Sutherland D, McClellan J S, Milner D, Soong W, Axon N, Sanders M, Hester A, Kao Y H, Poczatek T, Routt S, Pezzementi L
Division of Science and Mathematics, Birmingham-Southern College, Alabama 35254, USA.
J Exp Zool. 1997 Feb 15;277(3):213-29. doi: 10.1002/(sici)1097-010x(19970215)277:3<213::aid-jez3>3.0.co;2-r.
To obtain information about the evolution of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the vertebrates, we investigated the cholinesterase (ChE) activity of the cephalochordate amphioxus (Branchiostoma floridae and Branchiostoma lanceolatum). On the basis of evidence from enzymology, pharmacology, and molecular biology, we conclude that amphioxus possesses two ChE activities and two ChE genes. Two covalent inhibitors of cholinesterases were able to pharmacologically isolate the two activities as drug-sensitive ChE and drug-resistant ChE. Kinetically, in terms of substrate specificity, the drug-sensitive ChE resembles vertebrate AChE, and the drug-resistant ChE resembles the BuChE of cartilaginous and bony fish or the intermediate ChE of protostome invertebrates. We also used the polymerase chain reaction with degenerate oligonucleotide primers and genomic DNA to obtain clones of 1,574 and 1,011 bp corresponding to two cholinesterase genes from amphioxus, which we designated as ChE1 and ChE2. ChE2 codes for an enzyme with an acyl-binding pocket sequence, a portion of the protein that plays an important role in determining substrate specificity, typical of invertebrate ChE. ChE1, which contains a 503-bp intron, encodes a protein with a novel acyl binding site. Phylogenetic analysis of the sequences suggests that the two genes are a result of a duplication event in the lineage leading to amphioxus. We discuss the relevance of our results to the evolution of the cholinesterases in the chordates. Previously, we reported that amphioxus contained a single cholinesterase activity with properties intermediate to AChE and BuChE (Pezzementi et al. [1991] In: Cholinesterases: Structure, Function, Mechanism, Genetics and Cell Biology. J. Massoulié et al., eds. ACS: Washington, D.C., pp. 24-31).
为了获取有关脊椎动物中胆碱酯酶、乙酰胆碱酯酶（AChE）和丁酰胆碱酯酶（BuChE）进化的信息，我们研究了头索动物文昌鱼（佛罗里达文昌鱼和 lanceolatum 文昌鱼）的胆碱酯酶（ChE）活性。基于酶学、药理学和分子生物学的证据，我们得出结论，文昌鱼具有两种 ChE 活性和两个 ChE 基因。两种胆碱酯酶的共价抑制剂能够从药理学上分离出这两种活性，即药物敏感型 ChE 和药物耐药型 ChE。从动力学角度，就底物特异性而言，药物敏感型 ChE 类似于脊椎动物的 AChE，而药物耐药型 ChE 类似于软骨鱼和硬骨鱼的 BuChE 或原口无脊椎动物的中间 ChE。我们还使用了简并寡核苷酸引物和基因组 DNA 进行聚合酶链反应，以获得与文昌鱼的两个胆碱酯酶基因相对应的 1574 和 1011 bp 的克隆，我们将其命名为 ChE1 和 ChE2。ChE2 编码一种具有酰基结合口袋序列的酶，该序列是蛋白质中在决定底物特异性方面起重要作用的一部分，是无脊椎动物 ChE 的典型特征。含有 503 bp 内含子的 ChE1 编码一种具有新型酰基结合位点的蛋白质。对这些序列的系统发育分析表明，这两个基因是文昌鱼谱系中一次复制事件的结果。我们讨论了我们的结果与脊索动物中胆碱酯酶进化的相关性。此前，我们报道文昌鱼含有单一的胆碱酯酶活性，其性质介于 AChE 和 BuChE 之间（Pezzementi 等人 [1991] 在：《胆碱酯酶：结构、功能、机制、遗传学和细胞生物学》。J. Massoulié 等人编。美国化学学会：华盛顿特区，第 24 - 31 页）。
