Fan Tingjun, Zhang Yanan, Yang Lingling, Yang Xiuxia, Jiang Guojian, Yu Miaomiao, Cong Rishan
Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, Qingdao, PR China.
Comp Biochem Physiol B Biochem Mol Biol. 2009 Feb;152(2):144-9. doi: 10.1016/j.cbpb.2008.10.010. Epub 2008 Nov 5.
To determine effective activators of crab hemocyanin (Hc) and the properties of Hc-derived phenoloxidase (HdPO), Hc, for the first time, was purified from hemolymph of Charybdis japonica, and the properties of activated HdPO were studied by using L-DOPA as a substrate. Three distinct subunits were isolated, and each had a molecular mass of about 80, 75 and 70 kDa, respectively. SDS and HLS were much effective in conversion of Hc into HdPO whose PO activity was optimal at pH 7.0 and temperature of 40 degrees C. The Km value of the HdPO was 2.90 mM for L-DOPA and 7.33 mM for tyrosine. The PO activity of HdPO was most sensitive to 1-phenyl-2-thiourea, cysteine and ascorbic acid, and much sensitive to thio urea and sodium sulfite. Based on its inhibition characteristics and the substrate specificity, this HdPO could be classified as a kind of tyrosinase-type phenoloxidase. The PO activity of HdPO was also strongly inhibited by Cu(2+), Zn(2+), ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu(2+) on DETC-inhibited PO activity, indicate that the HdPO is a kind of copper-containing metalloenzyme. All these imply that the Hc, as an oxygen carrier, can be activated to have PO activities by SDS or HLS, and the activated HdPO has the properties of a tyrosinase-type copper-containing phenoloxidase. This study makes us to understand more easily the multifunctions of crustacean Hc in oxygen carrier and melaninization at certain stresses in host defence as well.
为了确定蟹血蓝蛋白（Hc）的有效激活剂以及血蓝蛋白衍生的酚氧化酶（HdPO）的特性，首次从日本蟳的血淋巴中纯化出Hc，并以L - 多巴为底物研究了激活的HdPO的特性。分离出三个不同的亚基，每个亚基的分子量分别约为80、75和70 kDa。SDS和HLS在将Hc转化为HdPO方面非常有效，其酚氧化酶活性在pH 7.0和40℃温度下最佳。HdPO对L - 多巴的Km值为2.90 mM，对酪氨酸的Km值为7.33 mM。HdPO的酚氧化酶活性对1 - 苯基 - 2 - 硫脲、半胱氨酸和抗坏血酸最敏感，对硫脲和亚硫酸钠也很敏感。基于其抑制特性和底物特异性，这种HdPO可归类为一种酪氨酸酶型酚氧化酶。HdPO的酚氧化酶活性也受到Cu(2+)、Zn(2+)、乙二胺四乙酸（EDTA）和二乙基二硫代氨基甲酸盐（DETC）的强烈抑制。EDTA、DETC和一些金属离子的实验结果，结合Cu(2+)对DETC抑制的酚氧化酶活性的完美恢复作用，表明HdPO是一种含铜金属酶。所有这些都表明，作为氧载体的Hc可以被SDS或HLS激活而具有酚氧化酶活性，并且激活的HdPO具有酪氨酸酶型含铜酚氧化酶的特性。这项研究使我们更容易理解甲壳类动物Hc在氧载体以及宿主防御中特定应激下黑色素形成方面的多种功能。
