Planta Med 2008; 74(5): 546-550
DOI: 10.1055/s-2008-1074502
Natural Products Chemistry
Original Paper
© Georg Thieme Verlag KG Stuttgart · New York

Isolation and Characterization of a Novel Trypsin Inhibitor from Fresh Lily Bulbs

Xiaoqing Zhang1 , Hexiang Wang1 , Tzi Bun Ng2
  • 1State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, P. R. China
  • 2Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, P. R. China
Further Information

Publication History

Received: September 6, 2007 Revised: February 11, 2008

Accepted: February 24, 2008

Publication Date:
10 April 2008 (online)

Abstract

A 17-kDa trypsin inhibitor was isolated from fresh lily bulbs with an isolation procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. Its N-terminal sequence displayed similarity to a short segment of the sequences of the Populus tremula trypsin inhibitor, a putative trypsin inhibitor from Arabidopsis thaliana and sporamin B from sweet potato. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC50 value of 1.3 μM. There was a stimulatory effect on macrophage production of nitric oxide. Unlike field bean trypsin inhibitor it did not inhibit [methyl-3H]thymidine incorporation by leukemia L1210 cells and MBL2 cells when tested up to 100 μM. In contrast to broad bean trypsin inhibitor, there was no inhibitory effect on HIV-1 reverse transcriptase when lily bulb trypsin inhibitor was tested up to 100 μM. The present report is one of the very few on bulbs in contrast to the voluminous literature on seeds.

