Takemoto D J
Department of Biochemistry, Kansas State University, Manhattan 66506, USA.
Acta Diabetol. 1998 Oct;35(3):145-9. doi: 10.1007/s005920050119.
Changes in the amounts and localization of protein kinase C (PKC) isoforms occur in galactosaemic lens epithelial cells. A link between PKC changes and myo-inositol depletion has been suggested. Raf-1, a component of a Ras pathway, is a substrate for PKC. Raf-1 levels were measured in galactosaemic lens epithelial cells grown with or without myo-inositol. Raf-1 levels were measured by densitometric scanning of Western blots from cells grown with or without 40 mmol/l galactose or 40 mmol/l galactose plus 1.0 micromol/l myo-inositol for 1, 3, 5 or 7 days. Scans were compared to those for PKCalpha, an isoform of PKC and to 14-3-3, a protein which binds to Raf-1. Cell growth was quantitated by thymidine incorporation. Raf-1 levels were decreased in bovine lens epithelial cells after 3, 5 or 7 days (33% of control) of growth in 40 mmol/l galactose. Addition of 1 micromol/l myo-inositol reversed this decrease at day 3, but not after 5 or 7 days of growth in 40 mmol/l galactose. PKCalpha and 14-3-3 levels were not affected by galactose. The decrease in Raf-1 was not a result of cell growth as measured by thymidine incorporation. These results suggest that Raf-1 levels are decreased during galactosaemia. This was only partially reversed by the addition of myoinositol.
半乳糖血症性晶状体上皮细胞中蛋白激酶C（PKC）亚型的含量和定位会发生变化。有人提出PKC变化与肌醇耗竭之间存在联系。Raf-1是Ras途径的一个组成部分，是PKC的底物。在添加或不添加肌醇的情况下培养的半乳糖血症性晶状体上皮细胞中测量Raf-1水平。通过对在添加或不添加40 mmol/l半乳糖或40 mmol/l半乳糖加1.0 μmol/l肌醇的条件下培养1、3、5或7天的细胞的蛋白质印迹进行光密度扫描来测量Raf-1水平。将扫描结果与PKC的一种亚型PKCalpha以及与Raf-1结合的蛋白质14-3-3的扫描结果进行比较。通过胸苷掺入来定量细胞生长。在40 mmol/l半乳糖中生长3、5或7天（为对照的33%）后，牛晶状体上皮细胞中的Raf-1水平降低。在第3天添加1 μmol/l肌醇可逆转这种降低，但在40 mmol/l半乳糖中生长5或7天后则不能。PKCalpha和14-3-3的水平不受半乳糖影响。Raf-1的降低不是通过胸苷掺入测量的细胞生长的结果。这些结果表明，半乳糖血症期间Raf-1水平降低。添加肌醇只能部分逆转这种情况。
