Bhanumathy C D, Balasubramanian A S
Neurochemistry Laboratory, Department of Neurological Sciences, Christian Medical College and Hospital, Vellore, India.
Biochem J. 1996 Apr 1;315 ( Pt 1)(Pt 1):127-31. doi: 10.1042/bj3150127.
Purified human serum butyrylcholinesterase after treatment with either of the metal chelators EDTA or NaCN was able to bind to a Zn(2+)-chelate-Sepharose affinity column and was eluted from the column by EDTA or imidazole. Prior EDTA treatment of the enzyme was essential for binding to this affinity column. The enzyme could be labelled with (65)Zn(2+) after EDTA treatment of the enzyme. Diethylpyrocarbonate modification of histidine residues in the EDTA-treated enzyme resulted in the abolition of both binding to the Zn(2+)-chelate-Sepharose column and labelling by (65)Zn(2+). Stoicheiometry of (65)Zn(2+) binding indicated approximately 0.85 mol of Zn(2+)/mol of subunit of the EDTA-treated enzyme. EDTA or NaCN treatment resulted in the loss of thermal stability of the enzyme at 37 degrees C which could not be reversed by Zn(2+). Whereas the cholinesterase activity of butyrlcholinesterase was not affected by EDTA, there was significant loss of its carboxypeptidase activity in the presence of EDTA, and the loss could be reversed by added ZnCl2. These results suggest the presence of a Zn(2+)-binding site on human serum butyrylcholinesterase and the involvement of histidine residues in the metal binding. The presence in human serum butyrylcholinesterase of a sequence HXXE...H found in many known Zn(2+)-containing enzymes supports these findings.
用金属螯合剂乙二胺四乙酸（EDTA）或氰化钠（NaCN）处理后的纯化人血清丁酰胆碱酯酶，能够与锌离子螯合琼脂糖亲和柱结合，并通过EDTA或咪唑从柱上洗脱下来。酶预先经EDTA处理对于与该亲和柱结合至关重要。酶经EDTA处理后可用锌-65（65Zn2+）进行标记。对经EDTA处理的酶中组氨酸残基进行焦碳酸二乙酯修饰，导致其与锌离子螯合琼脂糖柱的结合以及锌-65（65Zn2+）标记均被消除。锌-65（65Zn2+）结合的化学计量表明，经EDTA处理的酶每摩尔亚基约结合0.85摩尔锌离子（Zn2+）。EDTA或NaCN处理导致酶在37℃时热稳定性丧失，且不能被锌离子（Zn2+）逆转。虽然丁酰胆碱酯酶的胆碱酯酶活性不受EDTA影响，但在EDTA存在下其羧肽酶活性显著丧失，添加氯化锌（ZnCl2）可逆转这种丧失。这些结果表明人血清丁酰胆碱酯酶上存在锌离子（Zn2+）结合位点，且组氨酸残基参与金属结合。在人血清丁酰胆碱酯酶中存在许多已知含锌离子（Zn2+）酶中发现的HXXE...H序列，支持了这些发现。
