Eigenbrodt E, Fister P, Rübsamen H, Friis R R
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin und Tierzucht der Justus Liebig-Universität Giessen, FRG.
EMBO J. 1983;2(9):1565-70. doi: 10.1002/j.1460-2075.1983.tb01625.x.
Using chicken embryo fibroblasts infected with the NY68 transformation-defective temperature-sensitive mutant of Rous sarcoma virus, the phosphorylation and enzyme kinetic properties of enolase have been studied before, and at different stages after, the onset of transformation. A method for purification of enolase was developed, which minimized dephosphorylation. Two enolase (EC 4.2.1.11) isoenzymes were separated by isoelectric focussing revealing that it was the gammagamma form (pI 5.2-6.7) which had become phosphorylated at tyrosine residues after transformation. The phosphorylation of enolase in tyrosine occurred slowly after shift to the permissive temperature, rising from undetectable levels in phenotypically normal cells, to < 10% of the total phosphoamino acid after 3 h, and reaching 30-50% of the total phosphoamino acid by 16 h. Interestingly, the fraction of phosphorylated enolase molecules declined during transformation from 8% in normal cells to 5% by 16 h after temperature shift, due to a 3- to 5-fold increase in the total amount of enolase present in the transformed cultures. Although transformation had no apparent effect on the K0.5 of enolase (26 +/- 4 microM for 2-phosphoglycerate), its specific activity was reduced by about one third.
利用感染劳氏肉瘤病毒NY68转化缺陷型温度敏感突变体的鸡胚成纤维细胞，在转化开始前以及转化后的不同阶段，对烯醇化酶的磷酸化和酶动力学特性进行了研究。开发了一种纯化烯醇化酶的方法，该方法可最大程度减少去磷酸化。通过等电聚焦分离出两种烯醇化酶（EC 4.2.1.11）同工酶，结果显示，转化后在酪氨酸残基处发生磷酸化的是γγ形式（pI 5.2 - 6.7）。转移至允许温度后，烯醇化酶酪氨酸位点的磷酸化过程缓慢，从表型正常细胞中无法检测到的水平开始，3小时后升至总磷酸氨基酸的不到10%，到16小时时达到总磷酸氨基酸的30 - 50%。有趣的是，由于转化培养物中烯醇化酶总量增加了3至5倍，在转化过程中，磷酸化烯醇化酶分子的比例从正常细胞中的8%下降至温度转移后16小时的5%。尽管转化对烯醇化酶的K0.5（对2 - 磷酸甘油酸而言为26±4 microM）没有明显影响，但其比活性降低了约三分之一。
