Amino acid sequence of serine protease inhibitor CI-1 from barley. Homology with barley inhibitor CI-2, potato inhibitor I, and leech eglin

  • IB Svendsen
  • Sigurd Boisen
  • Jørn Hejgaard
Article

Abstract

Three molecular forms of a protein inhibitor of chymotrypsin and microbial alkaline proteases have been isolated from Hiproly high-lysine barley.

Automated Edman degradation of one of these inhibitor preparations (CI-1C) resulted in the following amino acid sequence (77 residues in total): Tyr-Pro-Glu-Pro-Thr-Glu-Gly-Ser-Ile-Gly-Ala-Ser-Gly-Ala-Lys-Thr-Ser-Trp-Pro-Glu-Val-Val-Gly-Met-Ser-Ala-Glu-Lys-Ala-Lys-Glu-Ile-Ile-Leu-Arg-Asp-Lys-Pro-Asn-Ala-Gln-Ile-Glu-Val-Ile-Pro-Val-Asp-Ala-Met-Val-Pro-Leu-Asn-Phe-Asn-Pro-Asn-Arg-Val-Phe-Val-Leu-Val (His, Lys, Ala, Thr, Thr, Val, Ala, Glx, Val, Ser, Arg) Val-Gly.

The inhibitor (CI-1) is homologous with another barley inhibitor (CI-2), with potato inhibitor I and with the elastase-cathepsin G inhibitor eglin from the leech Hirudo medicinalis (30–50% of the amino acid residues in identical positions). This established «family of cystine-independent inhibitors» also showed some sequence similarities with the cystine-free yeast proteinase B inhibitors 1 and 2. In the reactive site region homologies with the cystine-rich inhibitors of the «Kazal pancreas secretory inhibitor» and the «Streptomyces subtilisin inhibitor» families were observed.

Keywords

Hiproly barley chymotrypsin inhibitor subtilisin inhibitor 

Abbreviations

APNE

N-acetyl-d,l-phenylalanine-2-naphtylester

CI-1 and CI-2

chymotrypsin inhibitor 1 and 2 from barley (3)

LIE

Leech inhibitor eglin (17)

PI-I

potato inhibitor I (15)

Polybrene

hexadimethrine bromide

PTH

phenylthiohydantoin

SDS

sodium dodecyl sulfate

THEED

N,N,N′,N′,-tetrakis (2-hydroxyethyl)ethylenediamine

YIB

yeast inhibitor of protease B (11)

