BLUE LIGHT RESPONSES IN NITRATE REDUCTASE MUTANTS OF NEUROSPORA CRASSA

Abstract— The role of nitrate reductase in the blue light responses of photosuppression and phase shifting of circadian conidiation was studied in Neurospora crassa. The photoresponses, as assayed in three nitrate reductase mutants (nit-1,nit–2 and nit-3), showed no significant differences as compared to the responses in a strain which could utilize nitrate. In addition, the responses occur on a medium which (i) represses nitrate reductase activity due to the presence of ammonium ion and arginine or (ii) results in the production of an inactive enzyme due to the presence of tungsten. Nitrate reductase appears to be of no or secondary importance as a photoreceptor in the responses studied.     

NITRATE REDUCTASE—A KEY ENZYME IN BLUE LIGHT-PROMOTED CONIDIATION AND ABSORBANCE CHANGE OF NEUROSPORA

Abstract— Neurospora , when starved for several hours, can be stimulated by blue light to conidiate and also shows a blue light-inducible, flavin-mediated absorbance change near 425 nm due to photoreduc-tion of a h-type cytochrome. Under the same physiological conditions, nitrate reductase activity (NADPH-dependent nitrate reduction) decreases and the activity of its small subunit (methylviologen-to-nitrate reductase activity) increases. The increased activity is membrane associated (plasma membrane and mitochondria). The methylviologen-to-nitrate activity increase can also be brought about by treating mycelium briefly with diluted acetone. It is, therefore, interpreted as change within the nitrate reductase molecule (configuration change or separation of subunits with changed exposure of active sites). After acetone treatment the blue light-induced absorbance change can be observed immediately in the mycelium. Nitrate reductase with its flavin and protoheme prosthetic groups seems to be involved in both events, the induction of conidiation and in the described absorbance change.     

Separation of organic mixtures by pervaporation using crosslinked rubber membranes

Natural rubber (NR) and poly(styrene-co-butadiene) rubber (SBR) has been crosslinked with sulfur and accelerator with three different doses of varied accelerator to sulfur ratios to obtain three crosslinked membranes from each of these two rubbers (NR-1, NR-2 and NR-3 and SBR-1, SBR-2 and SBR-3). These six rubber membranes were used for pervaporative separation of toluene–methanol mixture up to 10 wt% of toluene in feed. It has been found that with increase in accelerator to sulfur ratio from membrane-3 to membrane-1, the vulcanization system shifts from conventional to efficient system resulting in higher degree of crosslink density and permeation selectivity. All of these membranes showed reasonably good range of flux (45.26 gm/m² h for NR-3 to 12.0 gm/m² h for SBR-1) and separation factor (162 for SBR-1 to 35.12 for NR-3) for 0.55 wt% of toluene in feed. Among these membranes NR-1 and SBR-1 with highest crosslink density showed maximum separation factor for toluene along with good flux. It has also been found that for comparable crosslink density SBR membranes showed better separation factor than NR membranes.     

THE CONTROL OF METABOLISM BY BLUE LIGHT IN Acetabularia mediterranea—II. SELECTIVE TRANSLATIONAL CONTROL AND DIFFERENTIAL DEGRADATION OF ENZYMES

Abstract— During prolonged continuous irradiation with red light the specific activity of uridine 5'-diphosphoglucose (UDPG) pyrophosphorylase (uridine 5'-triphosphate: glucose 1-phosphate uridylyl-transferase EC 2.7.7.9) decreased in Acetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva). Subsequent blue light restored the original activity within a comparatively short period of 3 to 4 days. Computer-aided quantitative evaluation of density labelling experiments showed that the synthesis of the enzyme was accelerated about four-fold during the period of activation by blue light. A similar increase in the rate of synthesis was found for hydroxypyruvate reductase (EC 1.1.1.81), a control enzyme that showed no blue light-dependent changes in the specific activity under these conditions. The increase in the rate of enzyme synthesis was caused by an overall stimulation of the cytosolic translation. Degradation of UDPG pyrophosphorylase was unaffected by blue light, while the half life of hydroxypyruvate reductase was shortened about two-fold compared to continuous red light. Thus, degradation of proteins appears to be selectively light dependent in Acetabularia.
Model calculations for enzyme amount and enzyme synthesis were carried out using the measurements of enzyme activity, rates of cytosolic protein synthesis, and degradation constants of the enzymes. Assuming that activities represented amounts of the given enzymes, these calculations indicated a selective activation of UDPG pyrophosphorylase synthesis by blue light since it did not coincide with the overall stimulation of protein synthesis in the cytosol, in contrast to hydroxypyruvate reductase.     

