Activities of enzymes in β-carboxylation reactions and of catalase in cell-free preparations from the symbiotic dinoflagellates Symbiodinium spp. from a coral, a clam, a zoanthid and two sea anemones

Cell-free extracts of cultured and freshly isolated symbiotic dinoflagellates, Symbiodinium spp, isolated from the stony coral Montipora verrucosa , the clam Tridacna maxima , the zoanthid Palythoa sp. and the sea anemone Aiptasia pulchella were assayed for the enzyme systems involved in β-carboxylation and photorespiration. Markedly different levels of phosphoenolpyruvate carboxylase (EC 4 . 1 . 1 .31; PEP-case) activity were demonstrated in extracts from the different algae. The activity of PEP-case in the algae from M. verrucosa was highest, being an order of magnitude higher than PEP-case in algae from Palythoa sp. and up to 25-fold higher than that in algae from A. pulchella and T. maxima . The algae from M. verrucosa also exhibited pyruvate-P i dikinase (EC 2 . 7 .9 .1) activity. When these data are combined with previous demonstrations of the existence of NAD malate dehydrogenase and NADP malate dehydrogenase (decarboxylating; ‘malic enzyme’) in these algae, the indications are that they possess functional β-carboxylation enzyme systems. Past demonstrations of the photoassimilation of 14 CO 2 into glycollate by Symbiodinium spp. indicated photorespiration. The demonstration for the first time of activity of catalase (EC 1 . 11 . 1 . 6) when viewed in light of previous demonstrations of other composite enzymes of the photorespiratory pathway (e.g. phosphoglycollate phosphatase, glycollate dehydrogenase), add further confirmation to the disputed existence of a functional photorespiratory system in these symbiotic dinoflagellates.

Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z

Euglena mitochondria contain both glyoxylate reductase (NADP+) and glycollate dehydrogenase to constitute the glycollate-glyoxylate cycle [Yokota & Kitaoka (1979) Biochem. J. 184, 189-192]. Euglena glyoxylate reductase (NADP+) was purified and its submitochondrial location was determined in order to elucidate the cycle. The purified glyoxylate reductase was homogeneous on polyacrylamide-gel electrophoresis. Difference spectra of the purified enzyme revealed that the enzyme was a flavin enzyme. The Mr of the enzyme was 82 000. The enzyme was specific for NADPH, with an apparent Km of 3.9 microM, and for glyoxylate, with an apparent Km of 45 microM. It was 30% as active with oxaloacetate as with glyoxylate. NADH and hydroxypyruvate did not support the activity at all. The optimum pH was 6.45. Submitochondrial fractionation of purified mitochondria showed that the enzyme was located in the intermembrane space and loosely bound to the outer surface of the inner membrane. These properties and the submitochondrial localization of NADPH-glyoxylate reductase facilitate the operation of the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase, which is tightly bound to the inner membrane of Euglena mitochondria.

Occurrence and operation of the glycollate–glyoxylate shuttle in mitochondria of Euglena gracilis Z

Both glyoxylate reductase (NADP+) and glycollate dehydrogenase were located exclusively in mitochondria in Euglena gracilis and constitute the glycollate–glyoxylate shuttle, whose existence in higher plants was thought doubtful, owing to different subcellular locations of the two enzymes. Disrupted Euglena mitochondria showed a glycollate-dependent NADPH oxidation, indicating actual operation of the shuttle in this protozoon.

Glycollate Oxidase and Glycollate Dehydrogenase in Marine Algae and Plants

Glycollate : dichlorophenolindophenol reductase, or glycollate dehydrogenase, was present in 15 green marine algae and one blue-green alga collected around Lizard Island, Australia. No enzyme activity was found in red or brown algae or zooxanthellae. Glycollate dehydrogenase was also found in the marine grasses Cymodocea and Thalassia, and this is the first report of it in higher plants. However, glycollate oxidase was present in the particulate fraction from Halophila, another marine monocotyldonous plant. On Lizard Island, the C3 plants, the C4 salt plants Distichlis and Salsola, and a crassulacean acid metabolism plant, Sesuvium all contained glycollate oxidase.

Oxidative phosphorylation during glycollate metabolism in mitochondria from phototrophic Euglena gracilis

Mitochondria were isolated by gradient centrifugation on linear sucrose gradients from broken cell suspensions of phototrophically grown Euglena gracilis. An antimycin A-sensitive but rotenone-insensitive glycollate-dependent oxygen uptake was demonstrated in isolated mitochondria. The partial reactions of glycollate-cytochrome c oxidoreductase and cytochrome c oxidase were demonstrated by using Euglena cytochrome c as exogenous electron acceptor/donor. Isolated mitochondria contain glycollate dehydrogenase and glyoxylate-glutamate aminotransferase and oxidize exogenous glycine. A P:O ratio of 1.7 was obtained for glycollate oxidation, consistent with glycollate electrons entering the Euglena respiratory chain at the flavoprotein level. The significance of these results is discussed in relation to photorespiration in algae.

The localization of glycollate-pathway enzymes in Euglena

Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-α-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae.