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Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Abstract. Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP+/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, Km for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.

Physiological and morphological responses of Lead or Cadmium exposed Chlorella sorokiniana 211-8K (Chlorophyceae)

The heavy metal pollution in soils and aquatic environments is a serious ecological problem. In the green-microalga Chlorella sorokiniana 211-8K (Chlorophyceae) exposed to ions of lead (Pb) and cadmium (Cd) we studied the metabolic responses to the toxicity of these two heavy metals. Our data indicate that both the pollutants alter the alga cell ultrastructure and its physiological characteristics (growth, photosynthesis, respiration, enzyme activities). The toxic effects of the two metals resulted time-dependent to the exposure. After 24 h of treatment with 250 μM Pb or Cd, photosynthesis was inhibited until to 77 and 86%, however respiration was strongly enhanced up to 300 and 350%, respectively. In the algal cells Pb or Cd exposure induced a reduction in the content of the total chlorophylls and a decrease of the soluble protein levels, significantly compromising the growth, particularly in cultures cadmium-treated. We report data on ultrastructural changes induced by the two heavy metals; they affected overall chloroplast ultrastructure of the alga. Most importantly, the O-acetyl-L-serine(thiol)lyase (OASTL) activity was appreciably increased after only 2 h of Cd exposure, indicating the existence of a link between the metal contamination and cysteine synthesis. Then, Chlorella sorokiniana cells seem to better tolerate high concentrations of Pb while appear to be more sensitive to Cd ions. These results provide some additional information that can lead to better understand consequences of heavy metal poisoning in microalgae.

Nitrate reductase and glutamine synthetase activities, nitrate and ammonia assimilation, in the unicellular alga Cyanidium caldarium

Nitrogen-limited continuous cultures of Cyanidium caldarium contained induced levels of glutamine synthetase and nitrate reductase when either nitrate or ammonia was the sole nitrogen source. Nitrate reductase occurred in a catalytically active form. In the presence of excess ammonia, glutamine synthetase and nitrate reductase were repressed, the latter enzyme completely. In the presence of excess nitrate, intermediate levels of glutamine synthetase activity occurred. Nitrate reductase was derepressed but occurred up to 60% in a catalytically inactive form.Cell suspensions of C. caldarium from nitrate- or ammonialimited cultures assimilated either ammonia or nitrate immediately when provided with these nutrients. In these types of cells, as well as in cells grown with excess nitrate, the rate of ammonia assimilation was 2.5-fold higher than the rate of nitrate assimilation. It is proposed that the reduced rate at which nitrate was assimilated as compared to ammonia might be due to regulatory mechanisms which operate at the level of nitrate reductase activity.

Glutamine synthetase activity, ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.Under physiological conditions nitrate reductase [NAD(P)H: nitrate oxidoreductase EC 1.6.6.2] is reversibly converted into an inactive enzyme upon addition of ammonia. In the presence of methionine sulphoximine, when glutamine synthetase activity is lost, nitrate reductase is no longer inactivated by ammonia. It is suggested that ammonia itself is not the actual effector of nitrate reductase inactivation.Concomitantly with the failure of nitrate reductase to undergo ammonia-inactivation, in the presence of methionine sulphoximine nitrate reduction is an uncontrolled process, thus, in media with nitrate ammonia continues to be produced and excreted into the external medium at a constant rate.

Ammonium assimilation by young plants of Hordeum vulgare in light and darkness: effects on respiratory oxygen consumption by roots.