References

  • 1 Banerji A P, Fernandes A O. Field bean protease inhibitor preparations, unlike methotrexate, can completely suppress Yoshida sarcoma tumor in rats.  Cell Biol Int. 1994;  18 1025-34
  • 2 Macedo M L, de Sa C M, Freire M D, Parra J R. A Kunitz-type inhibitor of coleopteran proteases, isolated from Adenanthera pavonina L. seeds and its effect on Callosobruchus maculates. .  J Agric Food Chem. 2004;  52 2533-40
  • 3 Devaraj V R, Manjunatha N H. Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab purpureus L.  J Protein Chem. 1999;  18 47-54
  • 4 Banerji A, Fernandes A, Bane S, Ahire S. The field bean protease inhibitor has the potential to suppress B16F10 melanoma cell lung metastasis in mice.  Cancer Lett. 1998;  129 15-20
  • 5 Fernandes A O, Banerji A P. Inhibition of benzopyrene-induced forestomach tumors by field bean protease inhibitor(s).  Carcinogenesis. 1995;  16 1843-6
  • 6 Fernandes A O, Banerji A P. The field bean protease inhibitor can effectively suppress 7, 12-dimethylbenz[a]anthracene-induced skin tumorigenesis in mice.  Cancer Lett. 1996;  104 219-24
  • 7 Troncoso M F, Cerda Zolezzi P, Hellman U, Wolfenstein-Todel C. A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis.  Arch Biochem Biophys. 2003;  411 93-104
  • 8 Banerji A, Fernandes A, Bane S. Treatment with field bean protease inhibitor can effectively repress ethylnitrosourea (ENU)-induced neoplasms of the nervous system in Sprague-Dawley rats.  Cancer Lett. 1998;  130 161-7
  • 9 Giri A P, Harsulkar A M, Ku M S, Gupta V S, Deshpande V V, Ranjekar P K. et al . Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds.  Phytochemistry. 2003;  63 523-32
  • 10 Birk Y, Applebaum Y. Effect of soybean trypsin inhibitors on the development and midgut proteolytic activity of Tribolium castaneum larvae.  Enzymol Acta Biocatalyst. 1960;  22 318-26
  • 11 Lanza A, Tava A, Catalano M, Ragona L, Singuaroli I, della Cuna F SR. et al . Effects of the Medicago scutellata trypsin inhibitor (MsTI) on cisplatin-induced cytotoxicity in human breast and cervical cancer cells.  Anticancer Res. 2004;  24 227-33
  • 12 Campos J E, Whitaker J R, Yip T T, Hutchens T W, Blanco-Labra A. Unusual structural characteristics and complete amino acid sequence of a protease inhibitor from Phaseolus acutifolius seeds.  Plant Physiol Biochem. 2004;  42 209-14
  • 13 Delgado-Vargas F, Lopez-Valdes H E, Valdes-Rodriguez S, Blanco-Labra A, Chagolla-Lopez A, Lopez-Valenzuela E J. Isolation and properties of a Kunitz-type protein inhibitor obtained from Pithecellobium dulce seeds.  J Agric Food Chem. 2004;  52 6115-21
  • 14 Deshimaru M, Hanamoto R, Kusano C, Yoshimi S, Terada S. Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds.  Biosci Biotechnol Biochem. 2002;  66 1897-903
  • 15 Di Ciero L, Oliva M L, Torquato R, Kohler P, Wederv J K, Novello J C. et al . The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.  J Protein Chem. 1998;  17 827-34
  • 16 Dodo H W, Viquez O M, Maleki S J, Konan K N. cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4.  J Agric Food Chem. 2004;  52 1404-9
  • 17 do Socorro M, Cavalcanti M, Oliva M L, Fritz H, Jochum M, Mentele R. et al . Characterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii. .  Biochem Biophys Res Commun. 2002;  291 635-9
  • 18 Ye X Y, Ng T B, Rao P F. A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans.  Biochem Biophys Res Commun. 2001;  289 91-6
  • 19 Wang H X, Ng T B. Isolation of lilin, a novel arginine- and glutamine-rich protein with potent antifungal and mitogenic activities from lily bulbs.  Life Sci. 2002;  70 1075-84
  • 20 Cheung S C, Li N H. Chinese medicinal Herbs of Hong Kong. Hong Kong; Commercial Press 1987
  • 21 Huang B, Ng T B, Fong W P, Wan C C, Yeung H W. Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.  Int J Biochem Cell Biol. 1999;  31 707-15
  • 22 Laemmli U K, Favre M. Gel electrophoresis of proteins.  J Mol Biol. 1973;  80 573-98
  • 23 Wong J H, Ng T B. Sesquin, a potent defensin-like antifungal peptide from beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase.  Peptides. 2005;  26 1120-6
  • 24 Lin X, Kaul S, Town C D, Bonito M I, Creasy T H, Haas B. et al .Arabidopsis thaliana chromosome III Bac T6K12 genomic sequence. Submitted to NCBI on Dec 8, 1999
  • 25 Ingvarsson P K. Molecular population genetics of herbivore-induced protease inhibitor genes in European aspen (Populus tremula L., Salicaceae).  Mol Biol Ecol. 2005;  22 1802-12
  • 26 Murakami S, Hattori T, Nakamura K. Structural difference in full-length cDNAs for two classes of sporamin, the major soluble protein of sweet potato tuberous roots.  Plant Mol Biol. 1986;  7 343-55
  • 27 Wong J H, Ng T B. Lunatusin, a trypsin-stable antimicrobial peptide from lima beans (Phaseolus lunatus).  Peptides. 2005;  26 2086-92
  • 28 Haq S K, Khan R H. Characterization of a proteinase inhibitor from Cajanus cajan (L.).  J Protein Chem. 2003;  22 543-54

Tzi Bun Ng

Department of Biochemistry

Faculty of Medicine

The Chinese University of Hong Kong

Shatin N.T.

Hong Kong

People's Republic of China

Phone: +852-2609-803

Fax: +852-2603-5123

Email: b021770@mailserv.cuhk.edu.hk

Hexiang Wang

State Key Laboratory for Agrobiotechnology and Department of Microbiology

China Agricultural University

Bejing P.R.

China

Phone: +8610-62132518

Fax: +8610-62132518

Email: hxwang@cau.edu.cnI