PURIFICATION, PROPERTIES AND PHOSPHORYLATION OF ANAEROBICALLY INDUCEDENOLASE IN ECHINOCHLOA PHYLLOPOGON AND E-CRUS-PAVONIS

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) activity is di fferentially induced by anoxia in the flood-tolerant species E. phyllo pogon (Stev.) Koss and the flood-intolerant species E. crus-pavonis (H .B.K.) Schult. To examine the regulation of enolase at the protein lev el, we purified the enzyme from both species to near homogeneity and c ompared their physico-chemical and catalytic properties. Enolase purif ied from E. phyllopogon exhibits optimal activity at pH 7.0, a K-m of 80 mu M for 2-PGA, a Q(10) of 1.97 and an E(a) of 12.3 kcal mol(-1). S imilarly, enolase from E. crus-pavonis exhibits optimal activity at pH 7.0, a K-m of 50 mu M for 2-PGA, a Q(10) of 2.04 and an E(a) of 12.9 kcal mol(-1). The enzyme from both species is thermostable (100% activ e after 15 min, 50 degrees C) and is a homodimer of 52.5 kDa subunits as resolved by SDS-PAGE and immunoblotting. 0E. phyllopogon enolase wa s phosphorylated in vitro using either [gamma-P-32]ATP or [gamma-P-32] GTP; however, enolase activity was neither stimulated nor inhibited by phosphorylation. Furthermore, addition of alkaline phosphatase had no effect on enolase activity. These findings suggest that factors other than phosphorylation regulate enolase activity under anaerobic stress . Likewise, since the properties of purified enolase from the two spec ies are almost identical, the differential induction of activity under anoxia cannot be ascribed to possible differences in catalytic functi ons between the two enzymes.