IDENTIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE AND RELATED ENZYMES FROM THE ENDOCYANOTIC ALGA CYANOPHORA-PARADOXA AND FROM PEA LEAVES

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophor a paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O2 or H2O2 and glyoxylate, respectively. Aerobically, the enzyme did not red uce the artificial electron acceptor dichlorophenol indophenol. Howeve r, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichloro phenol indophenol reductase activity was detectable. The leaf GO inhib itor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to t he flavin moiety of the active site of leaf GO, inhibited Cyanophora G O and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea G O cannot oxidize D-lactate. In contrast to GO from pea or other organi sms, the affinity of Cyanophora GO for L-lactate is very low (K(m) 25 millimolar). Another important difference is that Cyanophora GO produc ed sigmoidal kinetics with O2 as varied substrate, whereas pea GO prod uced normal Michaelis-Menten kinetics. It is concluded that there is c onsiderable inhomogeneity among the glycolate-oxidizing enzymes from C yanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-depen dent hydroxypyruvate reductase (HPR) and glyoxylate reductase activiti es were detected in Cyanophora. NADH-HPR was markedly inhibited by hyd roxypyruvate above 0.5 millimolar. Variable substrate inhibition was o bserved with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylat e reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate amino transferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.