Barley plants (Hordeum vutgare L.) grown for 10 d in nitrogen-free hydroponic culture, after a rapid initial phase absorbed supplied NH4 (+) at a constant rate of 15.1 ±1.2 μ mol h(-1) g(-1) f. wt in the light, arid at a rate of 13.81 ± 1.6 μ mol h(-1) g(-1) f. wt in darkness. Ammonium-grown plants assimilated NH4 (+) at a rate of 7.5 ± 0.33 μmol h-1 g(-1) f. wt and at a 50% lower rate in darkness. Nitrogen-free grown plants showed low concentrations of free amino acids in both root and shoot tissues. Supplying NH4 (+) caused an immediate increase in the concentration of free amino in the root tissues of both illuminated and darkened plants over a 120 mm period. The increase in concentration of glutamine then exhibited a lag period of 120 min, after which it resumed, but to a very small extent. Glutamine also accumulated in shoot tissue of illuminated plants at increasing rates, attaining a concentration which, 8 h after NH4 (+) supply, was 1.61-fold greater than that attained in the roots. In shoots of darkened plants, by contrast, the concentration of glutamine increased slowly and was always smaller than that in the root tissue. Overall formation of glutamine (in shoots and roots) occurred at decreasing rates during the first 4 h, and then at increasing rates. The increase was more pronounced in illuminated plants than in darkened plants, liven 24 h after NH4 (+) was supplied, glutamine content in root tissue was lower than that in shoot tissue. However, 48 h later, the concentrations of glutamine in root and shoot were similar, attaining values that were almost 47-fold (in root) and 134-fold (in shoot) greater than initial values. Significant levels of asparagine were detected in the root and in the shoot 24 h after adding NH4 (+) . These increased further during the succeeding period. Ammonium supply caused a transitory drop in the concentration of ATP in root tissue, along with noticeable transitory variations in glucose-6-P concentration. A permanent decrease in free glucose concentration was also detected. Addition of NH4 (+) caused 2- and 1.43-fold increases in respiratory oxygen consumption by roots of illuminated and darkened plants, respectively. Both in the light and in the dark, the root tissue accumulated methylammonium up to a concentration of 55-67 μmol h(-1) g(-1) f. wt. Methylammonium was never found in shoot tissue of either illuminated or darkened plants. Methylammonium stimulated respiration of root barley plants by a factor of 1.2. Regulatory aspects of NH4 (+) metabolism are discussed.

Nitrate and ammonium assimilation in algal cell-suspensions and related pH variations in the external medium, monitored by electrodes

Abstract Nitrate and ammonium utilization in cell suspension of the acidophilic thermophilic non-vacuolate alga Cyanidium caldarium and the related pH variations in the external medium, were measured using ion specific electrodes. Nitrate absorption and pH variations were also monitored. The acidophilic C. caldarium allowed experimentation under acidic conditions where CO 2 exchange did not affect pH measurements. Both ammonium and nitrate assimilation occured in dark and light conditions; however, the rate of assimilation was higher in the light than in the dark. The presence of oxygen was an absolute requirement. It is suggested that the uptake of both nitrate and ammonium are mediated by active mechanisms dependent on the energy charge of the cell. The assimilation of nitrate and ammonium was accompanied by an increase and a decrease, respectively, of the external medium pH. Under steady-state conditions, for each equivalent of ammonium incorporated there were 0.8–1 equiv. of protons released into the external medium and for each equivalent of nitrate absorbed there were 1–1.2 equiv. of protons absorbed from the external medium.

Changes in cysteine and O-acetyl-l-serine levels in the microalga Chlorella sorokiniana in response to the S-nutritional status

We analyzed the effects of deprivation and subsequent restoration of sulphate (S) in the nutrient solution on cysteine (Cys) and O-acetyl-L-serine (OAS) levels in Chlorella sorokiniana (211/8k). The removal of S from the culture medium caused a time-dependent increase in O-acetyl-L-serine(thiol)lyase (OASTL) activity and a decrease in soluble proteins content. The protein gel blot analysis was used to show that OASTL isoforms are located in the chloroplast and in the cytoplasm of S-starved cells. S-deprivation caused a decrease in the intracellular levels of Cys and glutathione (GSH) and an increase in serine (Ser) and OAS, reflecting an imbalance between sulphur and nitrogen assimilation. Re-supplying of sulphate to S-starved cells produced a decrease in OAS levels and concomitant rapid increase in Cys and GSH concentrations. The simultaneous addition of OAS and sulphate to S-starved cells did not further increase the concentration of Cys, suggesting the existence of a threshold level of intracellular Cys that is independent of the cellular concentration of OAS. Our findings that OAS is stored during S-starvation and that its quick decrease appears to be coupled with the increase of Cys levels upon re-supply of sulphate, imply that the central role that these two compounds play is in the regulation of sulphur-assimilating enzymes in response to the S status of the cell.

