Han Yaoping, Yu Haining, Yang Xinbo, Rees Huw H, Liu Jingze, Lai Ren
College of Life Sciences School of Heibei Normal University, Shijiazhuang, Hebei, 050016 China.
Comp Biochem Physiol B Biochem Mol Biol. 2008 Jan;149(1):58-62. doi: 10.1016/j.cbpb.2007.08.003. Epub 2007 Aug 14.
By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.
通过葡聚糖凝胶G-50凝胶过滤、Resource Q阴离子交换和C4反相高效液相色谱法，从臭蛙（滇蛙）的卵中鉴定并纯化出一种蛋白酶抑制剂蛋白（滇蛙丝氨酸蛋白酶抑制剂）。经SDS-PAGE分析，该蛋白在分子量标记14.4 kDa附近呈现单一条带。通过基质辅助激光解吸电离飞行时间质谱分析再次确认了该抑制剂蛋白的均一性及其分子量。基质辅助激光解吸电离飞行时间质谱分析给出该抑制剂蛋白的质荷比为14422.26，与SDS-PAGE结果吻合良好。该蛋白是一种丝氨酸蛋白酶抑制剂，可靶向多种蛋白酶，包括胰蛋白酶、弹性蛋白酶和枯草杆菌蛋白酶。滇蛙丝氨酸蛋白酶抑制剂抑制了胰蛋白酶、弹性蛋白酶和枯草杆菌蛋白酶的蛋白水解活性。它对胰蛋白酶、弹性蛋白酶和枯草杆菌蛋白酶的抑制常数（K(i)）分别为6.2×10(-8) M、2.7×10(-7) M和2.2×10(-8) M。这种丝氨酸蛋白酶抑制剂对革兰氏阳性菌枯草芽孢杆菌（ATCC 6633）具有抑菌作用。提示滇蛙丝氨酸蛋白酶抑制剂可能在抵抗害虫或病原体入侵中发挥防御作用。这是关于两栖动物卵中丝氨酸蛋白酶抑制剂及其直接防御作用的首次报道。
