Rassam Maysoon, Laing William A
Gene Technologies, The Horticultural and Food Research Institute of New Zealand, PB 92169, Auckland, New Zealand.
Phytochemistry. 2004 Jan;65(1):19-30. doi: 10.1016/j.phytochem.2003.09.019.
Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwifruit after inactivation of the abundant cortex cysteine proteinase actinidain. One major (KCPI1) and four minor cystatins were identified from Actinidia deliciosa ripe mature kiwifruit cortex as well as a seed KCPI from A. chinensis. The predominant cortex cystatin, KCPI1, inhibited clan CA, family C1 (papain family) cysteine proteinases (papain, chymopapain, bromelain, ficin, human cathepsins B, H and L, actinidain and the house dust mite endopeptidase 1), while cysteine proteinases belonging to other families, [clostripain (C11), streptopain (C10) and calpain (C2)] were not inhibited. Inhibition constants (K(I)) ranged between 0.001 nM for cathepsin L and 0.98 nM for endopeptidase 1. The K(I) (14 nM) for KCPI1 inhibiting actinidain is at least 2 orders of magnitude higher than for other plant proteinases measured. The cortex KCPI1 and a seed KCPI purified from seeds had the same N-terminal sequence (VAAGGWRPIESLNSAEVQDV). BLAST-matching the peptide sequence against an in-house generated Actinidia EST database, identified 81 cDNAs that exactly matched the measured KCPI1 peptide sequence. Peptide sequences of two other cortex KCPIs each exactly matched a predicted peptide sequence of a cDNA from kiwifruit. The predicted peptide sequence of KCPI1 of 116 amino acids encodes a signal peptide and does not contain cysteine. Without the signal peptide (mature protein), KCPI1 has a molecular mass of approximately 11 kDa, possesses the consensus sequence characteristic for the phytocystatins and shows the highest homology to a cystatin from Citrusxparadisi (52% identity). This is the first report of phytocystatins from the Ericales.
在丰富的皮层半胱氨酸蛋白酶猕猴桃蛋白酶失活后，从猕猴桃的皮层和种子中纯化出了猕猴桃半胱氨酸蛋白酶抑制剂（KCPIs）。从美味猕猴桃成熟果实的皮层中鉴定出一种主要的（KCPI1）和四种次要的半胱氨酸蛋白酶抑制剂，以及中华猕猴桃种子中的一种种子KCPIs。皮层中主要的半胱氨酸蛋白酶抑制剂KCPI1可抑制CA家族C1亚家族（木瓜蛋白酶家族）的半胱氨酸蛋白酶（木瓜蛋白酶、糜木瓜蛋白酶、菠萝蛋白酶、无花果蛋白酶、人组织蛋白酶B、H和L、猕猴桃蛋白酶以及屋尘螨内肽酶1），而属于其他家族的半胱氨酸蛋白酶[梭菌蛋白酶（C11）、链霉蛋白酶（C10）和钙蛋白酶（C2）]则不受抑制。抑制常数（K（I））范围在组织蛋白酶L的0.001 nM至内肽酶1的0.98 nM之间。KCPI1抑制猕猴桃蛋白酶的K（I）（14 nM）比所测定的其他植物蛋白酶至少高2个数量级。从种子中纯化出的皮层KCPI1和种子KCPIs具有相同的N端序列（VAAGGWRPIESLNSAEVQDV）。将该肽序列与内部生成的猕猴桃EST数据库进行BLAST比对，鉴定出81个与所测KCPI1肽序列完全匹配的cDNA。另外两种皮层KCPIs的肽序列分别与猕猴桃cDNA的预测肽序列完全匹配。KCPI1的116个氨基酸的预测肽序列编码一个信号肽，且不含半胱氨酸。去除信号肽（成熟蛋白）后，KCPI1的分子量约为11 kDa，具有植物半胱氨酸蛋白酶抑制剂的共有序列特征，与来自葡萄柚的半胱氨酸蛋白酶抑制剂具有最高的同源性（同一性为52%）。这是杜鹃花目植物半胱氨酸蛋白酶抑制剂的首次报道。
