Mutation and molecular evolution of squash family proteinase inhibitors

葫芦科植物种子中含有的南瓜家族抑制剂仅由28个氨基酸残基组成。因此，我们可以简单地通过追踪氨基酸序列之间的氨基酸替换来绘制系统发育树。从实验结果来看，苦瓜、葫芦、葫芦和东方腌瓜可作为抑制剂的纯化材料。通过对南瓜科抑制剂纯化方法的研究，明确了丙酮沉淀法、离子交换层析法和反相基质层析法是南瓜科抑制剂纯化的简便方法苦瓜中有4种抑制剂，葫芦中有2种抑制剂，3个来自葫芦和3个来自东方腌菜），其氨基酸序列测定如下：II RRCPRIWMECKRDSDCMAQCICVD-GFOGMCTI-III <ERGCPRILKQCKQDSDCPGECICMAHGFCGMCEI-I RICIWMECKRDSDCLAQCITI@VD-GHCG瓶内LLDTI-I <ERRCPRIYMECKHDSDCLADCVCLEHGICGLLDTI-II RRCPRIYMECKHDSDCLADCVCLEHGICGGourdLLTI-I <ERRCPRIYMECKHDSDCLADCVCLEHGICGGourdLLTI-I@CPRIYMECKHDSDCLADCVCLEHGICGGLLTI-II RRCPRIYMECKHDSDCLADCVCLEHGICGGLLTI-III ERRCPRIYMECKHDSDCLADCVCLEHGICGGGOri凉瓜CMCTI-I MCPKILMKCKQDSDCLLDCVCKE-GFCGCMCTI-II RMCPKILMKCKQDSDCLLDCVCKE-GFCGCMCTI-III <ERMCPKILMK CKQDSDCLLDCVCKE-GFCG

Squash family inhibitors which are included in cucurbitaceae seeds are composed of only 28 amino acid residues. Therefore, we could draw a phylogenetic tree simply by tracing substitutions of amino acids among amino acid sequences of inhibitors.For this purpose, many kinds of cucurbitaceous seeds were investigated for their proteinase inhibitory activities. From the results, bitter gourd, bottle gourd, gourd and oriental pickling melon were choosed for the purification of inhibitors. By the investigation of the purification method, it was clarified that acetone precipitation and chromatographies on ion-exchangers and reversed phase matrices were the simple and convenient method for the purification of squash family inhibitors.Total 12 inhibitors were isolated from 4 kinds of cucurbitaceous seeds (4 inhibitors from bitter gourd, 2 from bottle gourd, 3 froin gourd and 3 from oriental pickling inelon) and their amino acid sequences were determined as follows.Bitter gourdMCTI-I <ERRCPRILKQCKRDSDCPGECICMAHGFCGMCTI-II RRCPRIWMECKRDSDCMAQCICVD-GFOGMCTI-III <ERGCPRILKQCKQDSDCPGECICMAHGFCGMCEI-I RICPLIWMECKRDSDCLAQCICVD-GHCGBottle gourdLLDTI-I <ERRCPRIYMECKHDSDCLADCVCLEHGICGLLDTI-II RRCPRIYMECKHDSDCLADCVCLEHGICGGourdLLTI-I <ERRCPRIYMECKHDSDCLADCVCLEHGICGGLLTI-II RRCPRIYMECKHDSDCLADCVCLEHGICGGLLTI-III ERRCPRIYMECKHDSDCLADCVCLEHGICGGOriental pickling melonCMCTI-I MCPKILMKCKQDSDCLLDCVCKE-GFCGCMCTI-II RMCPKILMKCKQDSDCLLDCVCKE-GFCGCMCTI-III <ERMCPKILMKCKQDSDCLLDCVCKE-GFCG