References

  1. 1.
    Begg, G. &F. J. Morgan: A non-volatile buffer with improved performance in automated protein sequencing. FEBS Lett. 66, 243–245 (1976)PubMedCrossRefGoogle Scholar
  2. 2.
    Biedermann, K., U. Montali, B. Martin, I. Svendsen &M. Ottesen: The amino acid sequence of proteinase A inhibitor 3 from baker's yeast. Carlsberg Res. Commun. 45, 225–235 (1980)CrossRefGoogle Scholar
  3. 3.
    Boisen, S., C. Y. Andersen &J. Hejgaard: Inhibitors of chymotrypsin and microbial serine proteases in barley grains. Isolation, partial characterization and immunochemical relationships of multiple molecular forms. Physiol. Plant. 52, 167–176 (1981)CrossRefGoogle Scholar
  4. 4.
    Hejgaard, J. &S. Boisen: High-lysine proteins in Hiproly barley breeding: Identification, nutritional significance and new screening methods. Hereditas 93, 311–320 (1980)CrossRefGoogle Scholar
  5. 5.
    Hejgaard, J.: Isoelectric focusing of subtilisin inhibitors: Detection and partial characterization of cereal inhibitors of chymotrypsin and microbial proteases. Anal. Biochem. 116, 444–449 (1981)PubMedCrossRefGoogle Scholar
  6. 6.
    Jonassen, I.: Characteristics of Hiproly barley I. Isolation and characterization of two water-soluble high-lysine proteins. Carlsberg Res. Commun. 45, 47–58 (1980)CrossRefGoogle Scholar
  7. 7.
    Kato, I., W. J. Kohr &M. Laskowski, Jr.: Evolution of avian ovomucoids. In: Regulatory proteolytic enzymes and their inhibitors. Proc. 11th FEBS Meeting, Copenhagen 1977. S. Magnusson et al., eds., Pergamon Press, Oxford, pp. 197–206 (1978)Google Scholar
  8. 8.
    Kominami, E., H. Hoffschulte, L. Leuschel, K. Maier &H. Holzer: The substrate specificity of proteinase B from baker's yeast. Biochim. Biophys. Acta 661, 136–141 (1981)PubMedGoogle Scholar
  9. 9.
    Kulbe, K. D.: Micropolyamide thin-layer chromatography of phenyl-thiohydantoin amino acids (PTH) at a subnanomolar level. Anal. Biochem. 59, 564–573 (1974)PubMedCrossRefGoogle Scholar
  10. 10.
    Laskowski, M. Jr., &I. Kato: Protein inhibitors of proteinases. Ann. Rev. Biochem. 49, 593–626 (1980)PubMedCrossRefGoogle Scholar
  11. 11.
    Maier, K., H. Müller, R. Tesch, I. Witt &H. Holzer: Amino acid sequence of yeast proteinase B inhibitor 1. Comparison with inhibitor 2. Biochem. Biophys. Res. Commun. 91, 1390–1398 (1979)PubMedCrossRefGoogle Scholar
  12. 12.
    Martin, B., I. Svendsen &M. Ottesen: Use of carboxypeptidase Y for carboxy-terminal sequence determination in proteins. Carlsberg Res. Commun. 42, 99–102 (1977)CrossRefGoogle Scholar
  13. 13.
    Mikola, J. &E.-M. Suolinna: Purification and properties of an inhibitor of microbial alkaline proteinases from barley. Arch. Biochem. Biophys. 144, 566–575 (1971)PubMedCrossRefGoogle Scholar
  14. 14.
    Plunkett, G. &C. A. Ryan: Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities. J. Biol. Chem. 255, 2752–2755 (1980)PubMedGoogle Scholar
  15. 15.
    Richardson, M. &L. Cossins: Chymotryptic inhibitor I from potatoes: The amino acid sequences of subunits B, C, and D. FEBS Lett. 45, 11–13 (1974). (Corrigendum in FEBS Lett. 52, 161 (1975))PubMedCrossRefGoogle Scholar
  16. 16.
    Richardson, M., R. D. J. Barker, R. T. McMillan &L. M. Cossins: Identification of the reactive (inhibitory) sites of chymotryptic inhibitor I from potatoes. Phytochemistry 16, 837–839 (1977)CrossRefGoogle Scholar
  17. 17.
    Seemüller, U., M. Eulitz, H. Fritz &A. Strobl: Structure of the Elastase-Cathepsin G inhibitor of the leechHirudo medicinalis. Hoppe-Seyler's Z. Physiol. Chem. 361, 1841–1846 (1980)PubMedGoogle Scholar
  18. 18.
    Seemüller, U., H. Fritz &M. Eulitz: Eglin: Elastase-Cathepsin G inhibitor from leeches. Methods in Enzymology 80, 804–816 (1981)Google Scholar
  19. 19.
    Svendsen, I., B. Martin &I. Jonassen: Characteristics of Hiproly barley. III. Amino acid sequences of two lysine-rich proteins. Carlsberg Res. Commun. 45, 79–85 (1980)CrossRefGoogle Scholar
  20. 20.
    Svendsen, I., I. Jonassen, J. Hejgaard &S. Boisen: Amino acid sequence homology between a serine protease inhibitor from barley and potato inhibitor I. Carlsberg Res. Commun. 45, 389–395 (1980)CrossRefGoogle Scholar

Copyright information

© Carlsberg Laboratory 1982

Authors and Affiliations

  • IB Svendsen
    • 1
  • Sigurd Boisen
    • 2
  • Jørn Hejgaard
    • 2
  1. 1.Department of ChemistryCarlsberg LaboratoryCopenhagen Valby
  2. 2.Department of Biochemistry and NutritionTechnical University of DenmarkLyngby
  3. 3.National Institute of Animal ScienceCopenhagen V

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