INDEPENDENT EFFECTS OF PHYTOCHROME AND NITRATE ON NITRATE REDUCTASE AND NITRITE REDUCTASE ACTIVITIES IN MAIZE

Abstract— Involvement of phytochrome in the regulation of nitrate reductase (NR) and nitrite reductase (NIR) activities in excised, etiolated leaves of Zea mays (L.) variety 'Ganga-5' is demonstrated using low energy and high irradiance responses of phytochrome action. Photoreversibility by far-red light of red light stimulated increases in NR and NIR activities was lost by 2 h. Red light given to the leaves, when induction by NO^-₃, was saturated, further increased both enzyme activities. Even if red light was given 4–8 h before NO^-₃, it still increased both NR and NIR activities.     

PHOTOREDUCTION OF CYTOCHROME b551 OF PARTIALLY PURIFIED NEUROSPORA NITRATE REDUCTASE VIA ITS INTERNAL FLAVIN

Abstract— Nitrate reductases (NR) from NR-normal Neurospora crassa mutant albino band and from NR-defective mutantsnit–1 andnit–3 were isolated and partially purified in order to test the photo-reducibility of their cytochrome b ₅₅₇ via the NR-internal FAD. Photoreducibility with blue light of the isolated enzyme was observed as absorbance increase at 423, 524 and553–557 nm. It was independent of NADPH-nitrate reductase activity and could be induced if the dissociable FAD was not lost in the isolation procedure. The photoreduction of cytochrome b ₅₅₇ was readily reversible due the high autoxi-dation rate of this cytochrome. Therefore, anaerobic conditions are required for photoreduction with low light intensities. If aerobic conditions are applied, high intensities become necessary to overcome the simultaneous cytochrome h₅₅₇ oxidation.     

IRREVERSIBLE PHOTO IN ACTIVATION OF NITRATE REDUCTASE DURING THE FLAVIN-SENSITIZED PHOTOCHEMICAL ASSAY OF ITS CATALYTIC ACTIVITY

Abstract— The classic photochemical system in which flavin and EDTA act as photosensitizer and electron donor, respectively, has been employed for assaying in vitro the catalytic activity of Ankistrodesmus braunii nitrate reductase. When the photochemical assay is performed under air, but not in anaerobiosis, a considerable decay in the nitrite photoproduction rate is observed after 10–15 min. which is accompanied by a decrease of reduced methyl viologen-nitrate reductase activity. The first enzyme activity, i.e. diaphorase is photoinactivated even more quickly and does not present any initial lag phase. Some oxygen species (superoxide and/or hydrogen peroxide) are probably involved in the photoinactivating mechanism, which appears to be non-specific and irreversible.
The use of flavin/EDTA photosystems is therefore, very practical for the in vitro assay of nitrate reductase activity, although anaerobic conditions are required for optimum results. Since inactivation of the terminal activity is, however, relatively slow, anaerobiosis would not be required for assay times shorter than 10 min.     

PHOTOACTIVATION OF NITRATE REDUCTASE FROM NEUROSPORA CRASSA

Abstract— The photoactivation of nitrate reductase from Neurospora crassa was studied in partially purified extracts. The inactive enzyme [inactivated by reduction in the presence of potassium cyanide] could be reactivated by chemical oxidation with ferricyanide or by irradiation with blue light. The enzyme contains a short electron transfer chain consisting of flavin adenine dinucleotide, cytochrome b ₅₅₇ and molybdenum which normally transfers electrons from reduced pyridine nucleotide to nitrate. This overall activity, which was negligible in the inactive enzyme, was restored to approximately 70% of the ferricyanide control by irradiation. However, nitrate reduction using reduced methylviologen as reducing power, which was also negligible in the inactive enzyme, was photoactivated to 100%. The diaphorase activity of the enzyme mediated by the flavin adenine dinucleotide, which was fully active in the inactivated enzyme, was inhibited approximately 30% by the irradiation treatment. The action spectrum for photoactivation showed that a flavin was the photoreceptor chromophore. Photoactivation occurs only in the presence of oxygen.     

