Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: II. Assimilation of Ammonium by Anaerobic Cells

The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH₄⁺ in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH₄⁺ to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH₄⁺ assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH₄⁺ assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH₄⁺ assimilation under anoxia was via glutamine synthetase-glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH₄⁺ assimilation.     

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product

The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga.     

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: III. Alanine Is the Product of Anaerobic Ammonium Assimilation

We have determined the flow of ¹⁵N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of ¹⁵NH₄⁺ to aerobic or anaerobic cells. Under aerobic conditions, only a small proportion of the N assimilated was retained in the free amino acid pool. However, under anaerobic conditions almost all assimilated NH₄⁺ accumulates in alanine. This is a unique feature of anaerobic NH₄⁺ assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH₄⁺ assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH₄⁺ assimilation.     

Dark Ammonium Assimilation Reduces the Plastoquinone Pool of Photosystem II in the Green Alga Selenastrum minutum

The impact of dark NH₄⁺ and NO₃⁻ assimilation on photosynthetic light harvesting capability of the green alga Selenastrum minutum was monitored by chlorophyll a fluorescence analysis. When cells assimilated NH₄⁺, they exhibited a large decline in the variable fluorescence/maximum fluorescence ratio, the fluorescence yield of photosystem II relative to that of photosystem I at 77 kelvin, and O₂ evolution rate. NH₄⁺ assimilation therefore poised the cells in a less efficient state for photosystem II. The analysis of complementary area of fluorescence induction curve and the pattern of fluorescence decay upon microsecond saturating flash, indicators of redox state of plastoquinone (PQ) pool and dark reoxidation of primary quinone electron acceptor (Q_A), respectively, revealed that the PQ pool became reduced during dark NH₄⁺ assimilation. NH₄⁺ assimilation also caused an increase in the NADPH/NADP⁺ ratio due to the NH₄⁺ induced increase in respiratory carbon oxidation. The change in cellular reductant is suggested to be responsible for the reduction of the PQ pool and provide a mechanism by which the metabolic demands of NH₄⁺ assimilation may alter the efficiency of photosynthetic light harvesting. NO₃⁻ assimilation did not cause a reduction in PQ and did not affect the efficiency of light harvesting. These results illustrate the role of cellular metabolism in the modulating photosynthetic processes.     

Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum

In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH₄⁺ addition we found that: (a) N accumulated in glutamine while glutamate and α-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH₄⁺ addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH₄⁺ addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.     

Steady-State Chlorophyll a Fluorescence Transients during Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum

The assimilation of ammonium by the N-limited green alga Selenastrum minutum results in the suppression of photosynthetic electron flow from H₂O to CO₂ (6, 7, 18). In this study, results are presented which describe the correponding change in steady-state chlorophyll a fluorescence. The addition of ammonium resulted in a transient decline in fluorescence followed by a marked increase. Fluorescence did not return to control levels until the added ammonium had been assimilated. Analysis of the fluorescence transients showed that ammonium assimilation resulted in a rapid increase in nonphotochemical quenching (Q_e) peaking 10 to 15 seconds after ammonium addition. Q_e then decreased dramatically reaching a minimum value approximately 45 seconds following ammonium addition and returned to the control level only after the added ammonium had been assimilated. There were no effects of ammonium addition on photochemical quenching (Q_q) for approximately 10 to 15 seconds at which time both gross O₂ evolution (as measured by mass spectrometry) and Q_q declined. In the presence of d,l-glyceraldehyde or when cells were held at the CO₂ compensation point, the addition of ammonium resulted in a decline in Qe 10 to 15 seconds after addition. The Q_e peak and the Q_q decline were absent. These results imply that the transient increase in Q_e and the subsequent decline in Q_q may be attributed to the decline in Calvin cycle activity during ammonium assimilation. The decline in Q_e is apparently a direct result of ammonium assimilation. The observation that the Q_e peak precedes the Q_q decline would be consistent with the decreases in Calvin cycle carbon flow occurring at the kinase reactions prior to glyceraldehyde-3-phosphate dehydrogenase.     

Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum

A unique phosphoribulokinase (ADP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19) has been purified to homogeneity from the green alga Selenastrum minutum. The enzyme has a native molecular mass of about 83 kilodaltons and a native isoelectric point of 5.1. The enzyme consists of two different-sized subunits of 41 and 40 kilodaltons, implying that it is a heterodimer. This is the first report of a eukaryotic heterodimeric phosphoribulokinase. The in vivo existence of two nonidentical subunits of S. minutum phosphoribulokinase was confirmed by western blot analysis of crude protein extracts from trichloroacetic acid-killed cells. These two subunits were immunologically similar, as rabbit immunoglobulin G affinity purified against the 41 kilodalton subunit of S. minutum phosphoribulokinase (PRK) cross-reacts with the 40 kilodalton subunit and vice versa. Antibodies against S. minutum phosphoribulokinase also cross-react with the spinach enzyme. NH₂-terminal sequencing revealed that the two S. minutum PRK subunits shared a considerable degree of structure homology with each other and with the enzymes from spinach and Chlamydomonas reinhardtii, but not with PRK from Rhodobacter sphaeroides. There are, however, differences between the NH₂-terminal amino acid sequences of the two S. minutum PRK subunits, that imply that they are the products of separate genes or products of two different mRNAs spliced from a single gene.     

Ammonium Assimilation Requires Mitochondrial Respiration in the Light : A Study with the Green Alga Selenastrum minutum

Mass spectrometric analysis of O₂ and CO₂ exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH₄Cl resulted in nearly a 250% increase in the rate of TCA cycle CO₂ efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O₂ consumption in both light and dark. Anaerobiosis inhibited the CO₂ release caused by NH₄Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O₂ consumption in the light. This implied that the Mehler reaction did not play a significant role in O₂ consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions.     

