Biomedical subjects

N E Tolbert

Publications and source records attributed to N E Tolbert.

At least 19 recordsLinked to original sources

PubMed1996

Association of glycolate oxidation with photosynthetic electron transport in plant and algal chloroplasts.

Photosynthetic carbon metabolism is initiated by ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which uses both CO2 and O2 as substrates. One 2-phosphoglycolate (P-glycolate) molecule is produced for each O2 molecule fixed. P-glycolate has been considered to be metabolized exclusively via the oxidative photosynthetic carbon cycle. This paper reports an additional pathway for P-glycolate and glycolate metabolism in the chloroplasts. Light-dependent glycolate or P-glycolate oxidation by osmotically shocked chloroplasts from the algae Dunaliella or spinach leaves was measured by three electron acceptors, methyl viologen (MV), potassium ferricyanide, or dichloroindophenol. Glycolate oxidation was assayed with 3-(3,4)-dichlorophenyl)-1,1-dimethylurea (DCMU) as oxygen uptake in the presence of MV at a rate of 9 mol per mg of chlorophyll per h. Washed thylakoids from spinach leaves oxidized glycolate at a rate of 22 mol per mg of chlorophyll per h. This light-dependent oxidation was inhibited completely by SHAM, an inhibitor of quinone oxidoreductase, and 75% by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which inhibits electron transfer from plastoquinone to the cytochrome b6f complex. SHAM stimulated severalfold glycolate excretion by algal cells, Dunaliella or Chlamydomonas, and by isolated Dunaliella chloroplasts. Glycolate and P-glycolate were oxidized about equally well to glyoxylate and phosphate. On the basis of results of inhibitor action, the possible site which accepts electrons from glycolate or P-glycolate is a quinone after the DCMU site but before the DBMIB site. This glycolate oxidation is a light-dependent, SHAM-sensitive, glycolate-quinone oxidoreductase system that is associated with photosynthetic electron transport in the chloroplasts.

Journal Article↗

PubMed1995

The oxygen and carbon dioxide compensation points of C3 plants: possible role in regulating atmospheric oxygen.

The O2 and CO2 compensation points (O2 and CO2) of plants in a closed system depend on the ratio of CO2 and O2 concentrations in air and in the chloroplast and the specificities of ribulose bisphosphate carboxylase/oxygenase (Rubisco). The photosynthetic O2 is defined as the atmospheric O2 level, with a given CO2 level and temperature, at which net O2 exchange is zero. In experiments with C3 plants, the O2 with 220 ppm CO2 is 23% O2; O2 increases to 27% with 350 ppm CO2 and to 35% O2 with 700 ppm CO2. At O2 levels below the O2, CO2 uptake and reduction are accompanied by net O2 evolution. At O2 levels above the O2, net O2 uptake occurs with a reduced rate of CO2 fixation, more carbohydrates are oxidized by photorespiration to products of the C2 oxidative photosynthetic carbon cycle, and plants senesce prematurely. The CO2 increases from 50 ppm CO2 with 21% O2 to 220 ppm with 100% O2. At a low CO2/high O2 ratio that inhibits the carboxylase activity of Rubisco, much malate accumulates, which suggests that the oxygen-insensitive phosphoenolpyruvate carboxylase becomes a significant component of the lower CO2 fixation rate. Because of low global levels of CO2 and a Rubisco specificity that favors the carboxylase activity, relatively rapid changes in the atmospheric CO2 level should control the permissive O2 that could lead to slow changes in the immense O2 pool.

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PubMed1992

Plant dihydroxyacetone phosphate reductases : purification, characterization, and localization.

A cytosolic form of dihydroxyacetone phosphate (DHAP) reductase was purified 200,000-fold from spinach (Spinacia oleracea L.) leaves to apparent electrophoretic homogeneity. The purification procedure included anion-exchange chromatography, gel filtration, hydrophobic chromatography, and dye-ligand chromatography on Green-A and Red-A agaroses. The enzyme, prepared in an overall yield of 14%, had a final specific activity of about 500 mumol of DHAP reduced min(-1) mg(-1) protein, a subunit molecular mass of 38 kD, and a native molecular mass of 75 kD. A chloroplastic isoform of DHAP reductase was separated from the cytosolic form by anion-exchange chromatography and partially purified 56,000-fold to a specific activity of 135 mumol min(-1) mg(-1) protein. Antibodies generated in rabbits against the cytosolic form did not cross-react with the chloroplastic isoform. The two reductases were specific for NADH and DHAP. Although they exhibited some dissimilarities, both isoforms were severely inhibited by higher molecular weight fatty acyl coenzyme A esters and phosphohydroxypyruvate and moderately inhibited by nucleotides. In contrast to previous reports, the partially purified chloroplastic enzyme was not stimulated by dithiothreitol or thioredoxin, nor was the purified cytosolic enzyme stimulated by fructose 2,6-bisphosphate. A third DHAP reductase isoform was isolated from spinach leaf peroxisomes that had been prepared by isopycnic sucrose density gradient centrifugation. The peroxisomal DHAP reductase was sensitive to antibodies raised against the cytosolic enzyme and had a slightly smaller subunit molecular weight than the cytosolic isoform.

