Kleczkowski LA, Givan CV, Randall DD, Loboda T
Substrate specificity and activities of glyoxylate and hydroxypyruvate reductases from leaf extracts: differentiation by ammonium sulfate fractionation and by immunoprecipitation.
Physiologia Plantarum: 1988 74:763-769
Leaf extracts from seven monocotyledonous and dicotyledonous species
contained considerable levels of NADPH-dependent glyoxylate- and
hydroxypyruvate reductase activities. These activities ranged from 0.02
to 0.22 μmol (mg protein)1 min1.
For all plants tested, the glyoxylate reductase (GR) activity, assayed
with either NADPH or NADH, was sensitive to inhibition by
acetohydroxamate, a glycine analogue. Hydroxypyruvate reductase (HPR)
activities were unaffected by acetohydroxamate. Differential
precipitation of soluble leaf proteins of spinach, pea and barley by
ammonium sulfate (0–45% and 45–60% saturation) indicated the presence
of at least three distinct reductases, which differed in their
specificities for glyoxylate, hydroxypyruvate and NAD(P)H. For all
species, the NADH-dependent HPR-activity was almost completely
precipitated by low ammonium sulfate concentration (45%), while
precipitation of the NADPH-GR, NADH-GR and, to some extent, NADPH-HPR
activities required 60% ammonium sulfate. The NADPH-dependent GR and
HPR activities had high affinity for glyoxylate and hydroxypyruvate,
respectively, as indicated by low apparent Km values of 40–120 μM.
The occurrence of at least three distinct reductases utilizing
hydroxypyruvate and/or glyoxylate as substrate was supported by
antibody-precipitation studies using antibodies prepared against
NADH(NADPH)-HPR, the well-known peroxisomal enzyme that also shows
non-specific GR activity. These data are discussed with respect to
recent reports on the purification and characterization of
NADPH(NADH)-GR, and NADPH (NADH)-HPR, two cytosolic reductases, and the
role is assessed for these enzymes in reducing hydroxypyruvate and
glyoxylate that may be leaked from peroxisomes.
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