Effect of Light Quality on the Circadian Expression of Nitrate Reductase in the Red Macroalga Gracilaria tenuistipitata

A circadian rhythm in the activity of nitrate reductase (NR: EC 1.6.6.1) isolated from the marine red algae Gracilaria tenuistipitata is shown to be attributable to the daily oscillation of protein levels. The experiments reported here indicate that light quality has differential effects over NR expression. In extracts of algae grown under white light : dark, red light : dark and blue light : dark cycle, the activity of NR peaks during photophase, as does photosynthesis. Staining with a monoclonal antibody (NR10), raised against NR purified from Porphyra yezoensis, shows that the amount of protein changes by a factor of about 20, with a maximum occurring during photophase when algae are submitted to white and blue light. Red light changes the circadian rhythm of NR protein levels and also inhibits its night degradation. Illumination with blue light is able to restore the NR activity as well as its protein levels only when the light irradiance was the same of the white light. Surprisingly, the red light promoted 40% induction on NR activity under the same conditions.     

Circadian rhythm of a TCA cycle enzyme is apparently regulated at the translational level in the dinoflagellate Lingulodinium polyedrum

Previously, the authors have reported that intracellular amounts of several metabolic-related enzymes from the photosynthetic dinoflagellate Lingulodinium polyedrum(formerly Gonyaulax polyedra) showed a daily rhythm under a 12:12 h LD cycle. This led the authors to hypothesize that a circadian clock controls metabolism, including the tricarboxylic acid (TCA) cycle. In this study, the authors investigated daily changes in the levels of mRNA, protein, and enzyme activity of several metabolic enzymes during 12:12 h LD, 8:16 h LD, and constant light conditions. The NADP-dependent isocitrate dehydrogenase (NADPICDH) in the TCA cycle exhibited circadian changes of protein abundance and enzyme activity under all conditions, whereas its mRNA level remained constant throughout the cycle. These results indicate that the rhythm of NADPICDH is regulated by a circadian control of protein synthesis or modification rather than by message levels and suggest that the TCA cycle may be controlled by the circadian clock system.     

Oscillations in the Activities of Enzymes of Nitrate Reduction and Ammonia Assimilation in Glycine max and Zea mays

The activities of glutamate dehydrogenase (GDH), glutamine synthetase (GS), and nitrate reductase (NR) and the levels of soluble protein and NO^-₃ were assayed in soybean (Glycine max [L.] Merr.) leaves over a 48-h period with the initial 24 h under a light-dark cycle (LD 16:8) followed by 24 h of continuous light (LL). Plants had been entrained for 30 days under the LD regime. Maize (Zea mays) leaves (10 days old) under a LD 15:9 cycle were assayed only for NR and nitrite reductase (NiR). Data were subjected to frequency analysis by the least squares method to determine probabilities for cosine function periods (τ's) between 10 and 30 h. NR activities for both soybean and Zea leaves had 24 h τ's with P values < 0.05 indicating circadian periodicity. GDH in soybeans had a 24-h rhythm under LD conditions which lengthened under LL conditions. The 24-h rhythm of GDH displayed maximal activity toward the end of the dark period of the LD cycle whereas the highest activity of NR was early in the light period. Total soluble protein displayed a rhythm with a best fitting τ of greater than 24 h under both LD and LL. GDH, GS, NR, NO₃, and soluble protein in soybeans and NiR in Zea, all displayed that were ultradian (10–18 h), indicating that a τ of about one half a circadian periodicity may be a common characteristic of the enzymes of primary nitrogen metabolism in higher plants. These data also demonstrate that although both NR and GDH are circadian in their activity, the 24-h rhythm may be greatly influenced by ultradian oscillations in activity.     

Characterization of circadian oscillations in alanine dehydrogenase activity in non-dividing populations of Euglena gracilis (z)

Possible factors that could generate the circadian oscillations in alanine dehydrogenase (EC 1.4.1.1.) activity observed in cultures of non-dividing Euglena gracilis (Z) have been examined in an effort to learn more about the basic timekeeping mechanism of biological clocks. No differences in K_m, pH optimum or electrophoretic mobility could be demonstrated between enzyme extracted from the minimum part of the 24-h oscillation in activity and that extracted from the maximum part. Also, no evidence for the presence of activators or inhibitors was found in mixing experiments. The effect of cycloheximide on the rhythm was examined; it was shown that the oscillation ceases in the presence of the inhibitor, but that if the inhibitor is removed after 12 h, the rhythm resumes with no apparent change in phase. Analyses of gel scans of enzyme preparations partially purified by (NH₄)₂SO₄ fractionation and polyacrylamide gel electrophoresis indicated that there was more alanine dehydrogenase protein present at the maximum part of the cycle than there was at the minimum part. In view of these and other data, an operational model of a circadian biological clock is discussed.     

