Green Alga Selenastrum Minutum Research Articles

In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum.

Previously, we described two distinct classes of phosphoenolpyruvate carboxylase (PEPC) isoforms in the green alga Selenastrum minutum. Class 1 PEPC (PEPC1) is a homotetramer composed of 102 kDa subunits (p102), whereas Class 2 PEPCs exist as three large protein complexes (PEPC2-PEPC4) containing varying proportions of structurally dissimilar p102 and 130 kDa (p130) PEPC catalytic subunits. In the current study, a p102 calcium-independent protein kinase was shown to co-purify with PEPC1, but not PEPC2. However, the p130 subunit of PEPC2 was phosphorylated in vitro during its incubation in the presence of [gamma-(32)P]ATP and a clarified algal extract. Treatment of purified PEPC2 with protein phosphatase 2A(2) increased its apparent M(r) as judged by Superose 6 gel filtration chromatography. The presence of the protein phosphatase inhibitors NaF and microcystin-LR throughout PEPC purification significantly influenced the activity and structural organization of Class 2, but not Class 1, PEPC isoforms. The results are consistent with the notion that under the culture conditions employed: (i) Class 1 and Class 2 PEPC isoforms exist in vivo mainly in their dephosphorylated and phosphorylated forms, respectively, and (ii) phosphorylation of Class 2 PEPCs leads to a significant reduction in their activity and native M(r). We propose that protein kinase-mediated phosphorylation is involved in the control and structural organization of green algal PEPC.

Iron acquisition by the green alga Selenastrum minutum: Growth in iron-limited chemostats and effects of chelator stability constant

The unicellular green alga Selenastrum minutum was successfully cultured in an iron-limited chemostat system, under a wide range of iron-limited growth rates. Steady-state biomass was a function of the concentration of the limiting nutrient (Fe 3+ ) in the medium. Respiratory and photosynthetic activities both increased with increasing iron-limited growth rate of S, minutum, as did the amount of chlorophyll per cell. Contrary to a Strategy I green alga, Chlamydomonas reinhardtii, which displays greatly enhanced plasma membrane ferric chelate reductase activity as a result of iron limitation, cells of S. minutum displayed low activity of this enzyme under both iron-limited and iron-sufficient culture conditions. As well, the addition of BPDS, a strong chelator of Fe 2+ , to iron-limited S. minutum chemostat cultures had no discernable effect on culture biomass. These results strongly suggest that S. minutum does not use a Strategy I mechanism of iron acquisition. In contrast to C. reinhardtii, there was an inverse relationship between the stability constant of the ferric chelator (HEDTA, EDDHA, HBED) and steady state S. minutum culture biomass. However, the calculated free Fe 3+ levels provided a poor estimate of iron-limited S. minutum biomass, suggesting that S. minutum has the ability to directly access chelated Fe 3+ . There was no evidence for production of chelators or siderophores as a response to iron limitatio, possibly suggesting that Strategy II iron acquisition is also not involved. The experiments described in this paper represent the first description of a successful iron-limited chemostat culture system of a non-Strategy I organism, and clearly indicate that the iron acquisition mechanism of S. minutum differs from the wellcharacterized Strategy I system of C. reinhardtii.

