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 K(m) values of HPR-2 for hydroxypyruvate and glyoxylate were 0.7 and 1.1 millimolar, respectively, while the K(m) of GR-1 for glyoxylate was 0.07 millimolar. The K(m) 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 (K(i) 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.