Studies in vivo on the control by ammonia of nitrate reduction to nitrite in the unicellular alga Cyanidium caldarium

Abstract When intact cells of the acidophilic red alga Cyanidium caldarium, grown at pH 1.9 with nitrate as sole nitrogen source, are transferred to pH 7.5, they actively reduce nitrate to nitrite which accumulates in the external medium. At pH 7.5 nitrite is not further reduced to ammonia. Formation of nitrite from nitrate occurs in the light or in the dark, either in the presence or in the absence of oxygen. In cell suspensions supplied with ammonia in addition to nitrate, the formation of nitrite at pH 7.5 is strongly prevented. However ammonia is not able to prevent nitrite formation at pH 9.4. Nitrate reductase is 40% latent in cells resuspended at pH 7.5 in the presence of nitrate and up to 90% latent in cells resuspended in the presence of both nitrate and ammonia. It is proposed that the inhibition by ammonia of nitrate reduction to nitrite is based on the latent state that nitrate reductase exhibits in the presence of ammonia. In fact, at pH 9.4, when the production of nitrite is not inhibited by ammonia, nitrate reductase is only 14–25% latent even in cells resuspended in the presence of nitrate and ammonia.

Evidence for two transport systems for nitrate in the acidophilic thermophilic alga Cyanidium caldarium

In the unicellular non-vacuolate red alga Cyanidium caldarium nitrate uptake occurs through two specific permease systems which, on the basis of kinetic constants can be defined as low affinity system and high affinity system. The high affinity system is saturated at very low nitrate concentrations (<1 μM), whereas the low affinity system is saturated only at high nitrate concentrations (Km=0.45±0.10 mM). The low affinity system is present in cells growing under conditions of nitrogen limitation as well as in cells growing in excess nitrate. In contrast, the high affinity system is present only in cells growing under conditions of nitrogen limitation. The high affinity system works only at acid pH and is inactive at neutral pH. The low affinity system is active both at acid and at neutral pH.

Effects of sulfate-starvation and re-supply on growth, NH4+ uptake and starch metabolism in Chlorella sorokiniana

Chlorella sorokinianaShihira & Krauss, strain 211/8K resuspended in a illuminated mediumshowed continuous growth, and concomitantly NH4 +in the medium depleted at a constant rate. Upon sulfate removal, i) growthdeclined, ceasing within 5 h; ii) NH4 + depletionbecame almost undetectable over 2 h; and iii) photosynthetic capacity (Pc) wasreduced over 24 h by 80% in the light, but was reduced by only30% in darkness. Over 24 h of S starvation the chlorophyll content ofilluminated cells decreased by 50%, whereas that of darkened cellsdecreased by only 10%. Sulfate-deprivation over 24 h resulted in anoticeable increase in the starch content of the cell and a net increase inthe pools of glutamate, glutamine, serine and asparagine. Cysteine content, incontrast, was decreased. Sulfate addition to cells starved of S for 24 hprovoked a sudden time-dependent increase in Pc and in an immediate renewal ofgrowth in light but not in darkness. Sulphate supply also caused a suddenenhancement of respiratory oxygen consumption and a prompt degradation ofstarch. Starch was degraded at a higher rate in illuminated than in darkenedcells. After the S addition, depletion of NH4 + inthe medium occurred at slowly increasing rates; amino acids decreasednoticeably over 4 h, where in contrast, cysteine increased noticeably.