METABOLIC RESPONSES OF THE DIATOM ACHNANTHES BREVIPES (BACILLARIOPHYCEAE) TO NUTRIENT LIMITATION

The diatom Achnanthes brevipes C.A. Ag. was cultured in the presence of limiting concentrations of nitrogen (N) or inorganic phosphate (Pi). Growth, in terms of final yield, was more affected by N limitation than Pi limitation; N limitation had a greater effect also on protein and chlorophyll content. Carbohydrate concentrations increased under both nutrient starvation treatments, but N or Pi limitation had different effects. Total (intracellular plus extracellular) sugar content increased when cells were exposed to both types of nutrient limitation, but the extracellular polysaccharide fraction increased only in the presence of Pi starvation. Analyses were performed to identify the metabolic changes occurring in cells exposed to low phosphate because this was the main condition that affected carbohydrate extrusion. Activities of several enzymes involved in carbohydrate metabolism showed that under Pi limitation there was no activation of alternative reactions that were found to result in Pi liberation, instead of its consumption, in some higher plants and in the green alga Selenastrum minutum Naeg. Collins. Results showed that activities of pyruvate kinase, phosphorylating NAD‐dependent 3‐phosphate‐glyceraldehyde dehydrogenase, and 3‐phospho‐glycerate kinase were inhibited under Pi‐limited conditions compared with control cells, indicating limited glucose catabolism. Activity of uridine diphosphate glucose pyrophosphorylase, a key enzyme for the biosynthesis of the storage compound crysolaminarin, was also partly inhibited in Pi‐stressed cells. Our findings suggest that carbohydrate catabolism in A. brevipes is limited under Pi deficiency, whereas extracellular extrusion of carbohydrate is favored.

Interactions between Cu(II), Mn(II) and salicylhydroxamic acid in determination of algal peroxidase activity

Cells of the green alga Selenastrum minutum have a high capacity for O 2 consumption mediated by extracellular peroxidase. The rate of peroxidase-mediated O 2 consumption is greatly stimulated by the addition of salicylhydroxamic acid (SHAM), and the O 2 consumption rate is further enhanced by Mn(II) in the presence of SHAM; Mn(II) has no effect on the rate of O 2 consumption by cells in the absence of SHAM. Both in the presence and absence of Mn(II), SHAM-stimulated peroxidase-mediated cellular O 2 consumption is inhibited by Cu(II). In the absence of Mn(II), inhibition is probably via chelation of Cu(II) by SHAM. The combination of Mn(II) and SHAM results if O 2 consumption even in the absence of cells in a peroxidase-free system. This latter O 2 consumption is 50% inhibited by catalase or superoxide dismutase, and completely inhibited by low concentrations of Cu(II). In this latter case, Cu(II) is not acting by chelation of the SHAM. Contrary to some reports in the literature, in S. minutum there is no evidence that the inhibition of peroxidase by Cu(II) is solely due to superoxide dismutase-like properties of Cu(II). Furthermore, it is suggested that the combination of Mn(II) and SHAM should not be used to activate peroxidase activity due to the potential for high rates of non-enzymatic O 2 consumption.

Interactions between inorganic phosphate (Pi) assimilation, photosynthesis and respiration in the Pi‐limited green alga Selenastrum minutum*

ABSTRACTInorganic phosphate (Pi)‐limited chemostat cultures of the green alga Selenastrum minutum were employed to investigate interactions between Pi assimilation, respiration and photosynthetic processes. Changes in net and gross gas exchange rates indicated that O2 evolution decreases during photosynthetic Pi assimilation. Room temperature and 77K Chi a fluorescence measurements revealed that this photosynthetic suppression is correlated with a transition from state 1 to state 2. Substantial photosynthetic Pi uptake rates occur in the presence of DCMU and KCN. Additionally, the cellular ratio of ATP:NADPH increases following Pi enrichment, suggesting that the ratio of cyclic to linear electron flow is enhanced in response to the high energy requirements of Pi uptake. Net starch degradation was observed during photosynthetic Pi assimilation and the cellular pool size of 3‐phosphoglycerate increased; however, gross gas exchange parameters and cellular metabolite pool sizes indicated that mitochondrial respiration plays a smaller role during Pi assimilation in the light than it does in the dark. These observations were used to formulate a model depicting possible interactions between photosynthetic electron flow, photosynthetic and respiratory carbon metabolism and metabolite exchange between the chloroplast, cytosol and mitochondrion during photosynthetic Pi assimilation.

