Cheng Yuhui, Jiang Jingwei, Dong Ying, Zhou Zunchun
Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
Fish Shellfish Immunol. 2015 Nov;47(1):117-21. doi: 10.1016/j.fsi.2015.08.020. Epub 2015 Aug 24.
Three proteins with PO-like activities in the coelomocytes of sea urchin Strongylocentrotus nudus were identified using electrophoretic method and named as SnPO1, SnPO2 and SnPO3 according to their molecular mass from high to low. The SnPOs were characterized for substrate specificity and the effects of temperature, pH, divalent metal ions and inhibitors on PO activities. They showed oxidative activities to L-3,4-dihydroxyphenylalanine. (l-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine, which illustrated the three proteins had laccase-like PO activities. The optimum temperature for the activities of SnPO1, SnPO2 and SnPO3 was 75 °C, 70 °C, 40 °C, and the optimum pH was 7.0, 9.0, 8.0, respectively. The SnPOs were notably activated after being incubated in boiled water for 60 min, suggesting that the three proteins are thermophilic. The activity of SnPO1 was greatly enhanced by Cu(2+), Mn(2+) and Fe(2+) and inhibited by Pb(2+), Cd(2+), EDTA, DETC, sodium sulfite and ascorbic acid, but SnPO2 and SnPO3 were not obviously affected by Pb(2+) and Cd(2+), suggesting the three proteins are copper-containing, and the catalytic properties of SnPO1 might be different from those of SnPO2 and SnPO3. Taken together, SnPO1, SnPO2 and SnPO3 might play different roles in the immune and physiological processes of S. nudus.
利用电泳方法在光棘球海胆体腔细胞中鉴定出三种具有酚氧化酶（PO）样活性的蛋白质，并根据其分子量从高到低分别命名为SnPO1、SnPO2和SnPO3。对这些SnPOs的底物特异性以及温度、pH、二价金属离子和抑制剂对PO活性的影响进行了表征。它们对L-3,4-二羟基苯丙氨酸（L-DOPA）、多巴胺和对苯二酚表现出氧化活性，但不能氧化酪氨酸，这说明这三种蛋白质具有漆酶样PO活性。SnPO1、SnPO2和SnPO3活性的最适温度分别为75℃、70℃、40℃，最适pH分别为7.0、9.0、8.0。将SnPOs在沸水中孵育60分钟后，其活性显著增强，表明这三种蛋白质是嗜热的。SnPO1的活性被Cu(2+)、Mn(2+)和Fe(2+)大大增强，被Pb(2+)、Cd(2+)、EDTA、DETC、亚硫酸钠和抗坏血酸抑制，但SnPO2和SnPO3不受Pb(2+)和Cd(2+)的明显影响，这表明这三种蛋白质是含铜的，并且SnPO1的催化特性可能与SnPO2和SnPO3不同。综上所述，SnPO1、SnPO2和SnPO3可能在光棘球海胆的免疫和生理过程中发挥不同作用。
