扇贝碱性磷酸酶中Zn的作用

以华贵栉孔扇贝Chlamys nobilis(Reeve)为材料,获得聚丙烯酰胺凝胶电泳为单一蛋白区带的酶制剂。用凝胶过滤法测得该酶的分子量为159000,由20种氨基酸组成,共1118个氨基酸残基。每个酶蛋白含有4个Zn原子。将该酶的酶制剂制成脱Zn酶后,与相同浓度过度性两价金属离子作用,其活力回升以Zn最强,Co次之。若向脱Zn酶蛋白分别加入一定量Zn原子或Co原子,根据实验结果初步推测:每个酶蛋白的4个Zn原子中有2个Zn原子先与该酶结合,与酶的催化作用有密切关系;另外2个Zn原子与酶结合,则起维持酶结构的作用,与哺乳动物和Eschericha coli来源的碱性磷酸酶研究结果一致。此外,本实验采用可见吸收光谱和圆二色谱(CD)来确证Co能很好地取代该酶蛋白内的Zn,与Simpon报道相比较,得知当Co原子加入脱Zn酶时,先加2个Co~(2 )离子结合到该酶的结构部位,而后加2个Co~(2 )离子才结合到催化部位。

THE ROLE OF ZINC IN ALKALINE PHOSPHATASE FROM SCALLOPS

Alkaline phosphatase extracted from scallopsChlamys niblis(Reeve)] is purified to homogenity by chromatography on DEAE-cellulose.A nearly identical molecular weight, 159 000 was determined by Sephadex G-200 chromatography. The enzyme consists of 1 118 amino acid residues constituted by 20 different amino acids. The native enzyme contains 4.09 atoms of zinc per mol of protein. The Zn2 in alkaline phosphatase can be removed by EDTA-Na2, its potential can be restored by the addition of a variety of divalent transition metal ions to the apoenzyme, such as Zn24 and Co2 . Our results are the same as alkaline phosphatase from Bovine kidney and Eschericha coli. The enzyme contained 4 moles Zn per mole of protein, in which two of them were required for enzymatic activity and the other two for maintenance of the structure. Compared with the report of Simpon, we have been investigating by absorption spectrum and circular dichroism, and confirmed that the first 2g-atoms of Co bound to the apoenzyme results in a similar spectrum without producing enzymatic activity.Addition of further 2g-atoms of Co generate activity.