Purification and Properties of Four Phosphoenolpyruvate Carboxylase Isoforms from the Green AlgaSelenastrum minutum:Evidence That Association of the 102-kDa Catalytic Subunit with Unrelated Polypeptides May Modify the Physical and Kinetic Properties of the Enzyme

Four isoforms of phosphoenolpyruvate carboxylase (PEPC1, PEPC2, PEPC3, PEPC4) have been purified from the green algaSelenastrum minutum.PEPC1 is a homotetramer with a subunitMrof 102 kDa. PEPC2, PEPC3, and PEPC4 have respective nativeMrs of approximately 984, 1186, and 1590 kDa. SDS/PAGE analysis revealed that the latter three isoforms contain polypeptides havingMrs of 102, 73, 70, 65, and 61 kDa. Immunoblot analyses and CNBr cleavage patterns suggest that the 102-kDa polypeptide present in all four isoforms is the same PEPC catalytic subunit. Our data suggest that the three highMrs PEPC isoforms are heteromeric protein complexes consisting of the 102-kDa PEPC1 catalytic subunit and immunologically unrelated polypeptides. Attempts to measure other enzyme activities associated with the protein complexes gave negative results. However, PEPC1 had immunological, physical, and kinetic properties very different from those of the largerMrPEPC isoforms: (i) the anti-PEPC1 immune-serum was relatively inefficient for immunoprecipitating PEPC2, PEPC3, or PEPC4; (ii) immune-serum raised against a mixture of PEPC2, PEPC3, and PEPC4 had relatively weak immunoprecipitating activity toward PEPC1; (iii) PEPC1 was more heat sensitive than the other three isoforms; (iv) PEPC1 had a pH optimum of 9 versus 8.5 for the PEPC protein complexes; (v) the highMrPEPCs had greater apparent affinity for phosphoenolpyruvate compared to PEPC1 and (vi) PEPC1 activity was far more sensitive to metabolite activators (Gln and dihydroxyacetone phosphate) and inhibitors (Asp, Glu, 2-oxoglutarate and malate). We conclude that the interaction of the PEPC catalytic subunit with unrelated polypeptides is responsible for the observed differences between PEPC1 and the high molecular mass isoforms. We propose that this interaction possibly regulates PEPC activityin vivo.

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 μM) resulted in a stimulation of dark O2 consumption, while higher concentrations of azide (1–2 mM) 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 mM 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

Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

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 mM 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 μM and I50= 18.3 μM 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.

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.

PURIFICATION AND CHARACTERIZATION OF TWO FORMS OF PHOSPHOGLYCERATE KINASE FROM THE GREEN ALGA SELENASTRUM MINUTUM1

ABSTRACTWe report the first purification and characterization of a eukaryotic algal phosphoglycerate kinase (PGK), Two forms of PGK (PGK1 and PGK2) from the green alga Selenastrum minutum (Naeg.) Collins were purified to electrophoretic homogeneity with specific activities of 1100 and 1069 units · mg−1 protein, respectively. The portion of PGK1 and PGK2 (probably the cytosolic and chloroplastic forms, respectively) in this organism was estimated as 32 and 68%, respectively. PGK1 was more heat‐stable than PGK2. The Mr estimation for PGK1 and PGK2 by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and gel filtration indicated that they both were monomeric with a similar Mr of approximately 44 kDa. Antibodies raised against S. minutum PGK1 cross‐reacted with PGK2 as well as PGKs from prokaryotic and eukaryotic sources, suggesting that PGK1 was structurally and immunologically closely related to PGK2 and other PGKs, which was consistent with NH2‐terminal sequence analysis.Comparative kinetic and regulatory properties of PGK1 and PGK2 from S. minutum were investigated, Both forms exhibited hyperbolic kinetics with respect to both 3‐phosphoglycerate (3‐PGA) and Mg‐adenosine triphosphate2‐ (MgATP2‐) under the conditions tested and had similar Km values for each substrate (PGK1; Km (MgATP2‐) = 0.37 mM, Km(3‐PGA) = 0.59 mM; PGK2; Km(MgATP2‐) = 0.32 mM, Km(3‐PGA) = 0.46 mM). PGK1 and PGK2, however, differed significantly in several other kinetic properties. PGK2 had a broad pH optimum between 7.3 and 7.8, as compared to PGK1, with a pH optimum of 7.3 Mg2+ was the most efficient cofactor for both forms; it inhibited PGK1 but not PGK2 at higher concentrations (>10 mM). Other divalent cations (Mn2+, Zn2+, Co2+, Cd2+, and Ca2+) only partially replaced Mg2+ and were more effective for PGK1 than for PGK2, A wide range of metabolites was examined for regulatory properties. Energy charge was the most important factor in regulating the two forms of S. minutum PGK. These results were interpreted in light of the regulation of this kinase in response to the cell energy requirement and the need for glycolytic carbon flow to provide carbon skeletons for amino acid biosynthesis.

