BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF ALPHA-AMYLASE ISOENZYMES OF ARAUCARIA-ARAUCANA

Seven alpha-amylase isoenzymes present in quiescent seeds of the South American conifer Araucaria araucana were purified by affinity chromat ography and partially characterized. The molecular masses of these iso enzymes 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 CaCl2 concentrations. All isoe nzyme bands stained with periodic acid/dansylhydrazine, suggesting tha t they are glycoproteins. Electroblotting of the isoenzymes onto polyv inylidene difluoride membranes allowed determination of the amino acid composition and NH2-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, serin e, proline and glutamic acid/glutamine. The NH2-terminal sequences of the three isoenzymes are identical. Comparison of the amino acid compo sitions and the NH2-terminal sequence of these isoenzymes with the cer eal and Vigna radiata alpha-amylases demonstrated that there is no rel ation between them. However, polyclonal antibodies generated against b arley alpha-amylase cross-reacted with all the A. araucana-alpha-amyla ses. Peptide mapping analysis of the isoenzymes using cyanogen bromide suggests that there are genetic differences between them.