Journal of Molecular Evolution

, Volume 42, Issue 5, pp 560–569 | Cite as

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

  • Balaji Prakash
  • S. Selvaraj
  • M. R. N. Murthy
  • Y. N. Sreerama
  • D. Rajagopal Rao
  • Lalitha R. Gowda


Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

Key words

Bowman-Birk Inhibitors Sequence analysis Monocotyledon Dicotyledon Gene duplication Dolichos biflorus 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ambler RP (I972) Enzymic Hydrolysis with carboxypeptidases. Methods Enzymol 25:143–154Google Scholar
  2. Birk Y (1985) The Bowman-Birk inhibitor. Int J Pept Protein Res 25:113–131PubMedGoogle Scholar
  3. Bidlingmeyer BA, Cohen SA, Tarvin TL (1984) Rapid analysis of amino acids using pre-column derivatization. J Chromatogr 336:93–104PubMedGoogle Scholar
  4. Chang JY (1979) The destruction of serine and threonine thiohydantoins during the sequence determination of peptides by 4-N,N-dimethylaminoazobenzene 4′-isothiocyanate. Biochem Biophys Acta 578:175–187PubMedGoogle Scholar
  5. Chen P, Rose J, Love R, Wei CH, Wang BC (1992) Reactive sites of an anticarcenogenic Bowman-Birk Protease inhibitor are similar to other trypsin inhibitors. J Biol Chem 267:1990–1994PubMedGoogle Scholar
  6. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic acids Res 16:10881–10890PubMedGoogle Scholar
  7. Crestfield AM, Moore S, Stein WH (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem 238:622–627PubMedGoogle Scholar
  8. Desai N, Bourne P (1986) Protein and nucleic acid sequence information and analysis, PRONUC ver 4.4. Columbia University, New YorkGoogle Scholar
  9. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791Google Scholar
  10. Felsenstein J (1988) Phylogenies from molecular sequences: inference and reliability. Annu Rev Genet 22:521–565CrossRefPubMedGoogle Scholar
  11. Felenstein J (I989) PHYLIP—Phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  12. Hermodson MA, Erickson LH, Neurath H, Walsh KA (1973) Determination of the amino acid sequence of porcine trypsin in sequenator analysis. Biochemistry 12:3146–3153CrossRefPubMedGoogle Scholar
  13. Joubert FJ, Kruger H, Townshend GS, Botes DP (1979) Purification, some properties and the complete primary structures of two protease inhibitors (DE-3 and DE-4) from Macrotyloma axillare seed. Eur J Biochem 97:85–91CrossRefPubMedGoogle Scholar
  14. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 151:105–132Google Scholar
  15. Laskowski M Jr, Kato I (1980) Protein inhibitors of proteinases. Annu Rev Biochem 49:593–626CrossRefPubMedGoogle Scholar
  16. Lin G, Bode W, Huber R, Chi C, Engh RA (1993) The 0.25 run X-ray structure of the Bowman-Birk inhibitor from mung bean in ternary complex with porcine trypsin. Eur J Biochem 212:549–555CrossRefPubMedGoogle Scholar
  17. Lipman DJ, Pearson WR (1985) Rapid and sensitive protein similarity searches. Science 227:1435–1441PubMedGoogle Scholar
  18. Mahoney WC, Hermodson MA (1980) Separation of large denatured peptides by reverse phase high performance liquid chromatography. J Biol Chem 255:11199–11203PubMedGoogle Scholar
  19. Maki Z, Tashiro M, Sugihara N, Kanamori M (1980) Double headed nature of a trypsin inhibitor from rice bran. Agric Biol Chem 44:953–955Google Scholar
