Artículos de revistas
Biochemical Characterization And N-terminal Sequences Of Two New Trypsin Inhibitors From Copaifera Langsdorffii Seeds
Registro en:
Protein Journal. , v. 20, n. 1, p. 1 - 7, 2001.
15723887
2-s2.0-52549129095
Autor
Silva J.A.
Macedo M.L.R.
Novelle J.C.
Marangoni S.
Institución
Resumen
Two new trypsin inhibitors, TDI-I and TDI-II, were purified from the seeds of the native Brazilian tree Copaifera iangsdorffii (Caesalpinoideae, Leguminosae). The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on trypsin-Sepharose, and reversed-phase (RP) HPEC. RP-HPEC yielded two forms (TDI-I and TDI-II), as confirmed by isoelectric focusing, with pi values between 7.0 and 8.1. The molecular mass of the TDI forms was 24 kDa based on FPEC gel filtration on Superdex 75. Under reducing conditions in tricine SDS-PAGE the molecular masses of TDI-I and TDI-II were 12 and 10 kDa, respectively. The Ki values were 1.1 and 1.2 nM for TDI-I and TDI-II, respectively, and there was no inhibitory effect on chymotrypsin. Amino acid analysis revealed high levels of aspartic acid, glutamic acid, serine, glycine, proline, and lysine but low levels of methionine and aromatic amino acids in both inhibitors; the calculated molecular masses were 11,456 and 10,008 for TDI-I and II, respectively. Based on the N-terminal sequences of TDI-I and TDI-II, TDI-I belongs to the Kunitz family of trypsin inhibitors, whereas TDI-II showed no homology to any other protein. This observation suggests that TDI-II belongs to a new inhibitor subclass of low-molecular mass proteins in the subfamily Caesalpinoideae. © 2001 Plenum Publishing Corporation. 20 1 1 7 Batista, I.F.C., Oliva, M.L.V., Araujo, M.S., Sampaio, M.U., Richardson, M., Fritz, H., Sampaio, C.A.M., (1996) Phytochemistry, 41, pp. 1017-1022 Birk, Y., Applebaum, Y., (1994) Arch. Latinoam. Nutr., 44, pp. 26S-30S Bradford, M.M., (1976) Anal. Biochem., 72, pp. 248-254 Cronquist, A., (1988) The Classification of Flowering Plants, p. 396. , Houghton Mifflin, Boston Di Ciero, L., Oliva, M.L.V., Torquato, R., Kohler, P., Weder, J.K.P., Novello, J.C., Sampaio, C.A.M., Marangoni, S., (1998) J. Protein Chem., 17, pp. 827-834 Erlanger, B.F., Kolowsky, N., Cohen, N., (1961) Arch. Biochem. Biophys., 95, pp. 271-278 Ferrasson, F., Quillien, L., Gueguen, J., (1997) J. Agric. Food Chem., 45, pp. 127-131 Freitas, S.M., Mello, L.V., Suva, M.C.M., Vriend, G., Neshich, G., Ventura, M., (1997) FEBS Lett., 409, pp. 121-127 Hayashi, K., Takehisa, T., Hamato, N., Takano, R., Hara, S., Miyata, T., Kalo, H., (1994) J. Biochem., 116, pp. 1013-1018 Heinrikson, R.L., Meridith, S.C., (1984) Anal. Biochem., 136, pp. 60-65 Kalume, D.F., Sousa, M.V., Morhy, L., (1995) J. Protein Chem., 14, pp. 685-693 Kennedy, A.R., (1998) Pharmacol. Ther., 78, pp. 167-209 Kim, S.H., Hara, S., Hase, S., Ikenaka, T., Tdoa, H., Kitamura, K., Kaizuma, N., (1985) J. Biochem., 98, pp. 435-448 Kinight, C.G., (1986) Protease Inhibitors, pp. 23-51. , Barret, A. J., and Salvesen, G., eds., Eisevier, Amsterdam Kunitz, M., (1947) J. Gen. Physiol., 30, pp. 291-310 Laemmli, U.K., (1970) Nature, 227, pp. 680-685 Liener, I.F., (1995) J. Nutr., 125, pp. 744-759 Lorenzi, H., (1992) Arvores Brasileiras-Manual de Identificação e Cultiva de Plantas Nativas do Brasil, p. 1. , Editora Platarum LTDA Macedo, M.L.R., Xavier-Filho, J., (1992) J. Sci. Food Agric., 58, pp. 55-58 Marangoni, S., Toyama, M.H., Arantes, E.C., Giglio, J.R., Silva, C.A., Carneiro, E.C., Gonçalves, A.A., Oliveira, B., (1995) Biochim. Biophys. Acta, 1243, pp. 309-314 Norioka, S., Omichi, K., Ikenaka, T., (1982) J. Biochem., 91, pp. 1427-1434 Odani, S., Ikenaka, T., (1973) J. Biochem., 74, pp. 697-715 Oliva, M.L.V., Souza-Pinto, J.C., Batista, I.F.C., Araujo, M.S., Silveira, V.F., Auerswald, E.A., Mentele, R., Sampaio, C.A.M., (2000) Biochim. Biophys. Acta, 1477, pp. 64-74 Pando, L.A., Di Ciero, L., Novelle, J.C., Oliveira, B., Weder, J.K.P., Marangoni, S., (1999) IUBMB Life, 48, pp. 519-523 Richardson, M., (1991) Methods in Plant Biochemistry, Vol. 5, Amino Acids Proteins and Nucleic Acids, 5. , Rogers, L. J., ed., Academic Press, San Diego, California Schagger, H., Jagow, G.V., (1987) Anal. Biochem., 166, pp. 368-379 Sreerama, Y.N., Das, J.R., Rao, D.R., Gowda, L.R., (1997) J. Food Biochem., 21, pp. 461-477 Tamir, S., Kadner, S.S., Kaltz, T.H., (1996) Endocrinology, 127, pp. 1319-1323 Tanaka, A.S., Sampaio, M.U., Marangoni, S., Oliveira, B., Novello, J.C., Oliva, M.L.V., Fink, E., Sampaio, C.A.M., (1997) J. Biol. Chem., p. 378 Terada, S., Fujimura, S., Katayama, H., Nagasawa, M., Kimoto, E., (1994) J. Biochem., 115, pp. 392-396 Uriel, J., Berges, J., (1968) Nature, 218, pp. 578-580 Yoshikawa, M., Kiyohara, T., Iwasaki, T., Ishii, Y., Kimura, N., (1979) Agric. Biol. Chem., 43, pp. 787-793