A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis ... - UAM

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88 C. Aguirre et al. / Comparative Biochemistry and Physiology Part B 138 (2004) 81–89 inhibitors, such as, those present in tepary beans (Phaseolus acutifolius; Campos et al., 1997), amaranth (Amaranthus hypochondriacus; Valdes- ´ Rodrıguez ´ et al., 1993) and in quinoa (Chenopodium quinoa; Mendez-Moran, ´ ´ 1999). In the present report, a novel protease inhibitor was purified and characterized. The protein in this study was isolated from the seeds of chan, an ancient, not very widely used plant (Martınez, ´ 1959) known to be highly resistant to insect and fungal attack (Singh and Upadhyay, 1993; Fatope et al., 1995). HSTI shows some similarities with the Bowman–Birk family of inhibitors, such as its apparent molecular mass of 8700 Da, and an isoelectric point of 3.4. The inhibitor also had a high thermal stability, particularly at acidic pH, which decreased at higher pH values. However, its N-terminal sequence (18 amino acids) was not similar to other protease inhibitor sequences previously reported. Similarities were found in the circular dichroism spectrum with that of ubiquitin, whose molecular weight is also very similar. However, there is not enough information on the primary sequence of HSTI to establish structural similarities. The main characteristic of this new inhibitor is its specific activity against particular trypsin-like enzymes from insects. It was shown to recognize only two out of the seven tested enzymatic extracts from insects. One of them was the trypsin-like protease activity obtained from the insect P. truncatus, a very important pest of maize in Latin- America. In the last 10 years it has been the main constraint for maize production in central Africa. The other insect proteases inhibited by HSTI, were those from M. sexta. Acknowledgments We would like to express our gratitude to Elizabeth Mendiola-Olaya and Yolanda Rodrıguez ´ for their technical assistance. We also thank CON- ACYT for a Ph.D. fellowship to C.A., and for grant 41348-F; SIGHO project N8 19990201013 and Javier Luevano-Borroel ´ for his assistance in the insectary. References Alfonso-Rubi, J., Ortego, F., Castanera, ˜ P., Carbonero, P., Diaz, I., 2003. Transgenic expression of trypsin inhibitor CMe from barley in indica and japonica rice, confers resistance to the rice weevil Sitophilus oryzae. Transgenic Res. 12, 23–31. Altpeter, F., Diaz, I., McAuslane, H., Gaddour, K., Carbonero, P., Vasil, I.K., 1999. Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe. Mol. Breeding 5, 53–63. Andrade, M.A., Chacon, ´ P., Mertelo, J.J., Moran, ´ F., 1993. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 6, 383–390. 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