Heider H, Brodbeck U
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Biochem J. 1992 Jan 1;281 ( Pt 1)(Pt 1):279-84. doi: 10.1042/bj2810279.
Tetrameric detergent-soluble bovine caudate nucleus acetylcholinesterase (AChE) was reduced and alkylated under conditions in which at least 95% of initial activity is retained. This treatment alone did not result in monomerization of AChE, nor did it create a hydrophilic enzyme. However, in the presence of SDS the enzyme became monomerized. Incubation of AChE with trypsin in the presence of the reversible inhibitor edrophonium rendered the enzyme hydrophilic and led to catalytically active monomers being produced. SDS/PAGE of this preparation in non-reducing conditions revealed only a small decrease in the subunit molecular mass. N-Terminal sequencing of the enzyme, before and after trypsin treatment, yielded identical N-termini showing that the enzyme was monomerized subsequent to C-terminal tryptic cleavage. From our results, we conclude that the most C-terminal cysteine residue is involved in inter-subunit disulphide bonding as well as in the attachment of AChE to the membrane anchor. Furthermore, the C-terminal region in the primary structure provides an area for hydrophobic contacts between the different subunits and also between the subunits and the membrane anchor.
四聚体的可溶于去污剂的牛尾状核乙酰胆碱酯酶（AChE）在保留至少95%初始活性的条件下进行还原和烷基化处理。单独这种处理不会导致AChE单体化，也不会产生亲水性酶。然而，在十二烷基硫酸钠（SDS）存在的情况下，该酶会变成单体。在可逆抑制剂依酚氯铵存在下，用胰蛋白酶孵育AChE，使该酶具有亲水性，并导致产生具有催化活性的单体。在非还原条件下对该制剂进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS/PAGE）显示，亚基分子量仅略有下降。胰蛋白酶处理前后对该酶进行N端测序，得到相同的N端，表明该酶在C端经胰蛋白酶切割后单体化。从我们的结果可以得出结论，最C端的半胱氨酸残基参与亚基间二硫键的形成以及AChE与膜锚定物的连接。此外，一级结构中的C端区域为不同亚基之间以及亚基与膜锚定物之间的疏水接触提供了一个区域。
