Vischer U, Blondel B, Wollheim C B, Höppner W, Seitz H J, Iynedjian P B
Biochem J. 1987 Jan 1;241(1):249-55. doi: 10.1042/bj2410249.
We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.
我们分析了RIN-m5F胰岛素瘤细胞中己糖激酶同工酶组的表达模式。通过DEAE-纤维素色谱法分离出三种己糖激酶形式。最丰富的同工酶与大鼠脂肪组织中的己糖激酶II型共洗脱，其对葡萄糖的Km为0.15 mM，与脂肪组织中的酶相似。己糖激酶II型在很大程度上与RIN-m5F细胞中的颗粒亚细胞组分相关。通过离子交换色谱法分离出的另外两种己糖激酶是一种类似于脑己糖激酶I型的酶和葡萄糖激酶（或己糖激酶IV型）。通过以下特性将后一种同工酶鉴定为肝型葡萄糖激酶：与肝葡萄糖激酶从DEAE-纤维素和DEAE-葡聚糖共洗脱；对葡萄糖呈S形饱和动力学，半最大速度在5.6 mM，希尔系数（h）为1.54；被针对大鼠肝葡萄糖激酶产生的抗体抑制酶活性；经一维和二维凝胶电泳后的免疫印迹显示，表观分子量为56,500，pI为5.6；用胰凝乳蛋白酶和木瓜蛋白酶进行蛋白水解后，肽图与肝葡萄糖激酶的肽图相同。这些数据表明，编码肝葡萄糖激酶的基因在RIN-m5F细胞中表达，这一发现与朗格汉斯胰岛β细胞中存在葡萄糖激酶的间接证据一致。另一方面，RIN-m5F细胞和朗格汉斯胰岛中己糖激酶的总体模式明显不同，因为胰岛中似乎缺乏己糖激酶II型。在其他系统中也报道了肿瘤转化后己糖激酶表达的改变。
