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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。
K-pyruvate kinase (PyK) is purified from normal mouse liver and Hep A ascites hepatoma. When comparing PyK from these two different sources, the following indicators were found to be very close or exactly the same. These indices are: 1. Elute on similar concentrations of KCl on a phosphocellulose column. 2. Polyacrylamide gel electrophoresis mobility exactly the same, both mixed samples in different electrophoresis conditions can not be separated. The inactivation rate of the two enzymes incubated at 50 ℃ is basically the same, the half-inactivation time is 11.5 minutes. The pH-activity curve of the two enzymes almost completely coincide, the optimum pH is 7.4.5.The percentage inhibition of the two enzymes for each of the six kinds of amino acids is very close. Mg ~ (++) or serine activation of the two enzymes are basically the same. Both enzymes showed positive synergistic allosteric effect on PEP and ADP, and Hill’s coefficient (η_H) S_ (0.5) and K_ (0.5s) of PEP or ADP were also close to ten. Phenylalanine showed an allosteric inhibitory effect on both enzymes, and its η_H or apparent Ki were also basically the same. Alloxan showed no allosteric inhibitory effect. The Hill curves of the two enzymes were parallel to each other and almost coincided. (9) The molecular weights of Type K PyK in normal mouse liver and Hep A were determined to be 224,000 and 210,000 daltons, respectively, with dextran G-200 gel filtration, with a difference within experimental error. (10) Two-phase immunodiffusion identification, both enzymes and rabbit anti-rat type M PyK antibody precipitation reaction, and the precipitation line completely consistent. Summarizing the above results, it was demonstrated that K-PyK in liver cancer cells have the same properties as K-PyK in normal mouse liver and exhibit similar kinetics, suggesting that they are likely to have the same structure, which may be the same enzyme. The gene expression products of cancer cells are both the same as normal, indicating that changes in PyK isozyme activity in cancer cells may be due to gene regulatory disorders rather than changes in the base sequence in the DNA of the structural gene itself.