Purification and immunocharacterization oflong-chain acyl-CoA oxidase from loblolly pine megagametophytes

Acyl-CoA oxidase (EC 1.3.3.6), an enzyme that catalyses the first and rate limiting step of the β-oxidation spiral in plant glyoxysomes was purified to homogeneity from loblolly pine (Pinus taeda L.) megagametophytes isolated 9 to 11 d after imbibition at 30 °C. Homogeneity of the enzyme was confirmed by silver staining SDS-PAGE gels of peak enzymatic activity fractions from a molecular sieving column that was the last step of purification. The purification procedure included acetone extraction, heat treatment, ammonium sulphate precipitation and chromatography on three columns (phenyl-Sepharose, hydroxyapatite, and molecular sieving). The homogenous enzyme was purified 1 250-fold to a specific activity of 417.5 nkat·mg–1 protein. The enzyme was a homodimer with a native molecular mass of 150 kDa and a subunit molecular mass of 71 kDa. Polyclonal antibodies raised in rabbits were confirmed to be mono-specific for acyl-CoA oxidase by immunotitration and western blotting experiments. A western blot analysis of cell free extracts indicated that acyl-CoA oxidase was predominantly megagametophytic.