Mutation and molecular evolution of squash family proteinase inhibitors

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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