Purification and immunocharacterization of long-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 beta -oxidation spiral in plant glyoxysomes was purifi ed to homogeneity from loblolly pine (Pinus taeda L.) megagametophytes isol ated 9 to 11 d after imbibition at 30 degreesC. Homogeneity of the enzyme w as confirmed by silver staining SDS-PAGE gels of peak enzymatic activity fr actions from a molecular sieving column that was the last step of purificat ion. The purification procedure included acetone extraction, heat treatment , ammonium sulphate precipitation and chromatography on three columns (phen yl-Sepharose, hydroxyapatite, and molecular sieving). The homogenous enzyme was purified 1250-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 w ere confirmed to be mono-specific for acyl-CoA oxidase by immunotitration a nd western blotting experiments. A western blot analysis of cell free extra cts indicated that acyl-CoA oxidase was predominantly megagametophytic. (C) 2000 Editions scientifiques et medicales Elsevier SAS.