PLASTID CLASS-I AND CYTOSOL CLASS-II ALDOLASE OF EUGLENA-GRACILIS - PURIFICATION AND CHARACTERIZATION

The plastidic class I and cytosolic class II aldolases of Euglena grac ilis have been purified to apparent homogeneity. In autotrophically gr own cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg prote in by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of fou r identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)(2)SO4 fractionation, anion- exchange chromatography, chromatography on hydroxylapatite, and gel fi ltration. The native enzyme (molecular mass 80 kD) consisted of two id entical subunits of 38 kD. The K-m (fructose-1,6-bisphosphate) values were 12 mu M for the class I enzyme and 175 mu M for the class II enzy me. The class II aldolase was inhibited by 1 mM ethylenediaminetetraac etate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, R b+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored b y Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is pr oposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.