Biochemical Characterization And N-terminal Sequences Of Two New Trypsin Inhibitors From Copaifera Langsdorffii Seeds

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.20117Batista, 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. 1017-1022Birk, Y., Applebaum, Y., (1994) Arch. Latinoam. Nutr., 44, pp. 26S-30SBradford, M.M., (1976) Anal. Biochem., 72, pp. 248-254Cronquist, A., (1988) The Classification of Flowering Plants, p. 396. , Houghton Mifflin, BostonDi 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. 827-834Erlanger, B.F., Kolowsky, N., Cohen, N., (1961) Arch. Biochem. Biophys., 95, pp. 271-278Ferrasson, F., Quillien, L., Gueguen, J., (1997) J. Agric. Food Chem., 45, pp. 127-131Freitas, S.M., Mello, L.V., Suva, M.C.M., Vriend, G., Neshich, G., Ventura, M., (1997) FEBS Lett., 409, pp. 121-127Hayashi, K., Takehisa, T., Hamato, N., Takano, R., Hara, S., Miyata, T., Kalo, H., (1994) J. Biochem., 116, pp. 1013-1018Heinrikson, R.L., Meridith, S.C., (1984) Anal. Biochem., 136, pp. 60-65Kalume, D.F., Sousa, M.V., Morhy, L., (1995) J. Protein Chem., 14, pp. 685-693Kennedy, A.R., (1998) Pharmacol. Ther., 78, pp. 167-209Kim, S.H., Hara, S., Hase, S., Ikenaka, T., Tdoa, H., Kitamura, K., Kaizuma, N., (1985) J. Biochem., 98, pp. 435-448Kinight, C.G., (1986) Protease Inhibitors, pp. 23-51. , Barret, A. J., and Salvesen, G., eds., Eisevier, AmsterdamKunitz, M., (1947) J. Gen. Physiol., 30, pp. 291-310Laemmli, U.K., (1970) Nature, 227, pp. 680-685Liener, I.F., (1995) J. Nutr., 125, pp. 744-759Lorenzi, H., (1992) Arvores Brasileiras-Manual de Identificação e Cultiva de Plantas Nativas do Brasil, p. 1. , Editora Platarum LTDAMacedo, M.L.R., Xavier-Filho, J., (1992) J. Sci. Food Agric., 58, pp. 55-58Marangoni, 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. 309-314Norioka, S., Omichi, K., Ikenaka, T., (1982) J. Biochem., 91, pp. 1427-1434Odani, S., Ikenaka, T., (1973) J. Biochem., 74, pp. 697-715Oliva, 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. 64-74Pando, L.A., Di Ciero, L., Novelle, J.C., Oliveira, B., Weder, J.K.P., Marangoni, S., (1999) IUBMB Life, 48, pp. 519-523Richardson, M., (1991) Methods in Plant Biochemistry, Vol. 5, Amino Acids Proteins and Nucleic Acids, 5. , Rogers, L. J., ed., Academic Press, San Diego, CaliforniaSchagger, H., Jagow, G.V., (1987) Anal. Biochem., 166, pp. 368-379Sreerama, Y.N., Das, J.R., Rao, D.R., Gowda, L.R., (1997) J. Food Biochem., 21, pp. 461-477Tamir, S., Kadner, S.S., Kaltz, T.H., (1996) Endocrinology, 127, pp. 1319-1323Tanaka, 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. 378Terada, S., Fujimura, S., Katayama, H., Nagasawa, M., Kimoto, E., (1994) J. Biochem., 115, pp. 392-396Uriel, J., Berges, J., (1968) Nature, 218, pp. 578-580Yoshikawa, M., Kiyohara, T., Iwasaki, T., Ishii, Y., Kimura, N., (1979) Agric. Biol. Chem., 43, pp. 787-79

Enzymatic Characterization Of A Novel Phospholipase A2 From Crotalus Durissus Cascavella Rattlesnake (maracambóia) Venom

The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reversephase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40oC. The C. d. cascavella PLA2 required Ca2- for activity, but was inhibited by Cu2+ and Zn2+ and by Cu2 and Mg2 in the presence and absence of Ca2, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors. © 2000 Plenum Publishing Corporation.197603607Aleksiev, B., Tchorbanov, B., (1976) Toxicon, 14, pp. 477-485Azevedo-Marques, M.M., Cupo, P., Coimbra, T.M., Hering, S.E., Rossi, M.A., Laure, C.J., (1982) Toxicon, 23, pp. 631-636Bon, C., Changeux, J.P., Jeng, T.W., Fraenkel-Conrat, H., (1979) Eur. J. Biochem., 99, pp. 471-1181Breithaupt, H., (1976) Toxicon, 14, pp. 221-233Cho, W., Kezdy, F.J., (1991) Meth. Enzymol., 197, pp. 75-79Cupo, P., Azevedo-Marques, M.M., Hering, S.E., (1988) Trans. R. Soc. Trop. Med. Hyg., 82, pp. 924-929Faure, G., Bon, C., (1988) Biochemistry, 27, pp. 730-738Faure, G., Guillaume, J.L., Camoin, L., Saliou, B., Bon, C., (1991) Biochemistry, 30, pp. 8074-8083Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., (1994) Eur. J. Biochem., 223, pp. 161-164Habermann, E., Breithaupt, H., (1978) Toxicon, 16, pp. 19-30Hendon, R.A., Fraenkel-Conrat, H., (1971) Proc. Natl. Acad. Sei. USA, 68, pp. 1560-1563Holzer, M., Mackessy, S.P., (1996) Toxicon, 34, pp. 1149-1155Jarret, H.W., Cooksy, K.D., Ellis, B., Anderson, J.M., (1986) Anal. Biochem., 153, pp. 189-198Landucci, E.C.T., Condino-Neto, A., Ferez, A.C., Hyslop, S., Corrado, A.P., Novello, J.C., Marangoni, S., De Nucci, G., (1994) Toxicon, 32, pp. 217-226Landucci, E.C.T., Toyama, M., Marangoni, S., Oliveira, B., Cirino, G., Antunes, E., De Nucci, G., (2000) Toxicon, 38, pp. 199-208Martins, A.M.C., Monteiro, H.S.A., Junior, E.O.G., Menezes, D.B., Fonteies, M.C., (1988) Toxicon, 36, pp. 1441-1450Murakami, M., Hara, N., Kudo, I., Inoue, K., (1993) J. Immunol., 151, pp. 5675-5684Pieterson, W.A., Volwerck, J.J., De Haas, G.H., (1974) Biochemistry, 13, pp. 1439-1445Rosenfeld, G., (1971) Venomous Animals and Their Venoms, 2, pp. 345-362. , (Bucheri, W., and Buckley, E. E., eds.), Academic Press, New YorkRubsamen, K., Breithaupt, H., Habermann, E., (1971) NaunynSchmiedeberg's Arch. Pharmacol., 270, pp. 274-288Schagger, H., Von Jagow, G., (1987) Anal. Biochem., 166, pp. 368-379Toyama, M.H., Cameiro, E.M., Marangoni, S., Barbosa, R.L., Corso, G., Boschero, A.C., (2000) Biochim. Biophys. Acta, 1474, pp. 56-60Tu, A.T., Passey, R.B., Toom, P.M., (1970) Arch. Biochem. Biophys., 140, pp. 96-10

