Fazi A, Piacentini M P, Piatti E, Accorsi A
Istituto di Chimica Biologica Giorgio Fornaini, Università degli Studi, Urbino, Italy.
Prep Biochem. 1990;20(3-4):219-40. doi: 10.1080/00327489008050198.
We have developed a simple procedure for the purification of phosphoglucomutase (PGM) isozymes from human placenta of healthy women. The technique involves the ammonium sulfate fractionation, ion-exchange and dye-ligand chromatographies. By this method we obtained homogeneous isozyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci. The final specific activities were 1134.6-1441.8 units/mg for PGM1 forms and 40.2-46.5 units/mg for PGM2 forms. On SDS-polyacrylamide gel electrophoresis analysis, the final preparations gave a single protein band of 58,500 and 69,000 Mr for PGM1 and PGM2 isozymes, respectively. These forms have the same kinetic properties, but from the substrate specificity experiments we have found that PGM2 forms are more effective for catalyzing the phosphoribomutase and glucose 1,6-bisphosphate synthase reaction than PGM1 forms. All these properties are shared by the same isozymes previously isolated from human erythrocytes but in this procedure the use of human placenta for the PGM isozymes purification takes advantage of high specific activity of PGM in the extracts of this tissue as well as obtaining highly homogeneous protein suitable for studies at molecular level.
我们开发了一种从健康女性人胎盘中纯化磷酸葡萄糖变位酶（PGM）同工酶的简单方法。该技术包括硫酸铵分级分离、离子交换和染料配体色谱法。通过这种方法，我们获得了两个PGM1和PGM2基因座产物（“初级”和“次级”）的均一同工酶制剂。PGM1形式的最终比活性为1134.6 - 1441.8单位/毫克，PGM2形式的最终比活性为40.2 - 46.5单位/毫克。在SDS - 聚丙烯酰胺凝胶电泳分析中，最终制剂分别给出了PGM1和PGM2同工酶的单一蛋白条带，其分子量分别为58,500和69,000。这些形式具有相同的动力学性质，但从底物特异性实验中我们发现，PGM2形式在催化磷酸核糖变位酶和葡萄糖1,6 - 二磷酸合酶反应方面比PGM1形式更有效。所有这些性质与先前从人红细胞中分离的相同同工酶相同，但在这个方法中，用人胎盘来纯化PGM同工酶利用了该组织提取物中PGM的高比活性，以及获得适合分子水平研究的高度均一的蛋白质。
