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Biochemical and immunological characterization of α‐amylase isoenzymes of Araucaria araucana
Author(s) -
Acevedo Elba,
Cardemil Liliana
Publication year - 1994
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1994.tb06666.x
Subject(s) - isozyme , biochemistry , amino acid , biology , aspartic acid , affinity chromatography , asparagine , peptide sequence , polyclonal antibodies , ion chromatography , cyanogen bromide , amylase , isoelectric focusing , microbiology and biotechnology , enzyme , antibody , immunology , gene
Seven α‐amylase isoenzymes present in quiescent seeds of the South American conifer Araucaria araucana were purified by affinity chromatography and partially characterized. The molecular masses of these isoenzymes were 45.7, 47.0, 50.2, 51.2, 52.0, 53.5 and 55.2 kDa. The two main isoforms were separated from each other and from the rest of the isoenzymes by anion‐exchange chromatography using a linear gradient of 0 to 0.6 M NaCl and slightly different CaCl 2 concentrations. All isoenzyme bands stained with periodic acid/dansylhydrazine, suggesting that they are glycoproteins. Electroblotting of the isoenzymes onto polyvinylidene difluoride membranes allowed determination of the amino acid composition and NH 2 ‐terminal sequence of the 53.5‐, 50.2‐and 47.0‐kDa isoenzymes. Amino acid compositional analysis demonstrated that these enzymes are rich in glycine, aspartic acid/asparagine, alanine, serine, proline and glutamic acid/glutamine. The NH 2 ‐terminal sequences of the three isoenzymes are identical. Comparison of the amino acid compositions and the NH 2 ‐terminal sequence of these isoenzymes with the cereal and Vigna radiata α‐amylases demonstrated that there is no relation between them. However, polyclonal antibodies generated against barley α‐amylase cross‐reacted with all the A . araucana α‐amylases. Peptide mapping analysis of the isoenzymes using cyanogen bromide suggests that there are genetic differences between them.