Purification And Characterization Of A New Trypsin Inhibitor From Dimorphandra Mollis Seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30-60%), gel filtration, and ionexchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 ́ 10-9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors. ©2002 Plenum Publishing Corporation.208625632Batista, 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. 1017-1022Bernfeld, P., (1955) Methods in Enzymology, 1, 149p. , Collowick, S. P. and Kaplan, N. O., eds. Academic Press, New YorkBirk, Y., (1994) Arch. Latinoam. Nutricion, 44, pp. 26S-30SBradford, M.M., (1970) Anal. Biochem., 72, pp. 248-254Broze, G.J., Girard, T.J., Novotny, W.F., (1990) Biochemistry, 29, pp. 7539-7546DeClerck, Y.A., Imren, S., (1994) Eur. J. Cancer, 30 A, pp. 2170-2180Di Ciero, L., Oliva, M.L.V., Torquato, R., Köhler, P., Weder, J.K.P., Novelle, J.C., Sampaio, C.A.M., Marangoni, S., (1998) J. Protein. Chem., 17, pp. 827-834Erlanger, B.F., Kolowsky, N., Cohen, N., (1961) Arch. Biochem. 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The Complete Amino Acid Sequence Of A Trypsin Inhibitor From Bauhinia Variegata Var. Candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata Candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M, of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pi 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K, values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The Nterminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. Candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64). © 1998 Plenum Publishing Corporation.178827834Edman, P., Begg, G., (1967) Eur. J. Biochem., 1, pp. 80-91Heinrikson, R.L., Meridith, S.C., (1984) Anal. Biochem., 136, p. 65Kim, S.-H., Hara, S., Hase, S., Ikenaka, T., Toda, H., Kitamura, K., Kaizuma, N., (1985) J. Biochem., 98, pp. 435-448Knight, C.-G., (1986) Protease Inhibitors, pp. 23-51. , Barret, A. Jand Salvesen, G., eds, Elsevier, AmsterdamLaemmli, U.K., (1970) Nature, 227, p. 680Marangoni, S., Ghiso, J., Sampaio, S.V., Arantes, E.C., Giglio, J.R., Oliveira, B., Frangione, B.J., (1990) J. Protein Chem., 9, pp. 595-601Marangoni, S., Toyama, M.H., Arantes, E.C., Giglio, J.R., Suva, C.A., Caraeiro, E.C., Goncalves, A.A., Oliveira, B., (1995) Biochim. Biophys. Acta, 1243, pp. 309-314Oliva, M.L.V., Sampaio, M.U., Sampaio, C.A.M., (1987) Braz. J. Med. Biol. Res., 20, p. 767Richardson, M., (1977) Phytochemistry, 16, p. 159Richardson, M., (1991) Meth. Plant Biochem., 5, p. 259Ryan, C.A., (1973) Annu. Rev. Plant Physiol., 24, pp. 173-196Sampaio, C.A.M., Oliva, M.L.V., Sampaio, M.U., Batista, I.F.C., Buena, N.R., Tanaka, A.S., Auerswald, E.A., Fritz, H., (1996) Fmmunopharmacohgy, 32, pp. 62-66Sampaio, C.A.M., Sampaio, M.U., Prado, E.S., (1984) HoppeSeyler's Z. Physiol. Chem., 365, p. 297Weder, J.K.P., (1991) In Proceedings Ist Brazilian Congress on Proteins, pp. 29-55. , (Oliveira, Band Sgarbieri, V., eds.), Editera Unicamp, Campinas, BrazilWu, H.-C., Lin, J.-Y., (1993) J. Biochem., 113, pp. 258-263Xavier-Filho, J., Campos, F.A.P., (1989) In Protease Inhibitors and Toxicants of Plant Origin, 3, pp. 1-27. , Cheek, P. R., ed., CRC Press, Boca Raton, FloridaYamamoto, M., Hara, S., Ikenaka, T., (1983) J. Biochem., 94, pp. 849-86