PLANT PHYSIOLOGY , Vol 106, Issue 3 1137-1144, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)
B. Pelzer-Reith, S. Wiegand and C. Schnarrenberger
Institut fur Pflanzenphysiologie und Mikrobiologie, Freie Universitat Berlin, Konigin-Luise-Strasse 12-16 a, D-14195 Berlin, Germany
The plastidic class I and cytosolic class II aldolases of Euglena gracilis
have been purified to apparent homogeneity. In autotrophically grown 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 protein by anion-exchange
chromatography, affinity chromatography, and gel filtration. The native
enzyme (molecular mass 160 kD) consisted of four identical subunits of 40
kD. The class II aldolase was purified to a specific activity of 21
units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography,
chromatography on hydroxylapatite, and gel filtration. The native enzyme
(molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km
(fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and
175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1
mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5
mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity
up to 7-fold. After inactivation by EDTA, the activity could be partially
restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases
is proposed based on (a) activation/inhibition by Cys and (b) activation or
not by divalent ions.
