从野豌豆属（Rhynchosia sublobata (Schumach.) Meikle）的种子中纯化和表征 Bowman-Birk 和 Kunitz 同工酶抑制剂，野豌豆属是豇豆的野生近缘种。
Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea.
机构信息
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, Telangana, India.
Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, Telangana, India.
出版信息
Phytochemistry. 2019 Mar;159:159-171. doi: 10.1016/j.phytochem.2018.12.018. Epub 2019 Jan 9.
Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC = 24 ng) and RsKI (IC = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.
野花生Rhynchosia sublobata 是豇豆的野生近缘种，拥有防御性蛋白酶/蛋白酶抑制剂（PI）。通过使用 2-D 凝胶电泳和 Edman 降解方法对种子中分离出的胰蛋白酶特异性 PI（RsPI）进行柱层析分离，鉴定出 Rhynchosia sublobata 同时拥有 Bowman-Birk 同工型抑制剂（RsBBI）和 Kunitz 同工型抑制剂（RsKI）。基于它们在 TCA 和醋酸盐缓冲液中的不同溶解度，开发了一种从 RsPI 中快速分离 RsBBI 和 RsKI 的方法。MALDI-ISD 对 RsBBI 和 RsKI 的 N 端测序确定了 Rhynchosia sublobata 中存在 Bowman-Birk 和 Kunitz 同工型抑制剂。RsBBI（9216Da）和 RsKI（19412Da）分别通过 Western 印迹与抗 BBI 抗体和 MALDI-TOF 肽质量指纹分析显示出自身缔合模式。RsBBI 和 RsKI 的生化、生物物理和杀虫性质存在显著差异。RsBBI 抑制胰蛋白酶（Ki=128.5±4.5nM）和糜蛋白酶（Ki=807.8±23.7nM）的活性，而 RsKI（Ki=172.0±9.2nM）以非竞争性模式抑制胰蛋白酶的活性。RsBBI 的胰蛋白酶抑制剂（TI）和糜蛋白酶抑制剂（CI）活性在 100°C 下稳定。但是，RsBBI 在 3mM DTT 还原下完全失去其 TI 和 CI 活性。相反，RsKI 在 100°C 加热时失去 TI 活性，但在 3mM DTT 存在下保留超过 60%的 TI 活性。RsBBI 和 RsKI 的 CD 光谱研究表明它们的二级结构元件按以下顺序排列：无规卷曲>β-折叠/β-转角>α-螺旋。然而，RsKI 显示出可逆的变性中点（Tm）为 75°C。此外，RsBBI（IC=24ng）和 RsKI（IC=59ng）对 Achaea janata（AjGPs）和 Helicoverpa armigera（HaGPs）的胰蛋白酶样肠道蛋白酶的显著抑制活性表明它们分别作为管理 Achaea janata 和 Helicoverpa armigera 的潜在生物分子。
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