Activation of Respiration to Support Dark NO3− and NH4+ Assimilation in the Green Alga Selenastrum minutum

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO(3) (-) or NH(4) (+) assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH(4) (+) 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, NO(3) (-) 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 NO(3) (-) assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO(3) (-) assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH(4) (+) assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO(3) (-) 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.

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. NH(2)-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 NH(2)-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.

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

The impact of dark NH(4) (+) and NO(3) (-) assimilation on photosynthetic light harvesting capability of the green alga Selenastrum minutum was monitored by chlorophyll a fluorescence analysis. When cells assimilated NH(4) (+), 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 O(2) evolution rate. NH(4) (+) 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 NH(4) (+) assimilation. NH(4) (+) assimilation also caused an increase in the NADPH/NADP(+) ratio due to the NH(4) (+) 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 NH(4) (+) assimilation may alter the efficiency of photosynthetic light harvesting. NO(3) (-) 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.

The inorganic carbon requirements for nitrogen assimilation

In the green alga Selenastrum minutum (Naeg.) Collins the assimilation of NH4+ into the full suite of protein amino acids requires at least three separate and distinct inorganic carbon fixing reactions, catalyzed by the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), and carbamoyl phosphate synthetase. In this paper we examine the requirements for CO2 fixation of NH4+ assimilation in this organism. When grown under N-sufficient conditions, NH4+ assimilation is directly dependent upon photosynthetic CO2 fixation to provide carbon skeletons for amino acid synthesis. When cultured under N-limited conditions, the cells accumulate starch, which is then available for amino acid synthesis. This alleviates the requirement of photosynthetic CO2 fixation for NH4+ assimilation. N-limited cells, however, still exhibit a nonphotosynthetic CO2 requirement for N assimilation that is mediated through PEPC. This activity of PEPC increases during N assimilation to replenish TCA cycle intermediates consumed during amino acid synthesis. The in vivo activity of this enzyme is tightly regulated so that there are ~0.3 moles C fixed per mole N assimilated. In S. minutum PEPC is regulated primarily by the ratio of glutamine/glutamate, thus providing a mechanism by which primary NH4+ assimilation modulates the supply of carbon for amino acid biosynthesis. Activation of PEPC during NH4+ assimilation occurs in both the light and the dark. Key words: dissolved inorganic carbon, nitrogen assimilation, phosphoenolpyruvate carboxylase, photosynthesis, amino acid synthesis, respiration.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum

We have determined the flow of (15)N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of (15)NH(4) (+) 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 NH(4) (+) accumulates in alanine. This is a unique feature of anaerobic NH(4) (+) assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH(4) (+) assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH(4) (+) assimilation.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum

The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH(4) (+) 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 NH(4) (+) 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 NH(4) (+) 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 NH(4) (+) assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH(4) (+) 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 NH(4) (+) assimilation.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum

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.

Fructose 1,6-Bisphosphatase in the Green Alga Selenastrum minutum

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.

Regulation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum

Two isoforms of phosphoenolpyruvate carboxylase (PEPC) with very different regulatory properties were partially purified from the green alga Selenastrum minutum. They were designated PEPC(1) and PEPC(2). PEPC(1) showed sigmoidal kinetics with respect to phosphoenolpyruvate (PEP) whereas PEPC(2) exhibited a typical Michaelis-Menten response. The S(0.5)(PEP) of PEPC(1) was 2.23 millimolar. This was fourfold greater than the S(0.5)(PEP) of PEPC(2), which was 0.57 millimolar. PEPC(1) 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, PEPC(2) showed only 8% and 52% activation by glutamine and DHAP, respectively. The effects of glutamine and DHAP were additive. PEPC(1) was more sensitive to inhibition by glutamate, 2-oxoglutarate, and aspartate than PEPC(2). 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 NH(4+) 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 NH(4+) assimilation.