Purification, Properties and Phosphorylation of Anaerobically Induced Enolase inEchinochloa phyllopogonandE. crus-pavonis

摘要

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) activity is differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon(Stev.) Koss and the flood-intolerant speciesE. crus-pavonis(H.B.K.) Schult. To examine the regulation of enolase at the protein level, we purified the enzyme from both species to near homogeneity and compared their physico-chemical and catalytic properties. Enolase purified fromE. phyllopogonexhibits optimal activity at pH 7.0, aKmof 80μM for 2-PGA, a Q10of 1.97 and anEaof 12.3 kcal mol-1. Similarly, enolase fromE. crus-pavonisexhibits optimal activity at pH 7.0, aKmof 50μM for 2-PGA, a Q10of 2.04 and anEaof 12.9 kcal mol-1. The enzyme from both species is thermostable (100active after 15 min, 50°C) and is a homodimer of 52.5 kDa subunits as resolved by SDS-PAGE and immunoblotting.E. phyllopogonenolase was phosphorylated in vitro using either γ-32PATP or γ-32PGTP; 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 species are almost identical, the differential induction of activity under anoxia cannot be ascribed to possible differences in catalytic functions between the two enz