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PubMed1992

Two Polypeptides in the Inner Chloroplast Envelope of Dunaliella tertiolecta Induced by Low CO(2).

Unicellular green algae have a mechanism for concentrating dissolved inorganic carbon (DIC) only when grown in low CO(2). To find proposed transporter protein(s) for DIC, we isolated intact chloroplasts from Dunaliella tertiolecta cells, separated the chloroplast envelopes by isopyknic centrifugation, and separated their polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two peptides of apparent molecular masses of 45 and 47 kD were constituents of the inner chloroplast envelope only if the cells had been adapted to low CO(2) in the light or grown in low CO(2). These two low CO(2)-induced peptides appear to be part of the algal DIC pump.

Journal Article↗

PubMed1992

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae.

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O(2) evolution, dissolved inorganic carbon accumulation, and photosynthetic CO(2) fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO(2)-concentrating process. This COS inhibition can be attributed to inhibition of external HCO(3) (-) conversion to CO(2) and OH(-) by the carbonic anhydrase component of an active CO(2) pump. At a low pH of 5 to 6, COS stimulated O(2) evolution during photosynthesis by algae with low CO(2) in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO(2). Although COS had less effect on HCO(3) (-) accumulation at pH 9 by a HCO(3) (-) pump in Scenedesmus, COS reduced O(2) evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO(2) and H(2)S, its inhibition of the CO(2) pump activity and photosynthesis is not accurate, when measured by O(2) evolution, by NaH(14)CO(3) accumulation, or by (14)CO(2) fixation.

Journal Article↗

PubMed1991

The transport of glycolic acid by Chlamydomonas reinhardtii.

Evidence for a transport system for glycolate in Chlamydomonas was obtained. [14C]Glycolate was taken up rapidly, reaching an equilibrium in less than 2 s at 4 degrees C. Glycolate uptake was stimulated by valinomycin and high KCl or high KCl alone and inhibited by N-ethylmaleimide. This uptake was not dependent on temperature or pH in contrast to uptake of benzoate by diffusion which decreased by orders of magnitude with increasing external pH. Based on these data, a transporter for glycolate is proposed.

Benzoates↗

PubMed1990

Two Systems for Concentrating CO(2) and Bicarbonate during Photosynthesis by Scenedesmus.

Scenedesmus cells grown on high CO(2), when adapted to air levels of CO(2) for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO(2) from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O(2) evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO(2) pump with K(0.5) for O(2) evolution of less than 5 micromolar CO(2) was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO(3) (-) concentrating process between pH 8 to 10 lowered the K(0.5) (DIC) from 7300 micromolar HCO(3) (-) in high CO(2) grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO(3) (-) pump was inhibited by vanadate (K(i) of 150 micromolar), as if it involved an ATPase linked HCO(3) (-) transporter. The CO(2) pump was formed on low CO(2) by high-CO(2) grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO(3) (-) pump was partially activated on low CO(2) within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO(3) (-) pump at pH 9 had requirements similar to the activation of the CO(2) pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO(2)-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO(2) pump and in some algae an additional vanadate-sensitive plasmalemma HCO(3) (-) transporter for a HCO(3) (-) pump.

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PubMed1990

Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae.

Rates of photosynthetic O(2) evolution, for measuring K(0.5)(CO(2) + HCO(3) (-)) at pH 7, upon addition of 50 micromolar HCO(3) (-) to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K(1)(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO(2) uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O(2) evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO(3)), and the rate of (14)CO(2) fixation with 100 micromolar [(14)C] HCO(3) (-). Salicylhydroxamic acid inhibition of O(2) evolution and (14)CO(2)-fixation was reversed by higher levels of NaHCO(3). Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO(2) accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related.

Journal Article↗

PubMed1989

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

Amino Acid Sequence↗

PubMed1989

Changes in the Activity of the Chloroplastic and Cytosolic Forms of Dihydroxyacetone Phosphate Reductase during Maturation of Leaves.

