Subunit composition and oligomer stability of oat beta-glucosidase isozymes.
Oat beta-glucosidase (EC 3.2.1.21) has two isomeric forms, type I and type II, which are composed of 60 kDa peptides. To study the subunit composition and the stability of multimeric structure, the type I and II were purified from the primary leaves and coleoptiles of the etiolated oat seedlings where the isozymes are expressed organ-specifically. The monomers of the isozymes were isolated by urea-denatured gel electrophoresis followed by electroblotting. N-Terminal amino acid sequencing of the monomers indicated that the type I consisted of a peptide of ALESAKQVKPWQVPKRDWFP (As-Glu 1), and the type II having a peptide of ALESGKLKPWQIPKRDWFP (As-Glu 2) and As-Glu 1 in 1:1 ratio. The C-terminal amino acid of the As-Glu 1 was alanine and that of the As-Glu 2 was lysine. The As-Glu 2 was more negatively charged than the As-Glu 1. The type I isozyme is thus homomultimer of As-Glu 1 monomer and the type II heteromultimer of As-Glu 1 and As-Glu 2 monomers in 1:1 ratio. Partial denaturation of the multimers with urea and CaCl2 broke down the higher multimers to the lower multimers, which were in turn dissociated into homodimers and heterodimer. Denaturation study with urea and CaCl2 indicate that the higher multimers of the homooligomeric type I were more stable than those of the heterooligomeric type II and that hydrophobic interactions were important in the multimer formation. The homodimers were found to be more stable than the heterodimer. These results indicate that different combinations of the As-Glu 1 and As-Glu 2 monomers form the two isozymes of oat beta-glucosidase with different enzymatic properties and structural stability.
Kim
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YW
Department of Genetic Engineering, College of Natural Sciences, Kyungpook National University, Taegu 702-701, South Korea.
Kim
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D000595:Amino Acid Sequence; D018554:Avena / Q000201:enzymology*; D002122:Calcium Chloride / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017421:Sequence Analysis; D014508:Urea / Q000494:pharmacology; D001617:beta-Glucosidase / Q000737:chemistry*
D007527:Isoenzymes; D010940:Plant Proteins; D014508:Urea; D001617:beta-Glucosidase; D002122:Calcium Chloride