The anaerobic starch breakdown into end-products in the green alg Chiamydomonas reinhardtii F-60 has been investigted in the dark and in the light. The effects of 343,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) on the fermentation in the light have also been investigted.Anaerobic starch breakdown rnte (13.1 ± 3.5 micromoles C per milligram chlorophyll per hour) is increased 2-fold by FCCP in the dark. Light (100 watts per square meter) decreases up to 4-fold the dark rate, an inhibition reversed by FCCP. Stimulation of starch breakdown by the proton ionophore FCCP points to a pH-controlled rate-limiting step in the dark, while inhibition by light, and its reversal by FCCP, indicates a control by energy charge in the light.In the dark, formate, acetate, and ethanol are formed in the ratios of 2.07:1.07:0.91, and account for roughly 100% of the C from the starch.H2 production is OA3 mole per mole glucose in the starch. Glycerol >-lactate, and CO2 have been detected in minor amounts.In the light, with DCMU and FCCP present, acetate is produced in a 1:1 ratio to formate, and H2 evolution is 2.13 moles per mole glucose. When FCCP only is present, acetate production is lower, and CO2 and H2 evolution is 1.60 and 4.73 moles per mole glucose, respectively.When DCMU alone is present, CO2 and H2 photoevolution is higher than in the dark. Without DCMU, CO2 and H2 evolution is about 100% higher than in its presence. In both conditions, acetate is not formed. In all conditions in the light, ethanol is a minor product. Formate production is least affected by light.The stoichiometry in the dark indicates that starch is degraded via the glycolytic pathway, and pyruvate is broken down into acetyl- (3,19,20,28).Classical glycolysis followed by subsequent metabolism of the pyruvate to the various end-products has been proposed to account for the anaerobic catabolism of starch or glucose in the green algae (17,19,27). Except for H2 evolution, the effect of light on fermentative carbon flow has received little attention. This is principally due to the problems involved with photosynthetic fixation of the CO2 that might be evolved fermentatively. The first to attempt to resolve this question were Klein and Betz (20) who reported that light had no effect on the rate of starch breakdown or the pattern of fermentation in Chlamydomonas moewusii. But they used extremely low levels of light (160 lux (2) made use of C. reinhardtii F-60, a mutant characterized by an incomplete photosynthetic carbon reduction pathway but an intact photosynthetic electron transport chain, to monitor 'true' CO2 and H2 evolution. To account for their results, they proposed an involvement between anaerobic carbohydrate metabolism and the photosynthetic electron transport chain, implying that carbohydrate degradation is entirely or partially localized in the chloroplast. The purpose of our study was to establish for the first time a complete fermentative balance in C. reinhardtii F-60 between...

A method for cellular fractionation of Chiamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for ceil fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.The biochemistry and physiology of unicellular green algae have been studied intensively because these algae can be maintained easily under laboratory conditions. Cultures can be synchronized (21) and some strains grow heterotrophically as well as autotrophically and mixotrophically. Many similarities with the metabolism of higher plants have been established. The pathway of photosynthetic CO2 reduction was primarily elucidated with unicellular green algae. Enzymes of the glyoxylate cycle and glycolate metabolism, previously found in higher plants, could also be measured in algae, proving the general occurrence of these pathways in plants. Although it was possible to relate these pathways and cycles to cellular organelles in higher plants, very few investigations are reported on the compartmentation of metabolism in unicellular green algae. The first detailed study of localization of enzymes in a unicellular alga of the Chlamydomonas type was made by Kombrink and Wober (16) who were able to demonstrate the activity of starch-metabolizing enzymes in chloroplasts of Dunaliella marina. In their investigation, they used a new method involving DEAE-dextran for cell lysis.One reason for the difficulties in cell fractionation of unicellular