MOLYBDENUM COFACTOR BIOSYNTHESIS IN Neurospora crassa: BIOCHEMICAL CHARACTERIZATION OF PLEIOTROPIC MOLYBDOENZYME MUTANTS nit-7, nit-8, nit-9A,B and C*

Abstract— Available mutants of molybdenum cofactor (MoCo) biosynthesis of Neurospora crassa were studied for converting factor activity and for in vitro molybdate repair of nitrate reductase (NR) activity. Mutant nit-7 was found to contain an activity that fits the functional definition of converting factor activity in Escherichia coli. Its high molecular weight fraction converts a low molecular weight compound from nit-1 and nit-8 into biologically active molybdopterin (MPT). Like nit-1, mutant nit-8 is devoid of this activity. Mutants nit-9 A, B and C contain a protein-bound precursor form of MoCo, which is presumed to be MPT bound to apo-NR. It is converted into active MoCo as part of NR in the presence of reduced glutathione and high exogenous molybdate concentrations. The NR apoenzyme of nit-1 is needed to detect the total amount of MoCo after molybdate repair, because mutants nit-9 A, B and C build no detectable content of functional NR apoenzyme. Evidence is presented for the transfer of MPT from demolybdo-NR to free NR apoenzyme.     

PHOTOREGULATION OF NITRATE UTILIZATION IN GREEN ALGAE AND HIGHER PLANTS*

Abstract— Nitrate reductase from eukaryotes can be reversibly inactivated, blue light being an effective activating agent both in vitro and in vivo. Hydroxylamine proved to be a powerful inactivating agent of Ankistrodesmus braunii nitrate reductase. Irradiation with blue light of NH₂OH-inactivated nitrate reductase, specially in the presence of μM amounts of FAD, promoted the recovery of the enzyme activity. Similarly, photoexcited methylene blue reactivated spinach nitrate reductase. On the other hand, in vitro nitrate reductase is highly susceptible to photodynamic inactivation caused by singlet O₂. Aerobic incubation of the active spinach enzyme with either FMN or methylene blue under either blue or red light respectively led to its irreversible inactivation. Irradiation of frozen and thawed spinach leaf discs also promoted, in situ, an irreversible inactivation of nitrate reductase, provided that 6₂ was present in the incubation mixture. Thus, either in vitro or in situ, light can cause two quite different responses of nitrate reductase, its blue light-dependent photoactivation in a flavin sensitized reaction and its photodynamic inactivation in a singlet O₂-dependent process.     

BLUE LIGHT PHOTORECEPTION IN Neurospora ORCADIAN RHYTHM: EVIDENCE FOR INVOLVEMENT OF THE FLAVIN TRIPLET STATE

The mechanism of the photoreceptor acting on the circadian conidiation rhythm of Neurospora crassa was studied, with the following results: (1) the efficiency of 8-haloflavins as sensitizers increased with their triplet yields. (2) Phase shifts were not abolished by removal of oxygen prior to illumination. (3) Oxygen inhibited phase shifts when introduced into the cultures after light treatment. It is proposed that the blue light photoreceptor for the circadian clock of Neurospora crassa acts (1) from its triplet state, but (2) not via singlet oxygen; (3) signal transduction involves (an) oxygen-sensitive intermediate(s).     

Phototaxis in the cyanobacterium Synechocystis sp. PCC 6803: role of different photoreceptors

The second cyanobacterial phytochrome Cph2 from Synechocystis sp. PCC 6803 was suggested as a part of a light-stimulated signal transduction chain inhibiting movement toward blue light. Cph2 has the two bilin binding sites, cysteine-129 and cysteine-1022, that might be involved in sensing of red/far-red and blue light, respectively. Here, we present data on wavelength dependence of the phototaxis inhibition under blue light, indicating that Cph2 itself is the photoreceptor for this blue light response. We found that inhibition of blue-light phototaxis in wild-type cells occurred below the transition point of about 470 nm. Substitution of cysteine-1022 with valine led to photomovement of the cells toward blue light (cph2(-) mutant phenotype). Analysis of mutants lacking cysteine-129 in the N-terminal chromophore binding domain indicated that this domain is also important for Cph2 function or folding of the protein. Furthermore, putative blue-light and phytochrome-like photoreceptors encoded by the Synechocystis sp. PCC 6803 genome were inactivated in wild-type and cph2 knockout mutant background. Our results suggest that none of these potential photoreceptors interfere with Cph2 function, although inactivation of taxD1 as well as slr1694 encoding a BLUF protein led to cells that reversed the direction of movement under blue light illumination in mutant strains of cph2.     

Flavin-mediated photoinactivation of spinach leaf nitrate reductase involving superoxide radical and activating effect of hydrogen peroxide

Irradiation with white light of spinach leaf nitrate reductase (NR) in the presence of flavin mononucleotide (FMN) and ethylenediaminetetraacetic acid (EDTA) resulted in a gradual loss of the enzyme activity, measured with reduced methyl viologen as electron donor. Inactivation of NR was dependent on oxygen and was prevented by superoxide dismutase. On the contrary, the presence of catalase markedly enhanced the rate of inactivation. Nitrate showed a protective effect. NR previously inactivated by irradiation in the presence of FMN and EDTA was greatly reactivated by a short preincubation of the inactive enzyme with either ferricyanide or H₂O₂. These results suggest that spinach leaf NR is reversibly inactivated by photogenerated O₂ , while H₂O₂ has an activating effect.     

