PROENZYME OF MANDUCA-SEXTA PHENOL OXIDASE - PURIFICATION, ACTIVATION, SUBSTRATE-SPECIFICITY OF THE ACTIVE ENZYME, AND MOLECULAR-CLONING
PROENZYME OF MANDUCA-SEXTA PHENOL OXIDASE - PURIFICATION, ACTIVATION, SUBSTRATE-SPECIFICITY OF THE ACTIVE ENZYME, AND MOLECULAR-CLONING
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DOI：
10.1073/pnas.92.17.7764
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发表时间：
1995-08-15
影响因子：
11.1
通讯作者：
LAW, JH
中科院分区：
文献类型：
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作者：
HALL, M;SCOTT, T;LAW, JH
苯酚氧化酶 (PO) 作为酶原（苯酚氧化酶原，pro-PO）从天蛾幼虫的血淋巴中分离出来，并纯化至均质。 Pro-PO 在凝胶过滤上表现出 130,000 的 M(r)，在 SDS/PAGE 以及尺寸排阻 HPLC 上表现出两条表观 M(r) 接近 100,000 的条带。 pro-PO 的激活是通过角质层蛋白酶的特异性蛋白水解或通过浓度低于临界胶束浓度的洗涤剂氯化十六烷基吡啶来实现的。从幼虫血细胞 cDNA 文库中获得了 M. sexta pro-PO 的 cDNA 克隆。该克隆编码大约 80,000 Da 的多肽，包含两个铜结合位点，与节肢动物的 PO、血蓝蛋白和储存蛋白具有高度的序列相似性。 M. sexta pro-PO 与其他节肢动物 pre-PO 一起含有一小段氨基酸，其序列与 α-巨球蛋白和补体蛋白 C3 和 C4 的硫羟酸酯区域相似。
Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximate to 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximate to 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. sexta pro-PO, together with other arthropod pre-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.