Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism

Abstract

Protoplasts purified from pea (Pisum sativum L.) leaves were lysed and fractionated to assess the subcellular distribution of NADPH-dependent hydroxypyruvate reductase (NADPH-HPR) activity. Rate-zonal centrifugation and sucrose-gradient experiments demonstrated that most (about 70%) of the NADPH-HPR activity was located in the supernatant or cytosol fraction. Detectable, but relatively minor activities were associated with the chloroplast fraction (up to 10% on a chlorophyll basis when compared to the lysate) and with peroxisomes. The minor NADPH-HPR activity in the peroxisomes could be fully accounted for by the secondary NADPH-dependent activity of NADH-dependent HPR. The subcellular distribution of NADPH-HPR followed closely that previously determined for NADPH-dependent glyoxylate reductase (NADPH-GR), an enzyme localized predominantly in the cytosol of pea leaf protoplasts (CV Givan et al. 1988 J Plant Physiol 132: 593-599). Low activities of both NADPH-HPR and NADPH-GR were also found in purified chloroplasts prepared by mechanical homogenization of Pisum and Spinacia leaves. In pea and spinach chloroplasts, rates of both NADPH-HPR and NADPH-GR were lower than the activity of the NADH-dependent GR. The results are discussed in relation to a possible role for NADPH-HPR in the oxidative carbon pathway of photorespiration. Both NADPH-HPR and the GRs could function as auxiliary reactions to photorespiration, utilizing hydroxypyruvate and/or glyoxylate 'leaked' or otherwise exported from peroxisomes. NADPH-HPR function might be especially significant under conditions of limiting NADH supply to peroxisomes, with extraperoxisomal reduced pyridine nucleotide acting as the reductant.Hydroxypyruvate is a primary metabolite in the photorespiratory carbon pathway. Hydroxypyruvate formation in leaf cells occurs primarily in peroxisomes as a result ofthe transamination of serine by serine-glyoxylate aminotransferase (8). Serine-glyoxylate aminotransferase is accompanied by the peroxisomal

Identification of Hydroxypyruvate and Glyoxylate Reductases in Maize Leaves

“…The burst was attributed to nonspecific action of peroxisomal HPR-1, since it could be completely eliminated by preincubation with the antibodies against this enzyme (17). However, no burst was observed for the NADPH-dependent activities ofthe HPR when assayed in the peroxisomal fraction of pea leaf protoplasts (16) or for the purified peroxisomal enzyme from spinach (data not shown), which suggested an involvement ofsome additional factor(s) in this phenomenon. Based on this evidence, and finding that the burst strongly depends on the duration of NADPH-pretreatment (Fig.…”

“…allowing for some preincubation with NADPH (16)(17)(18). The burst was attributed to nonspecific action of peroxisomal HPR-1, since it could be completely eliminated by preincubation with the antibodies against this enzyme (17).…”

“…HPR-2 is localized largely in the cytosol ( 16) and, in analogy to the peroxisomal reductase, shows nonspecific rates with glyoxylate (18). NADPH(NADH)-GR is also mostly cytosol based (11,16), it is specific for glyoxylate (19), and should be considered as an important glycolate-generating reaction in leaves (17,19,20). Both enzymes have been proposed to serve as back-up reactions of the glycolate pathway, utilizing glyoxylate and hydroxypyruvate generated in the peroxisomes and 'leaked' or otherwise exported to the cytosol (1 1, [16][17][18][19][20].…”

“…HPR-2 is localized largely in the cytosol ( 16) and, in analogy to the peroxisomal reductase, shows nonspecific rates with glyoxylate (18). NADPH(NADH)-GR is also mostly cytosol based (11,16), it is specific for glyoxylate (19), and should be considered as an important glycolate-generating reaction in leaves (17,19,20).…”

“…NADPH(NADH)-GR is also mostly cytosol based (11,16), it is specific for glyoxylate (19), and should be considered as an important glycolate-generating reaction in leaves (17,19,20). Both enzymes have been proposed to serve as back-up reactions of the glycolate pathway, utilizing glyoxylate and hydroxypyruvate generated in the peroxisomes and 'leaked' or otherwise exported to the cytosol (1 1, [16][17][18][19][20]. The GR recycles glyoxylate back to the glycolate level, the latter is probably transported to the peroxisomes and converted to glycine, while glycerate formed by the cytosolic HPR may be directly exported to the chloroplasts to be metabolized to 3-phosphoglycerate by glycerate kinase (13).…”