Kondo H, Abe K, Nishimura I, Watanabe H, Emori Y, Arai S
Department of Agricultural Chemistry, University of Tokyo, Japan.
J Biol Chem. 1990 Sep 15;265(26):15832-7.
Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.
水稻半胱氨酸蛋白酶抑制剂（水稻半胱氨酸蛋白酶抑制剂-I）是水稻种子中的一种蛋白质类半胱氨酸蛋白酶抑制剂（胱抑素），是首个明确的植物源胱抑素。在本研究中，我们通过用水稻半胱氨酸蛋白酶抑制剂-I cDNA探针筛选，分离出了水稻种子中一种新型胱抑素（水稻半胱氨酸蛋白酶抑制剂-II）的cDNA克隆。新分离的cDNA克隆编码107个氨基酸残基，其序列与水稻半胱氨酸蛋白酶抑制剂-I的序列相似（同一性约为55%）。这些水稻半胱氨酸蛋白酶抑制剂没有二硫键，因此可归类为I类胱抑素；然而，其氨基酸序列与II类成员的序列更相似，而非I类成员。水稻半胱氨酸蛋白酶抑制剂-I和-II在两个方面有显著差异：1）它们对半胱氨酸蛋白酶的特异性；2）其mRNA在水稻种子成熟阶段的表达模式。水稻半胱氨酸蛋白酶抑制剂-I抑制木瓜蛋白酶的效果（Ki 3.0×10⁻⁸ M）比抑制组织蛋白酶H的效果（Ki 0.79×10⁻⁶ M）更好，而水稻半胱氨酸蛋白酶抑制剂-II抑制组织蛋白酶H的效果（Ki 1.0×10⁻⁸ M）比抑制木瓜蛋白酶的效果（Ki 0.83×10⁻⁶ M）更好。水稻半胱氨酸蛋白酶抑制剂-I的mRNA在开花后2周表达量最高，在成熟种子中未检测到，而水稻半胱氨酸蛋白酶抑制剂-II的mRNA在整个成熟阶段持续表达，在成熟种子中可清晰检测到。使用针对水稻半胱氨酸蛋白酶抑制剂-II的抗体进行的蛋白质印迹分析表明，与水稻半胱氨酸蛋白酶抑制剂-I一样，水稻半胱氨酸蛋白酶抑制剂-II也存在于成熟水稻种子中。因此，这两种水稻半胱氨酸蛋白酶抑制剂被认为参与了由不同蛋白酶引起的蛋白水解的调控。