Extracellular pH is under circadian control in Gonyaulax polyedra and forms a metabolic feedback loop

This study investigates the relationship between the circadian clock and metabolism based on recordings of the extracellular pH in cultures of the marine dinoflagellate, Gonyaulax polyedra. In light-dark cycles, pH of the medium rises during the light phase and declines in the dark. The amplitude of this pH-rhythm correlates with light intensity, indicating photosynthesis (and respiration) as the driving force. The recorded extracellular pH changes probably reflect the need to control intracellular pH in spite of pH-modifying reactions. The daily pH-changes are under control of the circadian clock because they continue to oscillate with a circa-24 h period in constant light, albeit with a smaller amplitude. Similar to other circadian output rhythms, the pH rhythm depends (amplitude and phase) on nitrate levels in the medium. Both the bioluminescence and the pH rhythm can also be shifted by extracellular pH-changes although Gonyaulax is rarely exposed to significant pH changes in its marine ecosystems (except for highly dense algal blooms). Because intracellular proton levels are both affecting circadian input and output they form a feedback loop with the Gonyaulax circadian system indicating complex interactions between metabolism and the circadian clock.     

Role of a luciferin-binding protein in the circadian bioluminescent reaction of Gonyaulax polyedra

A luciferin-binding protein (LBP), which binds and protects from autoxidation the substrate of the circadian bioluminescent reaction of Gonyaulax polyedra, has been purified to near homogeneity. The purified protein is a dimer with two identical 72-kDa subunits, and an isoelectric point of 6.7. LBP is a major component of the cells, comprising about 1% of the total protein during the night phase, but drops to only about 0.1% during the day. The luciferin is protected from autoxidation by binding to LBP, and one luciferin is bound per dimer at alkaline pH (Ka approximately 5 x 10(7) M-1). The protein undergoes a conformational change with release of luciferin at pH values below 7, concurrent with an activation of Gonyaulax luciferase. LBP thus has a dual role in the circadian bioluminescent system.     

ANALYSIS OF NUCLEAR MUTANTS OF CHLAMYDOMONAS DEFICIENT IN THE ACCUMULATION OF SPECIFIC CHLOROPLAST RNAS

Bioluminescence is broadly distributed in marine dinoflagellates and has been intensively studied in Lingulodinium ( Gonyaulax ) polyedra. In this species, bioluminescence is regulated in a circadian fashion; the enzyme (luciferase) and the luciferin (substrate)-binding protein are synthesized and degraded on a daily basis. Synthesis of both proteins is regulated at the level of translation. The L. polyedra luciferase gene is composed of three contiguous domains that are greater than 75% identical at the nucleic acid level. Possible explanations for the high degree of sequence conservation include: (1) the domains evolved through a recent duplication event; (2) the sequence similarity is maintained by a molecular process such as gene conversion; or (3) there is a functional role associated with the primary nucleic acid sequence, such as in the translational regulation of luciferase expression. The phylogenetic relationship of dinoflagellates predicted from 18S rDNA genes provides a framework for examining the molecular evolution of the regulation of luciferase expression and of genes encoding luciferase and the luciferin-binding protein. In particular, we are examining the evolution of the circadian rhythm of bioluminescence and of luciferase abundance, the presence/absence of the luciferin-binding protein, and the molecular structure of the luciferase gene. We anticipate that this approach will distinguish between regions of the luciferase molecule that are conserved for enzyme function versus those concerned with the regulation of protein expression. In addition, it will provide insight into the evolution of the regulatory processes and pathways.     

