PURIFICATION AND CHARACTERIZATION OF CATALASE FROM LOBLOLLY-PINE (PINUS-TAEDA L) MEGAGAMETOPHYTES

Catalase (EC 1.11.1.6) was purified to near homogeneity from isolated megagametophytes of germinated loblolly pine (Pinus taeda L.) seeds, a nd monospecific antibodies were elicited in rabbits. Following a proce dure that involved acetone extraction, (NH4)2SO4 fractionation, and fo ur chromatographic steps (i.e. DE-52 cellulose, Superdex-200, hydroxyl apatite, and phenyl-Sepharose CL-4B), catalase was purified about 140- fold to a final specific activity of 2215 mmol min-1 mg-1 of protein. Cotton isocitrate lyase antibodies were used, and protein immunoblots revealed that the resolution on hydroxylapatite and phenyl-Sepharose a llowed for the complete separation of catalase from contaminating isoc itrate lyase. The molecular masses of the native enzyme and its subuni t are 235 and 59 kD, respectively, indicating that the pine holoenzyme is a homotetramer. Loblolly pine catalase exists as multiple isoforms . When megagametophytes taken 7 d after imbibition at 30-degrees-C wer e extracted, subjected to nondenaturing isoelectric focusing, and stai ned for catalase activity, at least four catalase isoforms were observ ed, including one dominant form with an isoelectric point of 6.87. Pur ified pine catalase is not a glycoprotein and has a ratio of absorbanc e at 208 nm to absorbance at 405 nm of 1.5. When probed with loblolly pine catalase antibodies, protein blots of cell-free extracts from meg agametophytes of mature, stratified, and germinated loblolly pine seed s, the megagametophyte glyoxysomal fraction, and purified loblolly pin e catalase all revealed one immunoreactive 59-kD polypeptide. This ind icates that no detectable change in the enzyme's monomeric molecular m ass occurs during seed stratification and germination, early seedling growth, and purification.