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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。
Chlamys nobilis (Reeve) was used as the material to obtain polyacrylamide gel electrophoresis as a single protein band enzyme preparation. The enzyme has a molecular weight of 159,000 as measured by gel filtration and consists of 20 amino acids with a total of 1118 amino acid residues. Each enzyme protein contains 4 Zn atoms. The enzyme enzyme preparation made of de-Zn enzyme, with the same concentration of excess divalent metal ions, the vitality of the rebound to Zn strongest, followed by Co. According to the experimental results, it is presumed that if Zn atom or Co atom is added to the de-Zn enzyme protein, two Zn atoms among the four Zn atoms of each enzyme protein bind with the enzyme and the catalytic effect of the enzyme is close The other two Zn atoms bound to the enzyme, then the enzyme structure plays a role, consistent with the study of mammalian and Eschericha coli alkaline phosphatase. In addition, we used visible absorption spectroscopy and circular dichroism (CD) to confirm that Co could well replace Zn in this enzyme protein. Compared with Simpon’s report, we found that when Co atom added de-Zn enzyme, Co 2+ ions bind to the structural part of the enzyme, and then add two Co 2+ ions to the catalytic site.