固定化烟草核酮糖-1,5-二磷酸羧化酶/加氧酶的解离及重组作用

本文将共价偶联于CNBr活化的Sepharose 4B上的烟草核酮糖-1,5-二磷酸羧化酶/加氧酶用不同浓度尿素处理后,发现2—2.5mol/L尿素可将小亚单位解离下来而大亚单位仍偶联在载体上。3mol/L以上尿素可将大亚单位8聚体,L_8进一步解离为单体。因此,酶是通过大亚单位上的ε-氨基与载体相偶联的。小亚单位的解离量与酶活性的下降呈线性相关。将解离酶的尿素浓度稀释至0.5mol/L,解离的小亚单位几乎全部结合到残缺小亚单位的固相酶上,酶的活性也接近全部恢复。重组小亚单位量与酶活性增加亦呈线性相关。结果表明:除大亚单位外,小亚单位对维持酶的活性也有重要的作用。     

DISSOCIATION AND RECONSTITUTION OF IMMOBILIZED RIBULOSE-1, 5-BISPHOS PHATE CARBOXYLASE//OXYGENASE FROM TOBACCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco covalently coupled to CNBr-activated Sepharose 4B was treated with urea. Analysis by electrophoresis showed that the small subunit was dissociated at 2—2.5 mol/L urea, while the large subunit was still bound to matrix. The large subunit core, L_8, was further dissociated into monomer at 3 mol/L urea. It is suggested that RuBPCase is coupled to Sepharose by virtue of ε-NH_2 on a large subunit. The activity of the immobilized enzyme was inversely proportional to the amount of small subunit dissociated by urea. The dissociated small subunits were almost completely bound back to the S-depleted immobilized RuBPCase, if the urea concentration was diluted to 0.5 mol/L. The enzyme activity could be recovered nearly to 100％. The activity of the S-depleted enzyme was linearly correlated on the concentration of small subunits in solution. These results indicate that the small subunit plays an important role in the maintenance of RuBPCase activity.