Abstract
The wheat monomeric inhibitor WMAI-1 (syn. 0.28) produced inEscherichia coli using the pT7-7 expression ventor has the correct N-terminal sequence and the same electrophoretic mobility and specific activity towards the α-amylase from the insectTenebrio molitor as the native WMAI-1 isolated from wheat. This confirms that the native inhibitor is not glycosylated and contradicts claims that a putative glycosyl moiety was essential for inhibition. Thirteen mutants have been obtained at six different sites. Substitution of the highly conserved N-terminal S by the sequence ARIRAR increased the pre-incubation time required for maximum activity. A similar result was obtained by insertion of GPRLPW after position 4, while insertion of EPRAPW at the same position rendered the inhibitor inactive. The substitution D/EGPRL and insertions DGP or D, at position 58, produced complete inactivation. All other mutations had only minor effects on activity.
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Barber D, Sanchez-Monge R, Gomez L, Carpizo J, Armentia A, Lopez-Otin C, Juan F, Salcedo G: A barley flour inhibitor of insect α-amylase is a major allergen associated with baker's asthma disease. FEBS Lett 248: 119–122 (1989).
Boisen D: Characterization of the endosperm trypsin inhibitor in barley. Phytochemistry 15: 641 (1976).
Erlanger BF, Kokowsky N, Cohen W: The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95: 271–278 (1961).
García-Olmedo F, Salcedo G, Sanchez-Monge R, Gomez L, Royo J, Carbonero P: Plant proteinaceous inhibitors of proteinases and α-amylases. Oxford Surv Plant Mol Cell Biol 4: 275–334 (1987).
García-Olmedo F, Salcedo G, Sanchez-Monge R, Hernandez-Lucas C, Carmona MJ, Lopez-Fando JJ, Fernandez JA, Gomez L, Royo J, García-Maroto F, Castagnaro A, Carbonero P: Alpha-amylase/trypsin inhibitors and thionins. Possible defense proteins from barley. In: Shewry PR (ed) Barley: Genetics, Molecular Biology and Biotechnology. CAB International (in press) (1991).
Garnier J, Osguthorpe DJ, Robson B: Analysis of the accuracy and implication of simple methods for predicting secondary structure of globular proteins. J Mol Biol 120: 97–120 (1978).
Gomez L, Sanchez-Monge R, García-Olmedo F, Salcedo G: Wheat tetrameric inhibitors of insect α-amylases: alloploid heterosis at the molecular level. Proc Natl Acad Sci USA 86: 3242–3246 (1989).
Gomez L, Martin E, Hernandez D, Sanchez-Monge R, Barber D, del Pozo V, Andres B, Armentia A, Lahoz C, Salcedo G, Palomino P: Members of the α-amylase inhibitors family from wheat endosperm are major allergents associated with baker's asthma. FEBS Lett 261: 85–88 (1990).
Gomez L, Sanchez-Monge R, Lopez-Otin C, Salcedo G: Wheat inhibitors of heterologous α-amylases: characterization of major components from the monomeric class. Plant Physiol 96: 768–774 (1991).
Gutierrez C, Sanchez-Monge R, Gomez L, Ruiz-Tapiador M, Castañera P, Salcedo G: α-Amylase activities of agricultural insect pests are specifically affected by different inhibitor preparations from wheat and barley endosperm. Plant Sci 72: 37–44 (1990).
Jonassen I, Svendsen I: Identification of the reactive sites in two homologous proteinase inhibitors isolated from barley. Carlsberg Res Commun 47: 199–203 (1982).
Kashlan N, Richardson M: The complete amino acid sequence of a major wheat protein inhibitor of α-amylase. Phytochemistry 20: 1781–1784 (1981).
Laemli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lazaro A, Sanchez-Monge R, Salcedo G, Paz-Ares J, Carbonero P, García-Olmedo F: A dimeric inhibitor of insect α-amylase from barley. Cloning of the cDNA and identification of the protein. Eur J Biochem 172: 129–134 (1988).
Petrucci T, Sannia G, Parlamenti R, Silano V: Structural studies of wheat monomeric and dimeric protein inhibitors of alpha-amylase. Biochem J 173: 229–235 (1978).
Sanchez-Monge R, Gomez L, García-Olmedo F, Salcedo G: A tetrameric inhibitor of insect α-amylase from barley. FEBS Lett 207: 105–109 (1986).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors.Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Silano V: Alpha-amylase inhibitors. In: Kruger JE, Lineback D, Stauffer CE (eds) Enzymes and their Role in Cereal Technology, pp. 141–199. AACC, St Paul, MN (1987).
Silano V, Pooerio E, Buonocore V: A model for the interaction of wheat monomeric and dimeric protein inhibitiors with alpha-amylase. Mol Cell Biochem 18: 87–91 (1977).
Smith PK, Krohn RI, Hermanson ET, Maelia AK, Gastner FH, Provenzano MD, Fujimoto EK, Goeke NW, Olson BJ, Kenk DC: Measurement of protein using bicinchonimic acid. Anal Biochem 150: 76–85 (1985).
Tabor S, Ricardson CC: A bacteriophage T7 RNA polymerase promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82: 1074–1078 (1985).
Walsh BJ, Howden MEH: A method for the detection of IgE binding sequences of allergens based on a modification of epitope mapping. J Immunol Meth 121: 275–280 (1989).
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García-Maroto, F., Carbonero, P. & GArcía-Olmedo, F. Site-directed mutagenesis and expression inEscherichia coli of WMAI-1, a wheat monomeric inhibitor of insect α-amylase. Plant Mol Biol 17, 1005–1011 (1991). https://doi.org/10.1007/BF00037140
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DOI: https://doi.org/10.1007/BF00037140