ISOCITRATE LYASE FROM GERMINATED LOBLOLLY-PINE MEGAGAMETOPHYTES - ENZYME-PURIFICATION AND IMMUNOCHARACTERIZATION

The glyoxysomal enzyme isocitrate lyase (EC 4.1.3.1) was purified to h omogeneity from germinated loblolly pine (Pinus taeda L.) megagametoph ytes isolated 9 days after seed imbibition. During initial stages of t he purification, separation on DE-52 (diethylaminoethyl) cellulose res olved at least two separate forms of the enzyme (isocitrate lyase A an d B). Both forms are immunologically distinct, with isocitrate lyase A representing over 90% of total enzyme activity in the megagametophyte . Subsequently, form A was used for enzyme purification. Isocitrate ly ase A was purified from contaminating catalase by a procedure which in volved gel filtration, hydroxylapatite and octyl-Sepharose CL-4B hydro phobic interaction chromatography. Native isocitrate lyase A is a tetr amer with a molecular mass of 270 kDa, and when analyzed on 7% sodium dodecyl sulfate polyacrylamide gels, purified isocitrate lyase A migra ted as two distinct bands. Both polypeptides appear to represent indiv idual subunits of isocitrate lyase A with molecular masses of 62.5 and 64 kDa, respectively. Polyclonal antibodies raised against purified i socitrate lyase A were demonstrated to be monospecific by immunotitrat ion, immunoprecipitation, and protein blotting experiments. Both isoci trate lyase A subunits were antigentically cross-reactive, with the 62 .5 kDa subunit appearing as the dominant form at all stages examined. Protein blot analysis of various cell-free extracts including purified enzyme, isolated megagametophyte glyoxysomal fractions, and extracts from mature and germinated megagametophytes and embryos indicated no c hange in the enzymes monomeric molecular masses during These stages. P rotein blot experiments further indicated that isocitrate lyase A in b oth tile megagametophyte and embryo were physically similar, and that protein levels of both enzymes increased with the onset of lipid reser ve mobilization.