RHYTHMS IN BLUE-LIGHT-INDUCED CONIDIATION OF WILD TYPE AND A MUTANT STRAIN OF Trichoderma harzianum

Abstract— Trichoderma harzianum normally requires light for conidiation. Conidiation of colonies grown in continuous light does not appear to be rhythmic, but sharp banding patterns are formed under light/dark cycles. A single pulse of blue light produces a sharp band of conidia that forms where the growing edge of the mycelia is located at the time when the light is given. A period of about 24 h is required following the light pulse to produce mature conidia. During this time colonies are insensitive to further induction by light. The fluence required to produce 50% saturation varies by a factor of about 3 depending on when the pulse is given. This change of sensitivity is rhythmic with a period length of approximately 27 h when grown on medium containing deoxycholate.
The pattern of conidiation in a mutant strain (B119), which is able to form conidia in the dark, is rhythmic and the period length is dependent on the composition of the medium. Addition of deoxycholate to the medium increased the interval between dark bands from 12 to 24 h. The rhythmic banding is suppressed in constant light and a double banding pattern is produced in light/dark cycles. A pulse of blue light induces a band of conidia in this mutant, as in the wild type, but it also delays the reappearance of the dark banding pattern. The extent of this delay depends on when the pulse is given and, although the period length of the dark conidiation rhythm is affected by deoxycholate, the effect of blue light on its phase is not. Of the various rhythmic responses of Trichoderma studied here. the delay in reappearance of the dark banding pattern in B119 is the most promising for further detailed studies, for example of wavelength and temperature dependence.     

The opposed effect of 5-azacytidine and light on the development of reproductive structures in Neurospora crassa

Blue light inhibits the formation of asexual cycle spores (conidia) and stimulates the development of the sexual (female) reproductive structures (protoperithecia) in the nitrogen-starved mycelium of Neurospora crassa. The DNA methylation inhibitor, 5-azacytidine (3-300 microM), opposed the effect of light by suppressing the protoperithecia formation and stimulating a conidiation. The addition of 300 microM 5-azacytidine inhibited protoperithecia formation in the dark-cultivated mycelium by about two orders of magnitude and activated conidiation in the light-exposed mycelium by almost three orders of magnitude. Both in the dark-cultivated and the irradiated mycelium treated with various 5-azacytidine concentrations, the yield of conidia and protoperithecia demonstrated an inverse relationship. We suggest that DNA methylation and blue light are involved in the organism's selection of sexual or asexual reproductive cycle.     

Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE

Mammalian mitochondrial dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a diaphorase catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of DLDH diaphorase activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of DLDH, were resolved by nongradient BN-PAGE (9%), which was followed by diaphorase activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of DLDH diaphorase was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures DLDH dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of DLDH diaphorase activity in several rat tissues other than the brain, and the results indicated a similar level of DLDH diaphorase activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on DLDH diaphorase activity were evaluated, demonstrating that, with this method, DLDH diaphorase activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of DLDH dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial DLDH that is to be analyzed by mass spectral techniques in studying DLDH post-translational modifications.     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

Promotion of nano-anatase TiO2 on the spectral responses and photochemical activities of D1/D2/Cyt b559 complex of spinach

Previous researches approved that photocatalysis activity of nano-TiO₂ could obviously increase photosynthetic effects of spinach, but the mechanism of improving light energy transfer and conversion is still unclear. In the present we investigated effects of nano-anatase TiO₂ on the spectral responses and photochemical activities of D1/D2/Cyt b559 complex of spinach. Several effects of nano-anatase TiO₂ were observed: (1) UV–vis spectrum was blue shifted in both Soret and Q bands, and the absorption intensity was obviously increased; (2) resonance Raman spectrum showed four main peaks, which are ascribed to carotene, and the Raman peak intensity was as 6.98 times as that of the control; (3) the fluorescence emission peak was blue shifted and the intensity was decreased by 23.59%; (4) the DCPIP photoreduction activity showed 129.24% enhancement; (5) the oxygen-evolving rate of PS II was elevated by 51.89%. Taken together, the studies of the experiments showed that nano-anatase TiO₂ had bound to D1/D2/Cyt b559 complex, promoted the spectral responses, leading to the improvement of primary electron separation, electron transfer and light energy conversion of D1/D2/Cyt b559 complex.