Bontemps F, Hue L, Hers H G
Biochem J. 1978 Aug 15;174(2):603-11. doi: 10.1042/bj1740603.
The conversion of glucose into glucose 6-phosphate in an extract of isolated rat hepatocytes incubated in the presence of MgATP was studied spectrophotometrically at 340nm and also by a radiochemical procedure based on the release of (3)H from [2-(3)H]glucose. Both methods gave similar results. The glucose-saturation curve was sigmoidal and the shape of this curve was not influenced by the ionic composition of the incubation medium. The activity at 0.5mm-glucose was only 1-2% of V(max.), indicating a virtual absence of low-K(m) hexokinase in the preparation. The radiochemical method was also used for the determination of glucose phosphorylation by intact hepatocytes. The glucose-saturation curve was also markedly sigmoidal, but the s(0.5) (substrate concentration at half-maximal velocity) and the Hill coefficient were larger than in extracts of hepatocytes. These two parameters became smaller when cells were incubated in a medium in which Na(+) ions were replaced by K(+) ions. The increased rate of phosphorylation at low glucose concentration in a K(+) medium was accompanied by an increased rate of metabolite recycling between glucose and glucose 6-phosphate and also by an increased uptake of glucose. In both media phosphorylation of glucose was inhibited co-operatively by N-acetylglucosamine. Calculations indicate that this inhibition would reach 100% at saturation of the inhibitor, although at lower concentrations of N-acetylglucosamine it was smaller than expected from the known K(i) of N-acetylglucosamine for glucokinase. The rate of phosphorylation of glucose was proportional to the amount of glucokinase in hepatocytes from newborn rats and in conditions such as starvation and diabetes in which the total amount of glucokinase in the liver is decreased. In the same conditions, glucose 6-phosphatase activity was either normal or increased. It is concluded that the phosphorylation of glucose in isolated hepatocytes follows sigmoidal kinetics, which can be explained by the activity of glucokinase alone with no participation of low-K(m) hexokinase or of glucose 6-phosphatase.
在MgATP存在的情况下，对分离的大鼠肝细胞提取物中葡萄糖转化为葡萄糖6 - 磷酸的过程进行了研究。采用分光光度法在340nm波长下进行测定，同时也采用了基于[2 - (³H)]葡萄糖释放出(³H)的放射化学方法。两种方法得到了相似的结果。葡萄糖饱和曲线呈S形，且该曲线的形状不受孵育介质离子组成的影响。在0.5mmol/L葡萄糖浓度下的活性仅为V(max)的1 - 2%，表明该制剂中几乎不存在低K(m)己糖激酶。放射化学方法也用于完整肝细胞葡萄糖磷酸化的测定。葡萄糖饱和曲线也明显呈S形，但s(0.5)（最大速度一半时的底物浓度）和希尔系数比肝细胞提取物中的更大。当细胞在Na⁺离子被K⁺离子取代的介质中孵育时，这两个参数会变小。在K⁺介质中低葡萄糖浓度下磷酸化速率的增加伴随着葡萄糖与葡萄糖6 - 磷酸之间代谢物循环速率的增加以及葡萄糖摄取的增加。在两种介质中，N - 乙酰葡糖胺协同抑制葡萄糖的磷酸化。计算表明，在抑制剂饱和时这种抑制将达到100%，尽管在较低浓度的N - 乙酰葡糖胺时，其抑制作用比根据N - 乙酰葡糖胺对葡萄糖激酶已知的K(i)所预期的要小。葡萄糖磷酸化速率与新生大鼠肝细胞中葡萄糖激酶的量成正比，在饥饿和糖尿病等肝脏中葡萄糖激酶总量减少的情况下也是如此。在相同条件下，葡萄糖6 - 磷酸酶活性要么正常要么增加。结论是，分离的肝细胞中葡萄糖的磷酸化遵循S形动力学，这可以仅由葡萄糖激酶的活性来解释，而无需低K(m)己糖激酶或葡萄糖6 - 磷酸酶的参与。
