Nellaiappan K, Sugumaran M
Department of Biology, University of Massachusetts at Boston 02125, USA.
Comp Biochem Physiol B Biochem Mol Biol. 1996 Jan;113(1):163-8. doi: 10.1016/0305-0491(95)02047-0.
Melanization, and hence the participation of phenoloxidase, in defense mechanism of arthropods is well established. However, in the living fossil, horseshoe crab, it has been claimed that the prophenoloxidase system widely found in the hemolymph of most arthropods is absent. On the contrary, we present evidence for the presence of a prophenoloxidase system in the hemolymph of Limulus and a method to study its activation. Activation of prophenoloxidase was achieved by treatment with either the anionic detergent, SDS, or the cationic detergent, cetylpyridinium chloride. The detergents seemed to bind to the proenzyme below their critical micellar concentration and induce conformational changes that cause the activation of prophenoloxidase. In addition, a number of fatty acids and phospholipids also activated the prophenoloxidase. Proteases such as trypsin activated the enzyme only marginally. The approximate molecular weight of the proenzyme was found to be 70,000. Substrate specificity studies, product analysis and inhibition experiments revealed that the Limulus enzyme is a typical o-diphenoloxidase. The possible reasons for the failure to detect the phenoloxidase activity by earlier workers are discussed.
黑化作用以及由此而来的酚氧化酶参与节肢动物的防御机制已得到充分证实。然而，对于活化石鲎，有观点认为其缺乏大多数节肢动物血淋巴中广泛存在的前酚氧化酶系统。相反，我们提供了鲎血淋巴中存在前酚氧化酶系统的证据以及一种研究其激活的方法。通过用阴离子洗涤剂十二烷基硫酸钠（SDS）或阳离子洗涤剂十六烷基氯化吡啶处理可实现前酚氧化酶的激活。这些洗涤剂似乎在低于其临界胶束浓度时与酶原结合，并诱导构象变化从而导致前酚氧化酶的激活。此外，一些脂肪酸和磷脂也能激活前酚氧化酶。诸如胰蛋白酶之类的蛋白酶对该酶的激活作用很微弱。发现该酶原的近似分子量为70,000。底物特异性研究、产物分析和抑制实验表明，鲎的这种酶是典型的邻二酚氧化酶。文中还讨论了早期研究人员未能检测到酚氧化酶活性的可能原因。
