Walsh T A, Strickland J A
Biotechnology Laboratory, DowElanco, Indianapolis, Indiana 46268-1054, USA.
Plant Physiol. 1993 Dec;103(4):1227-34. doi: 10.1104/pp.103.4.1227.
The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.
在马铃薯（Solanum tuberosum L.）块茎细胞中发现的蛋白质晶体由一条85-kD的单一多肽组成。这种多肽是木瓜蛋白酶和其他半胱氨酸蛋白酶的抑制剂，能够同时结合多个蛋白酶分子（P. Rodis，J.E. Hoff [1984] Plant Physiol 74: 907-911）。我们对这种不同寻常的抑制剂进行了更详细的表征。用马铃薯木瓜蛋白酶抑制剂对木瓜蛋白酶活性进行滴定表明，每个抑制剂分子有八个木瓜蛋白酶结合位点。木瓜蛋白酶抑制的抑制常数（Ki）值为0.1 nM。用胰蛋白酶处理该抑制剂导致85-kD多肽断裂成一个32-kD多肽和五个10-kD多肽。32-kD和10-kD片段都保留了有效抑制木瓜蛋白酶的能力（对木瓜蛋白酶的Ki值分别为0.5和0.7 nM），木瓜蛋白酶结合的摩尔化学计量比分别为2至3:1和1:1。其他非特异性蛋白酶，如胰凝乳蛋白酶、枯草杆菌蛋白酶Carlsberg、嗜热菌蛋白酶和蛋白酶K，也将85-kD抑制剂多肽切割成功能性的22-kD和几个10-kD片段。通过反相高效液相色谱法纯化用胰蛋白酶消化马铃薯木瓜蛋白酶抑制剂得到的片段，并获得每个片段的N端氨基酸序列。这些序列的比较表明，片段具有高度同源性但并不相同。这些序列与半胱氨酸蛋白酶抑制剂胱抑素超家族成员的N端同源。因此，该抑制剂似乎由八个串联的胱抑素结构域通过蛋白水解敏感连接区相连组成。我们将该抑制剂称为马铃薯多胱抑素（PMC）。通过免疫印迹分析和木瓜蛋白酶抑制活性测定，发现PMC在马铃薯叶片中含量很高（高达0.6微克/克鲜重组织），在诱导与植物防御相关的其他蛋白酶抑制剂积累的条件下，它在叶片中积累。PMC可能具有类似的防御作用，例如保护植物免受利用半胱氨酸蛋白酶进行膳食蛋白质消化的植食性昆虫的侵害。
