Yellow Mutant of Chlorella vulgaris

An antiserum to tobacco glycolate oxidase has been prepared by injection of the purified enzyme into rabbits. Double gel diffusion tests between the antiserum and purified antigen and also with a crude tobacco preparation gave a single immunoprecipitation band. Crude extracts of Euglena gracilis Z Klebs, containing glycolate dehydrogenase, and of Chlorella vulgaris 211-llh/20, containing glycolate oxidase, also formed single bands with the tobacco antiserum. The algal bands were identical and showed partial identity with the tobacco band. The antiserum inhibited the glycolate oxidase activities of the tobacco and Chlorella extracts but did not affect Euglena glycolate dehydrogenase activity. All higher plants so far examined have been shown to contain an enzyme for the oxidation of glycolate. The enzyme, glycolate oxidase (glycolate: oxygen oxidoreductase E.C. 1.1.3.1.), is a FMN-flavoprotein and catalyzes the transfer of electrons from glycolate to 0,, forming glyoxylate and H.,O (1, 26, 27, 29). Artificial dyes such as DCPIP1 and methylene blue can function as electron acceptors in the reaction (3, 8), and the enzyme may, under certain conditions, donate electrons to quinones (5, 11, 12). Glycolate-oxidizing enzymes have also been detected in several unicellular green algae (3, 15, 19, 28). The enzyme prepared from Euglena gracilis Z. Klebs, Chlorella pyrenoidosa 211/8p, and Chiamydomonas reinhardtii Danglard (-) 90, all from the Cambridge Culture Collection, functions with DCPIP as electron acceptor but does not link to oxygen (3). Similar results were obtained with enzyme preparations from E. gracilis and Chlamydomonas reinhardtii (-) 90 from the Indiana Algal Culture Collection and with Acetabularia mediterranea (19, 20). Furthermore, a glycolate-oxidizing enzyme with no 02 requirement has been found in the blue-green algae Anabaena flos-aquae and an Oscillatoria species (9). No requirement for FMN was observed in the green algal and A. flos-aquae preparations, although a slight enhancement by FMN was noted with the Oscillatoria extract (3, 9, 20, 28). This enzyme found in the above algae is referred to in the 'Abbreviations: DCPIP: 2,6-dichlorophenolindophenol; DEAE: diethylaminoethyl. literature as glycolate dehydrogenase. Neither the higher plant nor the algal enzymes described show an absolute specificity for glycolate. The former can also oxidize L-lactate but not the D-isomer (1). The algal enzymes readily use D-lactate as a substrate but only slowly attack the L-form (10, 20). An algal glycolate-oxidizing enzyme showing a mixture of the criteria described has been characterized. Intact cells and enzyme preparations from green Chlorella vulgaris 211-llh (Algal Culture Collection, Gottingen) and the chlorophyllfree mutant Chlorella vulgaris 211-1 lh/20 (25) show a glycolate-dependent oxygen consumption, similar to that of higher plants (14). However, the addition of FMN does not affect enzyme activity in vitro, and both the nand L-lactate isomers are oxidized, at approximately equal rates (6, 14). The present investigation was carried out to characterize further plant glycolate-oxidizing enzymes from a serological approach, using an antiserum to the pure tobacco enzyme. Effects of this antibody to glycolate oxidase on immunoprecipitation and inhibition of the homologous enzyme are reported. The antibody has also been tested against enzyme preparations from E. gracilis, taken as an example of the algal glycolate dehydrogenase and against Chlorella vulgaris 211-1lh/20, which exhibits glycolate oxidase activity. MATERIALS AND METHODS Plant Material. Nicotiana tabacum L. cv. John Williams' Broadleaf was grown as described previously (23). Euglena gracilis Z. Klebs was cultured phototrophically with air (3) on the inorganic medium of Cramer and Myers (2). The yellow mutant Chlorella vulgaris 211-11 h/20 was grown as reported previously (14), on a glucose medium (13). Crude Enzyme Preparations. All procedures were performed at 0 to 4 C. Tobacco. One hundred grams of leaves (8-10 weeks old) were washed, and their midribs were removed. The leaves were homogenized with 200 ml of 50 mM tris-HCl buffer, pH 8.0, containing 0.25 M sucrose, for 30 sec in a Braun mixer. After filtration through a Schleicher and Schiill 6206 II 1/2 paper, the suspension was centrifuged at 600g for 20 min. The supernatant was then spun at 18,000g for 45 min, and the resulting supernatant was retained. Euglena. Exponential phase cells were harvested by centrifugation at 2,000g for 5 min, washed twice in 0.1 M K2HPO3, and disrupted by passage through a French pressure cell (18) at 4 tons/cm2. The extract was spun at 17,000g for 30 min, and the supernatant was decanted and used for experimental purposes.