Young or mature rosette leaves from spinach (Spinacia oleracea L.) plants growing in the field, in the greenhouse, or in a growth chamber under a regimen of 8 hours light and 16 hours dark contained 15 to 50 nanomoles per minute per gram wet weight of NADH:dihydroxyacetone phosphate reductase activity. Of this activity, 75 to 87% was the chloroplastic isoform and 25 to 13% was the cytosolic form. When plants were induced to senesce, as measured by stem elongation and flowering, the percentage of the two reductase isoforms in rosette or stem leaves changed to about 12% as the chloroplastic and 88% as the cytosolic isoform. The change in enzyme activity of the rosette leaves occurred within 3 days, before phenotypic changes were observed. Likewise, when plants senesced in continuous darkness, the percentage of chloroplastic to cytosolic reductase changed from 80:20% to 25:75% after 62 hours before changes in total protein or chlorophyll occurred. The ratio of activities did not change in the first 16 hours of darkness or overnight. In each case the change in ratio resulted from about a 75% decrease in activity of the chloroplastic isoform and up to 14-fold increase in cytosolic isoform. In spinach leaves purchased at a local market primarily only the cytosolic isoform remained. When plants were returned to normal day-nights, after 62 hours in continuous darkness, the activity of the chloroplastic isoform increased, but not to control levels after 3 days, while the cytosolic enzyme decreased within 1 day to normal day-night values. Changes in activity were not due to changes during in vitro assays in activation by thioredoxin for the chloroplastic isoform or fructose 2,6-phosphate for the cytosolic isoform.

Journal Article↗

PubMed1989

Isolation and Characterization of a Mutant of Chlamydomonas reinhardtii Deficient in the CO(2) Concentrating Mechanism.

A Chlamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO(2) concentrations but can grow on elevated CO(2). In a test cross, the high CO(2)-requirement for growth showed a 2:2 segregation. This mutant, designated CIA-5, had a phenotype similar to previously identified mutants that were defective in some aspect of CO(2) accumulation. Unlike previously isolated mutants, CIA-5 did not have detectable levels of the periplasmic carbonic anhydrase, an inducible protein that participates in the acquisition of CO(2) by C. reinhardtii. CIA-5 also did not accumulate inorganic carbon to levels higher than could be accounted for by diffusion. This mutant strain did not synthesize any of the four polypeptides preferentially made by wild type C. reinhardtii when switched from an environment containing elevated CO(2) levels to an environment low in CO(2). It is concluded that this mutant fails to induce the CO(2) concentrating system and is incapable of adapting to low CO(2) conditions.

Journal Article↗

PubMed1989

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme.

A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.

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PubMed1989

Variations in the Alternative Oxidase in Chlamydomonas Grown in Air or High CO(2).

Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O(2) uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O(2) uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O(2) uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO(2), the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO(2), which induced a CO(2) concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive.

Journal Article↗

PubMed1989

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella.

Neither Dunaliella cells grown with 5% CO(2) nor their isolated chloroplasts had a CO(2) concentrating mechanism. These cells primarily utilized CO(2) from the medium because the K((0.5)) (HCO(3) (-)) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K((0.5)) CO(2) was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO(2) concentrating mechanism was present that decreased the K(0.5) (CO(2)) to about 0.5 micromolar and K(0.5) (HCO(3) (-)) to 11 micromolar at pH 8. These K(0.5) values suggest that air-adapted cells preferentially concentrated CO(2) but could also use HCO(3) (-) from the medium. Chloroplasts isolated from air-adapted cells had a K((0.5)) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO(2). Chloroplasts from air-adapted cells, but not CO(2)-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO(2) concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO(2) concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO(2) concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.

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PubMed1989

Two isozymes of dihydroxyacetone phosphate reductase in dunaliella.

Two isoforms of dihydroxyacetone phosphate reductase were present in Dunaliella tertiolecta. The major form was located in the chloroplast and the minor form in the cytosol. The chloroplastic reductase eluted first from a DEAE cellulose column followed immediately by the cytosolic form. Both forms were unstable and cold labile. Addition of 5 millimolar dithiothreitol helped to stabilize the enzymes. The cytosolic isoform of DHAP reductase was detected only if the cells were in an active log phase of growth. Then its activity was 20 to 30% of the total reductase activity. When cell cultures entered late log phase of growth the activity of the cytosolic form of the enzyme disappeared, but the chloroplastic form remained. The cytosolic DHAP reductase from Dunaliella has some properties similar to the cytosolic isoform from spinach leaves. Detergents inhibited both enzymes. However, neither form of the algal dihydroxyacetone phosphate reductase was stimulated by fructose 2,6-bisphosphate. In Dunaliella the properties of the chloroplastic form were those expected for glycerol production for osmoregulation, whereas the cytosolic form, like the reductases in leaves, is more likely involved in glycerol phosphate formation for lipid synthesis.

