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. 1990 Oct;94(2):819–825. doi: 10.1104/pp.94.2.819

Enzymology of the Reduction of Hydroxypyruvate and Glyoxylate in a Mutant of Barley Lacking Peroxisomal Hydroxypyruvate Reductase 1

Leszek A Kleczkowski 1,2,3,2, Gerald E Edwards 1,2,3, Ray D Blackwell 1,2,3, Peter J Lea 1,2,3, Curtis V Givan 1,2,3
PMCID: PMC1077303  PMID: 16667783

Abstract

The use of LaPr 88/29 mutant of barley (Hordeum vulgare), which lacks NADH-preferring hydroxypyruvate reductase (HPR-1), allowed for an unequivocal demonstration of at least two related NADPH-preferring reductases in this species: HPR-2, reactive with both hydroxypyruvate and glyoxylate, and the glyoxylate specific reductase (GR-1). Antibodies against spinach HPR-1 recognized barley HPR-1 and partially reacted with barley HPR-2, but not GR-1, as demonstrated by Western immunoblotting and immunoprecipitation of proteins from crude leaf extracts. The mutant was deficient in HPR-1 protein. In partially purified preparations, the activities of HPR-1, HPR-2, and GR-1 could be differentiated by substrate kinetics and/or inhibition studies. Apparent Km values of HPR-2 for hydroxypyruvate and glyoxylate were 0.7 and 1.1 millimolar, respectively, while the Km of GR-1 for glyoxylate was 0.07 millimolar. The Km values of HPR-1, measured in wild type, for hydroxypyruvate and glyoxylate were 0.12 and 20 millimolar, respectively. Tartronate and P-hydroxypyruvate acted as selective uncompetitive inhibitors of HPR-2 (Ki values of 0.3 and 0.4 millimolar, respectively), while acetohydroxamate selectively inhibited GR-1 activity. Nonspecific contributions of HPR-1 reactions in assays of HPR-2 and GR-1 activities were quantified by a direct comparison of rates in preparations from wild-type and LaPr 88/29 plants. The data are evaluated with respect to previous reports on plant HPR and GR activities and with respect to optimal assay procedures for individual HPR-1, HPR-2, and GR-1 rates in leaf preparations.

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Selected References

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