Lee Kum Young, Zhang Rong, Kim Moon Suk, Park Ji Won, Park Ho Young, Kawabata Shun-ichiro, Lee Bok Luel
College of Pharmacy, Pusan National University, Jangjeon Dong, Korea.
Eur J Biochem. 2002 Sep;269(17):4375-83. doi: 10.1046/j.1432-1033.2002.03155.x.
To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+. This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+, a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and beta-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor.
为阐明昆虫前酚氧化酶（pro-PO）系统的生化激活机制，我们从黄粉虫幼虫的血淋巴中纯化出一种45 kDa的蛋白质并使其达到同质，然后克隆了其cDNA。这种45 kDa蛋白质的整体结构类似于果蝇的伪装丝氨酸蛋白酶同源物，后者是果蝇肌肉发育中的一个重要成分。这种黄粉虫伪装样丝氨酸蛋白酶同源物（Tm-mas）在C端区域含有一个胰蛋白酶样丝氨酸蛋白酶结构域，但活性位点三联体中的丝氨酸被甘氨酸取代，在N端区域有一个二硫键结合结构域。当将纯化的45 kDa Tm-mas与含有pro-PO和其他pro-PO激活因子的CM-Toyopearl洗脱液一起孵育时，所产生的酚氧化酶（PO）活性显示与Ca2+无关。这表明纯化的45 kDa Tm-mas是pro-PO激活因子的一种活化形式。当PO活性不明显时，在血淋巴中检测到了55 kDa的Tm-mas酶原形式。然而，当黄粉虫血淋巴与Ca2+一起孵育时，一种79 kDa的黄粉虫pro-PO和55 kDa的酶原Tm-mas分别转化为76 kDa的PO和45 kDa的Tm-mas，并具有可检测到的PO活性。此外，当黄粉虫血淋巴与Ca2+和β-1,3-葡聚糖一起孵育时，pro-PO向PO的转化以及55 kDa的酶原Tm-mas向45 kDa蛋白质的转化比仅存在Ca2+时更快。这些结果表明，通过有限的蛋白水解作用切割55 kDa 的Tm-mas酶原对于PO活性是必要的，并且Tm-mas是一种pro-PO激活辅因子。
