Heath R L, Barton P A, Simpson R J, Reid G E, Lim G, Anderson M A
Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria, Australia.
Eur J Biochem. 1995 May 15;230(1):250-7. doi: 10.1111/j.1432-1033.1995.tb20558.x.
A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease-sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.
一个编码40.3 kDa丝氨酸蛋白酶抑制剂（PI）前体的基因在观赏烟草（Nicotiana alata）的柱头中高水平表达。该前体在体内经蛋白水解加工，释放出5个约6 kDa的同源蛋白酶抑制剂以及两个侧翼肽段。这5种PI已从柱头中纯化出来，并通过N端测序、电喷雾质谱以及对胰凝乳蛋白酶或胰蛋白酶的抑制活性进行了鉴定。其中一种PI抑制胰凝乳蛋白酶，另外4种对胰蛋白酶活性最强。切割发生在前体分子中重复6次的连接区（EEKKND）。在植物中，最初的切割可能发生在天冬酰胺和天冬氨酸残基之间，随后的修剪形成参差不齐的末端。在体外，蛋白酶敏感的连接区被内蛋白酶Asp-N、Glu-C和Lys-C选择性切割，释放出对进一步蛋白水解消化具有抗性的完全活性的约6 kDa的PI。由重组杆状病毒产生的前体对胰凝乳蛋白酶的抑制作用比对胰蛋白酶更有效。2.6个胰蛋白酶分子/1个前体分子的化学计量比表明，需要加工才能激活或暴露所有4个胰蛋白酶抑制位点。