  20. Nagasue A, Fukamachi H, Ikenaga H, Funatsu G (1988) The amino acid sequence of barley rootlet trypsin inhibitor. Agric Biol Chem 52:1505–1514Google Scholar
  21. Needleman SB, Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48:443–453CrossRefPubMedGoogle Scholar
  22. Norioka S, Ikenaka T (1983) Amino acid sequences of trypsin chymotrypsin inhibitors (A-I,A- II,B-I and B-II) from peanut (Arachis hypogaea): a discussion on molecular evolution of legume Bowman-Birk type inhibitors. J Biochem 94:589–599PubMedGoogle Scholar
  23. Odani S, Koide T, Ono T (1986) Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type. J Biochem 100:975–983PubMedGoogle Scholar
  24. Prakash B, Murthy MRN, Sreerama YN, Rama Sarma PR, Rajagopal Rao D (1994) Crystallization and preliminary X-ray diffraction studies on a trypsin/chymotrypsin double headed inhibitor from horse gram. J Mol Biol 235:364–366PubMedGoogle Scholar
  25. Rama Sarma PR, Rajagopal Rao D (1991) Nature of tryptic /chymotryptic inhibitor from horse gram (Dolichos biflorus) Indian. J Biochem Biophys 28:418–424Google Scholar
  26. Ryan CA (1981) Proteinase inhibitors, a comprehensive treatise. In: Stumpf PK, Conn EE (eds) The Biochemistry of Plants, vol 6. Academic Press, New York, p 351–371Google Scholar
  27. Schwartz RM, Dayhoff MO (1978) Matrices for detecting distance relationships. In: Dayhoff MO (ed) Atlas of protein sequence and structure. Washington DC. National Biomedical Research Foundation, 5:353–358Google Scholar
  28. Suzuki A, Yamane T, Ashida T, Norioka S, Hara S, Ikenaka T (1993) Crystallographic refinement of Bowman-Birk type protease inhibifor A-II from peanut (Arachis hypogaea) at 2.3 Å resolution. J Mol Biol 234:722–734CrossRefPubMedGoogle Scholar
  29. Tashiro M, Hashino K, Shiozaki M, Ibuki F, Maki Z (1987) The complete amino acid sequence of rice bran trypsin inhibitor. J Biochem 102:297–306PubMedGoogle Scholar
  30. Tashiro M, Asao T, Hirata C, Takahashi K, Kanamori M (1990) The complete amino acid sequence of a major trypsin inhibitor from seeds of foxtail millet (Setaria italica). J Biochem 108:669–672PubMedGoogle Scholar
  31. Tsunogae Y, Tanaka I, Yamane T, Kikkawa J, Ashida T, Ishikawa C, Watanabe K, Nakamura S, Takahashi K (1986) Structure of trypsin binding domain of Bowman-Birk type protease inhibitor and its interaction with trypsin. J Biochem 100:1637–1646PubMedGoogle Scholar
  32. Waxadal MJ, Konigsberg WH, Henley WL, Edelman GM (1968) The covalent structure of a human γ-immunoglobulin—II. Isolation and characterization of the cyanogen bromide fragments. Biochemistry 7:1959–1966Google Scholar
  33. Yang CV, Paulay E. Krazer HS, Hilschmann N (1981) Chromatography and rechromatography in high performance liquid chromatography of peptide mixtures: the complete primary structure of an immunoglobulin L-chain of k-type, subgroup I (Bence-Jones protein Den). Hoppe Seylers Z Physiol Chem 362:1131–1146PubMedGoogle Scholar

Copyright information

© springer-Verlag New York Inc 1996

Authors and Affiliations

  • Balaji Prakash
    • 1
  • S. Selvaraj
    • 1
  • M. R. N. Murthy
    • 1
  • Y. N. Sreerama
    • 2
  • D. Rajagopal Rao
    • 2
  • Lalitha R. Gowda
    • 2
  1. 1.Molecular Biophysics UnitIndian Institute of ScienceBangaloreIndia
  2. 2.Department of Biochemistry and NutritionCentral Food Technological Research InstituteMysoreIndia

Personalised recommendations