【摘要】 从正常小鼠肝和Hep A 腹水肝癌中纯化了K 型丙酮酸激酶（PyK）。当比较这两种不同来源的PyK 时,发现下列各项指标均极为接近或完全相同。这些指标是:1.在磷酸纤维素柱上被相近浓度的KCl 洗脱下来。2.聚丙烯酰胺凝胶电泳迁移率完全一致,两者混合样品在不同的电泳条件下均不能分开。3.两酶在50℃保温时的失活速度基本相同,半失活时间均为11.5分。4.两酶的pH 活性曲线几乎完全重合,其最适pH 均为7.4。5.六种氨基酸中的每一种对两酶的抑制百分率十分接近。Mg⁺⁺或丝氨酸对两酶的激活也基本一致。6.两酶对底物PEP 和ADP 均呈正协同别构效应,不论PEP 或ADP 的Hill 氏系数（η_H）S_0.5和K_0.5s也十两分接近。7.苯丙氨酸对两酶均呈别构抑制效应,其η_H或表观Ki 也基本相同。8.ATP 对酶均呈别构抑制效应,两酶的Hill 氏曲线互相平行,且几乎重合。（9）用葡聚糖G-200凝胶过滤法测得正常小鼠肝和Hep A 中K 型PyK 的分子量分别为224,000及210,000道尔顿,其差别在实验误差范围内。（10）用双相免疫扩散鉴定,两酶都能和兔抗大鼠M 型PyK 抗体起沉淀反应,且沉淀线完全吻合。总结上述结果,证明肝癌细胞中K-PyK 与正常小鼠肝脏中的K-PyK 具有相同的性质,表现相似的动力学,提示它们很可能具有相同的结构,也即可能是同一种酶。癌细胞的基因表达产物既与正常相同,说明癌细胞中PyK 同工酶活性的改变可能是由于基因调控的失常而不是结构基因本身DNA 中碱基顺序的改变。更多还原

【Abstract】 Type K pyruvate kinase （PyK） was purified from normal mouse liver and HepA ascites hepatoma.When two type K PyKs of different origin were compared,theywere very much similar to each other or exactly the same with respect to the followingparameters.（1） They were eluted by closely similar concentrations of KCl from phosphocellu-lose columns.（2） They showed the same mobility on polyacrylamide gel electrophoresis;amixed sample could not be separated from each other under differentconditions of electrophoresis.（3） They had the same velocity of inactivation when incubated at 50℃,the halftime of inactivation for both enzymes being 11.5 minutes.（4） The pH vs activity curve of the two enzymes almost entirely overlapped,the optimum pH of both being 7.4.（5） The rates of inhibition by each of 6 amino acids and the rates of activation by Mg⁺⁺ or serine of the two enzymes were at the same or nearly the same.（6） The two enzymes showed positive cooperative allosteric kinetics with respectto both substrates,PEP and ADP.Not only the value of the Hill coefficient（η_H） but also K_0.5s and S_0.5 of the two enzymes were very close to each other.（7） Phenylalanine showed allosteric inhibitory kinetics for both enzymes andthe η_H or apparent Ki were nearly identical.（8） ATP also showed allosteric inhibitory kinetics for the two enzymes.Hillcurves for the two enzymes were parallel and almost overlapped.（9） The molecular weights of K type PyK isolated from normal mouse liver andHep A were 224,000 and 210,000 respectively as determined by SephedexG-200 gel filtration.The difference was within experimental error.（10） By using dimensional immunodiffusion method it was revealed that bothenzymes could precipatate with rat M-PyK antibody from the rabbit withthe ends of two precipitation lines fused into each other.It is concluded from the above results that type K PyK of Hep A displayed thesame characters as and similar kinetics with the enzyme from normal mouse liver,suggesting that the two enzyme structures are probably indentical.Since the productof gene expression of the cancer cell is identical with that of the normal,the changeof PyK isozyme activities in cancer seems to be due to the abnormal control of geneexpression,and not to changes in base sequence of the structural gene involved.更多还原