In vitro assay and light regulation of nitrate reductase in red alga Gracilaria chilensis

Nitrate reductase (NR) is the first enzyme in the nitrogen assimilation pathway. The in vitro NR activity of Gracilaria chilensis was assayed under different conditions to reveal its stability and biochemical characteristics, and an optimized in vitro assay is described. Maximal NR activities were observed at pH 8.0 and 15 degrees C. The apparent Km value for NADH was 8 microM and for nitrate 680 microM. Crude extracts of G. chilensis stored at 4 degrees C showed a 50% decrease of NR activity after 24 h. The highest NR activity value (253.20+/-2.60 x 10(-3) U g(-1)) was obtained when 100% von Stosch medium (500 microM NO3-) was added before extraction of apical parts. Algae under light:dark cycles of 12:12h exhibited circadian fluctuation of NR activity and photosynthesis with more than 2 times higher levels in the light phase. No evidence of endogenous diel rhythm controlling NR activity or photosynthesis was observed. Light pulses lasting 10 or 60 min during the darkness increased the NR activity by 30% and 45%, respectively. The results indicate that NR and photosynthesis are regulated mainly by light and not by a biological clock.     

Postnatal development of TRH and TSH rhythms in the rat

We previously observed that under a 12-hour light/12-hour dark schedule (lights off at 19.00 h), adult male Sprague-Dawley rats showed a circadian rhythm for serum thyroid-stimulating hormone (TSH) with a zenith near midday. In the present work, the ontogenesis of serum TSH rhythm was determined as well as pituitary TSH variations. In addition, hypothalamic and blood TRH were measured in these rats aged 15, 25, 40 and 70 days when sacrificed. As from the first age studied (15 days), a hypothalamic thyrotropin-releasing hormone (TRH) circadian rhythm was present. The mesor and the amplitude of this hypothalamic TRH rhythm increased while the rats were growing up, in contrast with the decrease observed for these parameters as far as blood TRH circadian rhythm is concerned. The time of the acrophase moved from 17.32 h in the 15-day-old rats to 13.57 h in the 70-day-old rats, being constantly in phase opposition with the blood TRH acrophase. The low amplitude pituitary TSH circadian rhythm detected in the young rat disappeared in the adult while, in contrast, the serum TSH rhythm became consistent to reach the well-characterized circadian midday peak in the 70-day-old rats.     

Exploring the signaling pathway of circadian bioluminescence

Bioluminescence in the unicellular dinoflagellate Gonyaulax polyedra represents an excellent model for studying a circadian controlled process at the biochemical and molecular levels. There are three key components involved in the bioluminescence reaction: the enzyme, luciferase, its substrate, luciferin, and a luciferin-binding protein (LBP), which sequesters the substrate at p^H 7.5 and thus prevents it from reacting with the enzyme. All components are tightly packed together in organdies, designated scintillons. The entire bioluminescent system is under circadian control with maximum amounts in the night. For both proteins circadian control is exerted at the translational level. In case of Ibp mRNA a small interval in its 3'untranslated region serves as a cis -acting element to which a trans -factor binds in a circadian manner. The binding activity of this factor decreases at the beginning of the night phase, when synthesis of LBP starts, and it increases al the end of the night, when synthesis of LBP stops indicating that it functions as a clock-controlled represser.     

The Relation Between the Denatured Nitrate Reductase and the Nitrate Reductase-Inhibiting Protein

