Bedoya F J, Meglasson M D, Wilson J M, Matschinsky F M
Anal Biochem. 1985 Feb 1;144(2):504-13. doi: 10.1016/0003-2697(85)90147-2.
Glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.1) plays a pivotal role in hepatic glucose metabolism and serves as the glucose sensor in pancreatic islet beta-cells. Biochemical studies of this enzyme are complicated by the cellular heterogeneity of the liver and the pancreas and because the presence of hexokinases (ATP:D-hexose 6-phosphotransferases, EC 2.7.1.1) seriously interferes with currently available analytical procedures. A radiometric assay was designed to deal with these problems. It is based on the liberation of 3H2O from D-[2-3H(N)]glucose 6-phosphate, the product of the glucokinase reaction, using exogenous phosphoglucose isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). Interference by hexokinases was largely eliminated by using glucose 6-phosphate as inhibitor and the sensitivity of the assay was greatly increased by using small volumes with the oil well procedure. The assay was sufficiently sensitive to detect about 1 pg of glucokinase. It thus allowed the application of quantitative histochemical procedures to the study of intralobular hepatic glucokinase profiles and the pancreatic beta-cell glucose sensor. The quantitative histochemical procedures were sufficiently sensitive and reliable for measuring important kinetic constants of glucokinase (i.e., the Km and the Hill number) in microscopic samples of tissue.
葡萄糖激酶（ATP：D - 葡萄糖6 - 磷酸转移酶，EC 2.7.1.1）在肝脏葡萄糖代谢中起关键作用，并作为胰岛β细胞中的葡萄糖传感器。由于肝脏和胰腺的细胞异质性，以及己糖激酶（ATP：D - 己糖6 - 磷酸转移酶，EC 2.7.1.1）的存在严重干扰了现有的分析程序，对这种酶的生化研究变得复杂。设计了一种放射性测定法来处理这些问题。它基于从葡萄糖激酶反应的产物D - [2 - ³H（N）]葡萄糖6 - 磷酸中释放³H₂O，使用外源性磷酸葡萄糖异构酶（D - 葡萄糖 - 6 - 磷酸酮醇异构酶，EC 5.3.1.9）。通过使用6 - 磷酸葡萄糖作为抑制剂，己糖激酶的干扰在很大程度上得以消除，并且通过油井程序使用小体积样品大大提高了测定的灵敏度。该测定法足够灵敏，能够检测到约1 pg的葡萄糖激酶。因此，它允许将定量组织化学程序应用于研究肝小叶内葡萄糖激酶分布和胰腺β细胞葡萄糖传感器。定量组织化学程序对于测量组织微观样品中葡萄糖激酶的重要动力学常数（即Km和希尔系数）足够灵敏且可靠。
