protein phosphatase by xylulose 5-P showed a highly sigmoidal saturation curve. The rate of formation of xylulose 5-P in the perfused liver showed a lag period of approximately 2 min, and after 4 min its concentration reached 10 microM, the minimum concentration necessary for the activation of the protein phosphatase. We conclude that the mechanism of glucose-induced Fru 2,6-P2 synthesis was not due to increased Fru 6-P as generally thought but occurred as a result of dephosphorylation of Fru 6-P,2-kinase:Fru 2,6-Pase. Moreover, the dephosphorylation was enhanced by increased xylulose 5-P, which activated a specific protein phosphatase. The results suggest a mechanism for coordinated regulation of glycolysis and the pentose shunt pathway that is mediated by xylulose 5-P.
研究了葡萄糖对饥饿大鼠肝脏中果糖-2,6-二磷酸（Fru 2,6-P2）的影响。当肝脏用高糖（40 mM）灌注时，肝脏中的己糖磷酸立即增加，并在2分钟内达到最大值，但Fru 2,6-P2在4分钟的延迟期后呈线性增加。果糖-6-磷酸-2-激酶的激活和果糖-2,6-二磷酸酶的失活也表现出类似的延迟期。葡萄糖灌注10分钟后对双功能酶的磷酸含量测定表明，90%的酶处于去磷酸化形式，而对照肝脏酶只有10%被去磷酸化。对用高糖或正常糖（5.6 mM）灌注的肝脏粗提物进行比较表明，高糖肝脏中蛋白质磷酸酶活性高50%，该酶使双功能酶去磷酸化。提取物的亚细胞分级分离表明，蛋白质磷酸酶的激活发生在细胞质中。细胞质部分脱盐导致蛋白质磷酸酶活性损失50%。分离出细胞质中的低分子量激活剂，并通过各种化学和酶学方法将其鉴定为木酮糖-5-磷酸。木酮糖-5-磷酸对蛋白质磷酸酶的激活表现出高度的S形饱和曲线。灌注肝脏中木酮糖-5-磷酸的形成速率显示出约2分钟的延迟期，4分钟后其浓度达到10 microM，这是激活蛋白质磷酸酶所需的最低浓度。我们得出结论，葡萄糖诱导的Fru 2,6-P2合成机制并非如通常所认为的那样是由于果糖-6-磷酸增加，而是由于果糖-6-磷酸-2-激酶：果糖-2,6-二磷酸酶的去磷酸化所致。此外，木酮糖-5-磷酸增加增强了去磷酸化，木酮糖-5-磷酸激活了一种特异性蛋白质磷酸酶。结果提示了一种由木酮糖-5-磷酸介导的糖酵解和戊糖分流途径协调调节的机制。
