Mellati A A, Yücel M, Altinörs N, Gündüz U
Biology Department, Middle East Technical University, Ankara, Turkey.
Clin Biochem. 1993 Oct;26(5):383-8. doi: 10.1016/0009-9120(93)90114-l.
The M2-type pyruvate kinase was purified from human meningioma by ammonium sulfate precipitation, followed by ion exchange and affinity chromatography. The specific activity of the purified enzyme was 33.4 U/mg with a yield of 6.5%. The enzyme gave a single band with 63,000 +/- 2000 Da upon SDS polyacrylamide gel electrophoresis. On cellulose acetate electrophoresis zymograms, the purified enzyme (M2) showed a single band, while crude extracts gave two broad bands corresponding to pyruvate kinase isozymes. The pI value of purified enzyme was found to be 6.9. With phosphoenol pyruvate as substrate the purified enzyme showed sigmoidal kinetics, while in the presence of 0.6 mM fructose 1,6-diphosphate as modulator it gave a hyperbolic saturation curve with a Km value of 0.53 mM.
通过硫酸铵沉淀，随后进行离子交换和亲和色谱法，从人脑膜瘤中纯化出M2型丙酮酸激酶。纯化酶的比活性为33.4 U/mg，产率为6.5%。该酶在SDS聚丙烯酰胺凝胶电泳上呈现出一条分子量为63,000 +/- 2000 Da的条带。在醋酸纤维素电泳酶谱上，纯化酶（M2）显示出一条单一的条带，而粗提物则给出了两条对应于丙酮酸激酶同工酶的宽带。纯化酶的pI值为6.9。以磷酸烯醇丙酮酸为底物时，纯化酶呈现出S形动力学，而在存在0.6 mM果糖1,6 -二磷酸作为调节剂的情况下，它给出了一条双曲线饱和曲线，Km值为0.53 mM。
