ABSTRACT
alpha-Amylases from germinated maize, oats, rice, and sorghum were isolated by glycogen precipitation and hydrophobic interaction chromatography. Several methods were used for the detection of glycoproteins, including barley alpha-amylase isozymes purified as previously described and using the rice alpha-amylase as a positive control for glycosylation. Affinoblotting using concanavalin A, immunoblotting using a xylose-specific serum which reacts with complex N-linked glycans, and endo-beta-N-acetylglucosaminidase H treatment of amylases gave negative results for maize, oats, sorghum, and barley. However, after deglycosylation with trifluoromethanesulfonic acid, the molecular weight of one maize alpha-amylase constituent was clearly decreased. The same result was obtained after beta-elimination in mild conditions. Together these results indicated probable O-linked glycosylation of one maize alpha-amylase when barley, oats, and sorghum alpha-amylases did not appear to be glycosylated. Chemical deglycosylation of rice alpha-amylase resulted in the production of two polypeptides with different molecular weights.
Subject(s)
Plants/enzymology , alpha-Amylases/metabolism , Chromatography, Gel , Concanavalin A , Edible Grain/enzymology , Electrophoresis, Polyacrylamide Gel , Hordeum/enzymology , Kinetics , Oryza/enzymology , Plant Lectins , Species Specificity , Zea mays/enzymology , alpha-Amylases/isolation & purificationABSTRACT
Monoclonal antibodies (MAbs) to barley alpha-amylase I have been produced, purified and characterized. Six are specific for the alpha-amylase I isoform, while one also reacts with alpha-amylase II. All but one recognize the antigen in solution. Topology of the MAbs was investigated by additivity in ELISA, which led us to subdivide them into four groups. An immunoassay was developed using one of these MAbs, to measure alpha-amylase I activity in an extract containing both isoforms.
Subject(s)
Edible Grain/enzymology , Hordeum/enzymology , alpha-Amylases/analysis , Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity , Epitopes/analysis , Hybridomas , Immunoassay , Immunosorbent Techniques , Isoenzymes/analysis , Isoenzymes/immunology , Mice , Mice, Inbred BALB C , alpha-Amylases/immunologyABSTRACT
Trichorzianines A, membrane active peptides of the peptaibol class, were isolated from cultures of the mould Trichoderma harzianum. Trichorzianines A were separated into pure components by HPLC on octadecyl bonded and SiO2 phases successively. Nine trichorzianines A (IIa, IIIa, IIIb, IIIc, IVb, Vb, VIa, VIb and VII) were isolated from the complex microheterogeneous mixture. Their N-terminal amino acid is acetylated, the C-terminal amino alcohol is either tryptophanol or phenylalaninol, 7 to 8 of the 19 residues are alpha-aminoisobutyric acid. Gas chromatography on a chiral phase showed isovaline to have the D-configuration and all the other optically active amino acids and amino alcohols to have the L-configuration. The amino acid sequences were determined from their positive ion FAB mass spectra which exhibited the preferential cleavage of the Aib 12-Pro 13 amide bond as a main fragmentation. The resulting fragments subsequently underwent amide bond ruptures that generated two series of abundant acylium ions which enabled direct determination of the 1-19 sequence. The relative position of the isomeric amino acids in the sequence of trichorzianine AVII was assigned from analysis of the N- and C-terminal oligopeptides yielded by its selective acidic hydrolysis. The microheterogeneity of trichorzianines A results mainly from single or multiple substitution of amino acids at the specific positions 5, 14, 16 and 19.
