Sritanyarat Wannapa, Pearce Gregory, Siems William F, Ryan Clarence A, Wititsuwannakul Rapepun, Wititsuwannakul Dhirayos
Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Phytochemistry. 2006 Aug;67(15):1644-50. doi: 10.1016/j.phytochem.2005.12.016. Epub 2006 Jan 24.
Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.
已从巴西橡胶树（Hevea brasiliensis clone RRIM 600）乳胶的C血清中分离出三种同功抑制物，并将其纯化至同质，分别命名为HPI - 1、HPI - 2a和HPI - 2b。这三种抑制物具有相同的氨基酸序列（69个残基），但三种形式的质量分别测定为14,893±10、7757±5和7565±5，这表明在乳胶采集过程中蛋白质发生了翻译后修饰。一种加合物可被还原剂去除，经测定为谷胱甘肽，而另一种加合物不能被还原剂去除且尚未被鉴定。抑制蛋白的N末端被乙酰化的丙氨酸封闭，但通过对来自内肽酶C消化片段的Edman降解和质谱对去封闭抑制物进行完整氨基酸序列分析，证实这三种同功抑制物源自单一蛋白质。该蛋白质的氨基酸序列与GenBank中报道的cDNA推导序列在两个位置不同。编码该抑制物的基因是创伤诱导型的，是蛋白酶抑制剂马铃薯抑制剂I家族的成员。该抑制物强烈抑制枯草杆菌蛋白酶A，微弱抑制胰蛋白酶，不抑制胰凝乳蛋白酶。反应位点P(1)和P(1)'处的氨基酸残基分别确定为Gln45和Asp46，这是马铃薯抑制剂I家族成员反应位点上很少报道的残基。氨基酸序列比较表明，HPI同功抑制物与马铃薯抑制剂I家族的抑制剂具有33%至55%的同一性（50 - 74%的相似性）。这些同功抑制物的特性表明它们可能在乳胶中对病原体和/或食草动物起到防御作用。