The anaerobic photodissimilation of acetate by Chlamydomonas reinhardii F-60 adapted to a hydrogen metabolism was studied utilizing manometric and isotopic techniques. The rate of photoanaerobic (N2) acetate uptake was approximately 20 moles per milligram chlorophyll per hour or one-half that of the photoaerobic (air) rate. Under N2, cells produced 1.7 moles H2 and 0.8 mole CO2 per mole of acetate consumed. Gas production and acetate uptake were inhibited by monofluoroacetic acid (MFA), 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and by H2. Acetate uptake was inhibited about 50% by 5% H2 (95% N2 (3) suggested that acetate increased gas production by consuming ATP which regulated the unspecified sequence of reactions giving rise to CO2 and H2. Healey (18) modifying a mechanism put forward by Jones and Myers (20) to explain the Kok effect in blue-green algae, proposed a flow of electrons from acetate via the citric acid cycle into PSI resulting in the photoevolution of H2 from reduced Fd. The operation of an anaerobic and light-dependent citric acid cycle which affects the stoichiometric conversion of acetate to CO2 and H2 had been documented in the photosynthetic purple bacteria ( 13).The present communication summarizes the results of a detailed investigation of the anaerobic photometabolism of acetate by C. reinhardii F-60, with reference to stoichiometry of gas (CO2 and H2) production, incorporation into cellular components, and sensitivity of the process to a variety of inhibitors. The stoichiometric relationships observed, together with the isotopic distribution following assimilation of ["4C]acetate, constitute strong evidence for the conclusion that anaerobic carbon oxidation occurs in part through the reactions of the glyoxylate and citric acid cycles. MATERIALS AND METHODSAlgal Growth Conditions. Chlamydomonas reinhardii (Dangeard) F-60, a mutant strain with an incomplete photosynthetic carbon reduction cycle but with an intact photosynthetic electron transport chain, was obtained from R. K. Togasaki, Indiana University. Cells were grown in batch cultures on an acetatesupplemented medium (14)

Preparations of TPN-linked nonreversible D-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible D-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The Plant tissues contain three enzymes for the oxidation of D-G3P' (12). Two of these catalyze a Pi-dependent, reversible oxidation of D-G3P to 1,3-diP-glycerate; one enzyme is DPNspecific and found in both photosynthetic and nonphotosynthetic tissues, while the other uses TPN and is most probably confined to the chloroplast. Both enzymes have been extensively studied (29). The third enzyme, which is TPN-specific, oxidizes D-G3P to glycerate-3-P in a reaction which does not require Pi and is not reversible. Evidence for this enzyme activity in green leaves was reported by leaves. Recently, Jacob and d'Auzac (17) have purified the enzyme from the latex of Hevea brasiliensis. In this laboratory, the partially purified enzyme from pea shoots was inhibited strongly by L-G3P (19). It is possible that the scarcity of reports on the enzyme is related to the presence of this inhibitor in commercial preparations of the substrate D-G3P.In the present investigation, the significance of this nonreversible D-G3P dehydrogenase to plant carbohydrate metabolism has been evaluated. A preparation of pea shoot enzyme, free of reversible Pi-dependent D-G3P dehydrogenase activity, has now been obtained, and the properties have been compared with those of the same enzyme partially purified from several other plant tissues. In addition, a relatively specific assay for the determination of the enzyme activity in cell-free extracts is described. Changes in the enzyme activity during the germination of peas and castor beans and the distribution of the enzyme in representative tissues were examined.The properties and in vivo localization of the three D-G3P dehydrogenases were compared, and the results indicate that, in some tissues, a major portion of triose-P oxidation may be mediated by the nonreversible enzyme.MATERIALS AND METHODS Materials. Pea (Pisum sativum L. var. Progress No. 9), maize (Zea mays L. var. Early Fortune), peanuts (Arachis hypogea L. var. Jumbo Virginia), and castor bean (Ricinus communis L.) were grown under natural lighting in a glasshouse at 16 to 18 C or in the dark at 22 C.The following chemicals and enzymes were obtained from the Sigma Chemical Company: ATP, dihydroxyacetone-P, DPN, D-erythrose-4-P, D-fructose-1 ,6-diP, DL-G3P, D-glycerate-3-P, P-glycolate, P-hydroxypyruvate, D-ribose-5-P, TPN, TPNH, G3P dehydrogenase, 3-P-glycerate kinase, and triose-P isomerase. Chloroacetol-P was a gift of Dr. F. C. Hartman through the courtesy of Dr. Louise Anderson. All chemicals were prepared as sodium salts at the recommended pH before use.L-G3P was prepared according to the method of Venkataraman and Racker (38). The D-G3P present in 0.65 mmole of DL-G3P was converted to glyce...

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