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

The assimilation of NH₄⁺ causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH₄⁺ assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.     

Fructose 1,6-Bisphosphatase in the Green Alga Selenastrum minutum: I. Evidence for the Presence of Isoenzymes

Two isoforms of fructose 1,6-bisphosphatase are present in the green alga Selenastrum minutum. The isoenzymes can be separated with ionexchange chromatography or acid precipitation. The stability of the two isoenzymes differ largely. The acid insoluble enzyme exhibits properties similar to that of the enzyme from the chloroplasts of higher plants, i.e. an alkaline pH optima in the absence of reductant, a lower affinity for substrate, strong inhibition by phosphate, and a low sensitivity to fructose-2,6-bisphosphate and AMP. The more abundant form of the enzyme exhibits several properties indicative of heterotrophic fructose 1,6 bisphosphatases, i.e. a high affinity for substrate and sensitivity toward fructose-2,6-bisphosphate and AMP. but is absolutely dependent on a reductant for stability and activity. Evidence is provided indicating that previously reported purification protocols cause inactivation of one of the isoenzymes which could lead to the erroneous conclusion that algae have a single fructose 1,6-bisphosphatase isoenzyme.     

Activation of Respiration to Support Dark NO(3) and NH(4) Assimilation in the Green Alga Selenastrum minutum

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO₃⁻ or NH₄⁺ assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH₄⁺ was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO₃⁻ addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO₃⁻ assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO₃⁻ assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH₄⁺ assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO₃⁻ assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis.     

Significance of Phosphoenolpyruvate Carboxylase during Ammonium Assimilation: Carbon Isotope Discrimination in Photosynthesis and Respiration by the N-Limited Green Alga Selenastrum minutum

The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO₂ fixation and respiratory CO₂ release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and α-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C₃ organism.     

Molecular, Kinetic, and Immunological Properties of the 6-Phosphofructokinase from the Green Alga Selenastrum minutum: Activation during Biosynthetic Carbon Flow

The ATP:d-fructose-6-phosphate 1-phosphotransferase (PFK) from Selenastrum minutum was purified to homogeneity. The purified plastid enzyme had a specific activity of 180 micromoles per milligram of protein per minute. It is a homomer with a subunit molecular weight of 70,000. The smallest enzymatically active form of the protein is a homotetramer of 280,000 daltons. The enzyme can, however, aggregate into different active forms, the largest of which has a molecular weight of more than 6 × 10⁶. The pH optimum, regardless of aggregation state, is 7.25. The enzyme exhibits sigmoidal kinetics with respect to fructose-6-phosphate and hyperbolic kinetics with respect to ATP. Phosphate changes the sigmoidal fructose-6-phosphate saturation kinetics to hyperbolic. Phosphoenolpyruvate, 3-phosphoglycerate, 2-oxoglutarate, malate, citrate and ATP all inhibit the enzyme. The ratios of phosphoenolpyruvate and/or 3-PGA to phosphate are probably the most important factors regulating PFK activity in vivo. The enzyme cross-reacts with several antisera against both cytosolic and plastidic PFKs as well as against native potato pyrophosphate dependent phosphofructokinase suggesting that the algal PFK represents an evolutionarily primitive form.     

Regulation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum: Properties Associated with Replenishment of Tricarboxylic Acid Cycle Intermediates during Ammonium Assimilation

Two isoforms of phosphoenolpyruvate carboxylase (PEPC) with very different regulatory properties were partially purified from the green alga Selenastrum minutum. They were designated PEPC₁ and PEPC₂. PEPC₁ showed sigmoidal kinetics with respect to phosphoenolpyruvate (PEP) whereas PEPC₂ exhibited a typical Michaelis-Menten response. The S_0.5(PEP) of PEPC₁ was 2.23 millimolar. This was fourfold greater than the S_0.5(PEP) of PEPC₂, which was 0.57 millimolar. PEPC₁ was activated more than fourfold by 2.0 millimolar glutamine and sixfold by 2.0 millimolar dihydroxyacetone phosphate (DHAP) at a subsaturating PEP concentration of 0.625 millimolar. In contrast, PEPC₂ showed only 8% and 52% activation by glutamine and DHAP, respectively. The effects of glutamine and DHAP were additive. PEPC₁ was more sensitive to inhibition by glutamate, 2-oxoglutarate, and aspartate than PEPC₂. Both isoforms were equally inhibited by malate. All of these metabolites affected only the S_0.5(PEP) not the V_max. The regulatory properties of S. minutum PEPC in vitro are discussed in terms of (a) increased rates of dark carbon fixation (shown to be catalyzed predominantly by PEPC) and (b) changes in metabolite levels in vivo during enhanced NH₄₊ assimilation. Finally, a model is proposed for the regulation of PEPC in vivo in relation to its role in replenishing tricarboxylic acid cycle intermediates consumed in NH₄₊ assimilation.     

Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH₄⁺ assimilation on exogenous CO₂. N-sufficient cells were only able to assimilate NH₄⁺ maximally in the presence of CO₂ and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH₄⁺ assimilation. These results indicate that NH₄⁺ assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO₂ fixation. N-limited cells assimilated NH₄⁺ both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO₂ fixation was not required for NH₄⁺ assimilation. Using CO₂ removal techniques reported previously in the literature, we were unable to demonstrate CO₂-dependent NH₄⁺ assimilation in N-limited cells. However, employing more stringent CO₂ removal techniques we were able to show a CO₂ dependence of NH₄⁺ assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO₂ requirements for NH₄⁺ assimilation. The first is as a substrate for photosynthetic CO₂ fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.     

Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum

The rate of NH₄⁺ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH₄⁺ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H¹⁴CO⁻₃ into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C₃ organism.     

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.     

Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.