Wheat seedlings (Triticum aestivum var. Feng-chan 3 ) were grown on water or KNO3 medium at 24℃. Before the second leaf had grown out, the shoots of the seedlings were cut down and ground with a little quartz sand. The homogenates were filtered through a layer of nylon cloth before centrifugation at 10000g for15 min. The supematant fraction was collected (crude nitrate reductase). Isolation and purification of nitrate reductase (NR) were according to Sherrard et al with a bit modifications. Ammonium sulfate was added to the crude NR and the enzyme protein was precipitated between 20%—40% saturation. After column chromatography on Sephadex G-25, the protein was then subjected to further purification by affinity chromatography on a blue dextran-Sepharose 4B column. The fraction in the NADH (0.1 mM) eluate was the highly purified enzyme. The activity of the isolated NR was assayed in vitro according to the standard method, Nitrate reductase-inhibiting protein (NRIP) was isolated and purified according to Wallace with a little modifications. After fractional precipitation by ammonium sulfate, the protein precipitating between 20%–40% saturation was collected and dissolved in distilled water. Column chromatography on Sephadex G-100 and DEAE (DE-11) cellulose was separately used. After dialysis, condensation of the highly purified NRIP was carried out. Antiserum against NR was prepared by injecting 2 mL purified NR protein (88 nmol NO2-/30 min/0.2 mL) into a rabbit five times with an interval of 10 days. For all five injections, the enzyme was mixed with complete Freund's adjuvant. Bleeding was taken 30 days after the first injection. Antiserum against NRIP was prepared in the same way mentioned above, but purified NRIP was used instead of NR. Rocket immunoelectrophoresis was performed by the method described by Funkhouser. Agarose gels (1.5% W/V). which contained 30 mM Tris and 12.3 mM meleate (pH 8.6) and 0.2% (V/V) crude antiserum were placed on a glass plate. Wells were cut along one side of the plate and filled with 10, 20, 30, 40 μ 1of antigen. Electrophoresis was carried out at 3 mA, 10 V for 2 h at 4 ℃. The antigen-antibody reaction resulted in the formation of rocket shaped immunoprecipitates. After washing overnight in PBS the rockets were visualized by staining with coomassie blue. The procedure of immunodiffusion and immunoelectrophoresis was according to that of Clausen. Nitrate reductase is a very unstable enzyme, Our former paper showed that the crude NR lost its enzyme activity by about one half, after it had been maintained at room temperature for 30 min. In order to study the stability of NR. crude NR was prepared and kept at room temperature. After the enzyme activity had been completely lost, it was added to a fresh NR preparation with high activity. The inhibition effect of denatured enzyme was revealed according to the difference between plus or minus denatured enzymes. About 70%–80% NR activities were lost in the preparation to which 0.1 ml denatured enzyme had been added instead of 0.1 ml H2O. Therefore we think that the denatured enzyme itself behaved like an inhibiting protein of NR. Wallace demonstrated that there was an inactivating enzyme of NR in maize roots. Some characteristics of the enzyme investigated in several labs. According to Wallace's methods we got a purified NR-inactivating-protein (NRIP). Furthermore, a purified NR was obtained by an affinity-chromatography method (table 1). Single of either NR or NRIP appeared on the chromatography and their Rm were the same (fig. 2). It might conclude that the NRIP and denatured NR are the similar protein. The highly purified NR protein incubated for several hours at room tempetature also became an inhibitor (table 2). We, therefore, infer that the activated NR could be converted to NRIP at room temperature. Antiserum against NR was prepared by injecting purified NR into rabbit, and antiserum against NRIP was prepared by injecting purified NRIP. The anti-NR antibody and the anti-NRIP antibody were prepared as reagents to study the immunological relation between these two proteins. The antibody of NR gave a single precipitate band against purified NRIP and the antibody of NRIP had a similar precipitate band against purified NR (fig. 3 and 4). Rocket immunoelectrophoresis was performed. The antiserum against NR were added to agarose gel and 4 wells were filled with different amount of NRIP. The height of the rockets was increased with the amount of NRIP (fig. 5). All these results show the identity of the denatured NR and NRIP. The percent of inhibition of NRIP depended upon the concentration of NADH in the reaction mixture. Fig. 6 shows that the NRIP was a competitive inhibitor. The inhibitor and NR both competed for the same cofactor NADH. The percentage of inhibition was decreased when the concentration of NADH in the reaction system was increased. According to this result, we suggest that the NR protein has two active sites. One site binds with nitrate and the other with NADH. When the site bound with nitrate is damaged or changed, the enzyme protein can not catalyze nitrate reduction. However, the site binding with NADH is less labile and not affected by incubation at room temperature, therefore NADH can still be bound on the denatured NR protein. If the concentration of NADH in this reaction system is limited, the nitrite formation decreases. This explains how the effect of NRIP can be overcome in the reaction system at higher concentration of NADH.     

Inhibitors of serine/threonine phosphoprotein phosphatases alter circadian properties in Gonyaulax polyedra.

Protein serine/threonine phosphatases were implicated in the regulation of circadian rhythmicity in the marine dinoflagellate Gonyaulax polyedra based on the effects of three inhibitors specific for protein phosphatases 1 and 2A (okadaic acid, calyculin A, and cantharidin). Chronic exposure to okadaic acid resulted in a significant period lengthening, as measured by the bioluminescent glow rhythm, whereas cantharidin and calyculin A caused large phase delays but no persistent effect on period. Short pulses of the phosphatase inhibitors resulted in phase delays that were greatest near subjective dawn. Unlike 6-dimethylaminopurine, a protein kinase inhibitor, okadaic acid, calyculin A, and cantharidin did not block light-induced phase shifts. The inhibitors tested also increased radiolabeled phosphate incorporation into Gonyaulax proteins in vivo and blocked protein phosphatase 1 and 2A activities in Gonyaulax extracts. This study indicates that protein dephosphorylation catalyzed by protein serine/threonine phosphatases is necessary for proper functioning of the circadian system.