猪骨骼肌磷酸葡萄糖异构酶的提纯及性质研究

FURIFICATION AND PROPERTIES OF GLUCOSE-6-PHOSPHATE ISOMERASE FROM ANIMAL MUSCLES

本文报道猪骨骼肌磷酸葡萄糖异构酶（ＧＰＩ．ＥＣ ５．３．１．９）的提纯及其性质。设计了四步提纯法（稀盐溶液抽提、有机溶媒沉淀、透析、葡聚糖Ｇ－１００柱层析），对猪的五种不同解剖部位的骨骼风进行了ＧＰＩ的提纯。结果均得到聚丙烯酰胺凝胶电泳（ＰＡＧＥ）图谱为一条带的产品。提纯效果以猪股二头肌为例：提纯１０．６８倍、活力回收３２．３７％，比活力１．６×１０５单位。对提纯酶性质的研究表明；猪肌提纯的ＧＰＩ由三个亚基组成。分子量约为１２０Ｋｄ，等电点为 ６． ６，最适ｐＨ８．５，最适温度 ３５℃，米氏常数：６．０２ｍｍｏｌ／Ｌ，活化能为３２３７８．４焦耳／Ｋ，ｍｏｌ。免疫电泳实验证实猪肌提纯酶具抗原性。其免疫血清与自牛、兔、鸡、鱼的骨骼肌用同法提纯的ＧＰＩ有交叉免疫反应。免疫血清且对原酶液有抑制作用。猪肌ＧＰＩ与其它数种动物肌肉之ＧＰＩ的ＰＡＧＥ行为、混合电泳行为，等电聚焦行为、氨基酸组成等各方面基本相同，可以认为这是猪肌ＧＰＩ与其它数种动物肌肉的ＧＰＩ为同源蛋白质的佐证。

The furification and properties of glucose-6-phosphate isomerase (GPI) from animal muscles were investigated. A concise 4-step furification procedure was designed and proceeded with good reproducibility. Hign purity GPI was obtained. For example,GPI of M. biceps femoris from pig was purified to 10. 86 folds with specific activity 1. 0 ×105 U/mg,a single band on PAGE pattern, M. W. about 120Kd,being composed of 3 sub-units, isoelectric point 5. 6, optimum temperature 35℃, optimum pH 8. 5 Michaelis-Menton constant 6.02mmol/L and activation energy 32378. 4 joules/k, mol. The GPI which was purified from pig muscle had antigenic property as identified by immunoelectrophoretic method with rabbit immune serum. And this serum had cross immune reaction with GPI purified from rabbit, bovine,chick and fish muscles. These immunological properties along with the results that a single band in PAGE was observed with mixture of purified GPI from pig and bovine muscles; and the close similarities among the values of pI and amino acid compositions of GPI from different animal muscles suggest that GPI from pig and Other animal muscles may be originally homologous proteins.