PLANT PHYSIOLOGY , Vol 103, Issue 2 477-483, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Purification and Characterization of Catalase from Loblolly Pine (Pinus taeda L.) Megagametophytes
R. T. Mullen and D. J. Gifford
Department of Botany, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
Catalase (EC 1.11.1.6) was purified to near homogeneity from isolated
megagametophytes of germinated loblolly pine (Pinus taeda L.) seeds, and
monospecific antibodies were elicited in rabbits. Following a procedure
that involved acetone extraction, (NH4)2SO4 fractionation, and four
chromatographic steps (i.e. DE-52 cellulose, Superdex-200, hydroxylapatite,
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 allowed for the
complete separation of catalase from contaminating isocitrate lyase. The
molecular masses of the native enzyme and its subunit 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[deg]C were extracted, subjected to
nondenaturing isoelectric focusing, and stained for catalase activity, at
least four catalase isoforms were observed, including one dominant form
with an isoelectric point of 6.87. Purified pine catalase is not a
glycoprotein and has a ratio of absorbance 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 megagametophytes of mature, stratified,
and germinated loblolly pine seeds, the megagametophyte glyoxysomal
fraction, and purified loblolly pine catalase all revealed one
immunoreactive 59-kD polypeptide. This indicates that no detectable change
in the enzyme's monomeric molecular mass occurs during seed stratification
and germination, early seedling growth, and purification.