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PubMed1988

Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.

Trypsin rapidly inactivated the catalytic activities of spinach ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], but the stoichiometry of binding of the reaction-intermediate analogue carboxyarabinitol bisphosphate was only slightly reduced after proteolysis. Electrophoretic analysis indicated that several forms of the large subunit were generated during proteolysis but that the small subunit was resistant. Three tryptic peptides were isolated and characterized after digestion of the activated enzyme; the tryptic-sensitive sites were identified at Lys-8, Lys-14, and Lys-466 of the large subunit. Tryptic digestion of the enzyme complexed with the reaction-intermediate analogue released only two peptides by hydrolysis at Lys-8 and at Lys-14. The loss of susceptibility of Lys-466 to trypsin may be the result of a conformational change that limits the accessibility of the carboxyl-terminal region after binding of the reaction-intermediate analogue. Analysis of the amino-terminal tryptic peptide by composition and fast atom bombardment mass spectrometry demonstrated that the actual amino-terminal residue is proline at position 3 of the DNA-deduced sequence and that this proline is blocked with an N-acetyl moiety. Thus, posttranslational processing of the chloroplast-encoded large subunit of the enzyme must occur to remove Met-1 and Ser-2 and to acetylate the amino terminus.

Acetylation↗

PubMed1988

Dihydroxyacetone phosphate reductase in plants.

Two forms of dihydroxyacetone phosphate reductase are present in spinach, soybean, pea, and mesophyll cells of corn leaves. An improved homogenizing medium was developed to measure this activity. The enzyme was detectable only after dialysis of the 35 to 70% saturated (NH(4))(2)SO(4) fraction and the two forms were separated by chromatography on either DEAE cellulose or Sephacryl S-200. About 80% of the reductase was one form in the chloroplast and the rest was a second form in the cytosol as determined by chromatography and by fractionation of subcellular organelles. The amount of activity detectable in the chloroplast fraction was 10.7 micromoles of dihydroxyacetone phosphate reductase per hour per milligram chlorophyll from spinach leaves and 4.9 from pea leaves. The chloroplast form eluted first from DEAE cellulose and, being smaller, it eluted second from Sephacryl S-200. Activity of the chloroplast form was stimulated 3- to 5-fold by the addition of 1 millimolar dithiothreitol or 50 microgram reduced Escherichia coli thioredoxin or 4 micrograms spinach thioredoxin to the assay mixture. This stimulation was not observed with monothiols. Activity of the cytosolic form was not affected by either reduced thioredoxin or dithiothreitol.

Journal Article↗

PubMed1988

Differential inhibition and activation of two leaf dihydroxyacetone phosphate reductases : role of fructose 2,6-bisphosphate.

The chloroplastic and cytosolic forms of spinach (Spinacia oleracea cv Long Standing Bloomsdale) leaf NADH:dihydroxyacetone phosphate (DHAP) reductase were separated and partially purified. The chloroplastic form was stimulated by dithiothreitol, reduced thioredoxin, dihydrolipoic acid, 6-phosphogluconate, and phosphate; the cytosolic isozyme was stimulated by fructose 2,6-bisphosphate but not by reduced thioredoxin. End product components that severely inhibited both forms of the reductase included lipids and free fatty acids, membranes, and glycerol phosphate. In addition, two groups of inhibitory peptides were obtained from the fraction precipitated by 70 to 90% saturation with (NH(4))(2)SO(4). Chromatography of this fraction on Sephadex G-50 revealed a peptide peak of about 5 kilodaltons which inhibited the chloroplastic DHAP reductase and a second peak containing peptides of about 2 kilodaltons which inhibited the cytosolic form of the enzyme. Regulation of the reduction of dihydroxyacetone phosphate from the C(3) photosynthetic carbon cycle or from glycolysis is a complex process involving activators such as thioredoxin or fructose 2,6-bisphosphate, peptide and lipid inhibitors, and intermediary metabolites. It is possible that fructose 2,6-bisphosphate increases lipid production by stimulating DHAP reductase for glycerol phosphate production as well as inhibiting fructose 1,6-bisphosphatase to stimulate glycolysis.

Journal Article↗