Blue-light reactivation of spinach nitrate reductase inactivated by acetylene or cyanide. Effects of flavins and oxygen

Nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) of spinach ( Spinacia oleracea L.) leaves, inactivated in vitro by acetylene, was reactivated by irradiation with blue light. Red + infrared, green or white light of the same irradiance were less effective. The dehydrogenase activity of the nitrate reductase complex was not required for pliotoreactivation. Photoreactivation of cyanide-inactivated nitrate reductase was greatly enhanced by the addition of 1 and 20 μ of either FMN or FAD; however, flavins showed a much smaller stimulatory effect on photoreactivation of acetylene-inactivated enzyme. The effect of flavins was higher under anaerobic conditions. This might imply the direct ievolvement of excited flavins in the photoreactivation mechanism. Besides promoting photoreactivation, blue light irradiation led simultaneously to a gradual inactivation of the enzyme especially under air and 20 μ FMN, eventually abolishing the recovered activity of the enzyme.     

Postreplication repair of DNA in chick cells: studies using photoreactivation.

During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick embryo fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split by an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultraviolet-irradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.     

Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions

Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions. J. Bacteriol. 92:144-147. 1966.-T4r mutations were induced by ultraviolet irradiation of extracellular phage particles, using a phage mutant, v, which is particularly susceptible to photoreactivation. Most of the induced r mutations could be subsequently photoreversed intracellularly with white light. Ultraviolet irradiation induces both transitions and sign mutations, and both were susceptible to photoreversal. The results suggest that two very different types of mutational lesions may arise from a common type of photochemical lesion.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

Blue-light-control of the uptake of amino acids and of ammonia in Chlorella mutants

In non-photosynthetic, yellow or colourless mutant cells of Chlorella kessleri , grown with nitrate as sole nitrogen source, blue light inhibited the uptake of the amino acids glycine, proline and arginine and of ammonia in growing cells, while it enhanced the uptake of these amino acids in resting cells. On the other hand, in cells grown with ammonia as the only nitrogen source without nitrate reductase activity, blue light did not influence the uptake of amino acids and of ammonia in growing cells, while it enhanced the uptake of amino acids in resting cells. Addition of methionine sulphoximine, a potent inhibitor of glutamine synthetase, to growing cells, resulted in intracellular ammonia-accumulation and inhibition of uptake of glycine and of ammonia. For the colourless mutant, blue light was shown to activate purified nitrate reductase. These results indicate that in the mutant cells of Chlorella examined, uptake of ammonia seems to be influenced by nitrate reductase and the uptake of amino acids was influenced by both nitrate reductase and an unknown blue-light-receptor(s). The uptake of urea in mutant cells is not influenced by the irradiation with blue light. Uptake of glycine was also increased after addition of glucose (hexose) in the dark. Because blue light is known to enhance the breakdown of starch, a reaction producing glucose for oxidative degradation in the algae used, the role of glucose (hexose) in the blue light-affected uptake of amino acids is discussed.     

DNA repair after ultraviolet irradiation of ICR 2A frog cells. Pyrimidine dimers are long acting blocks to nascent DNA synthesis.

The ability of ICR 2A frog cells to repair DNA damage induced by ultraviolet irradiation was examined. These cells are capable of photoreactivation but are nearly totally deficient in excision repair. They have the ability to convert the small molecule weight DNA made after irradiation into large molecules but do not show an enhancement in this process when the UV dose is delivered in two separate exposures separated by a 3- or 24-h incubation. Total DNA synthesis is depressed and low molecular weight DNA continues to be synthesized during pulse-labeling as long as 48 h after irradiation. The effects of pyrimidine dimer removal through exposure of UV irradiated cells to photoreactivating light indicate that dimers act as the critical lesions blocking DNA synthesis.     

Synthesis of Nitrate Reductase in Chlorella: I. EVIDENCE FOR AN INACTIVE PROTEIN PRECURSOR

Synthesis of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris was studied under inducing conditions, i.e. with cells grown on ammonia and then transferred to nitrate medium. Cycloheximide (but not chloramphenicol) completely inhibited synthesis of the enzyme, but only if it was added at the start (i.e. at the time of nitrate addition) of the induction period. Cycloheximide inhibition became less effective as induction by nitrate proceeded. Enzyme from small quantities of culture (1 to 3 milliliters of packed cells) was purified to homogeneity with the aid of blue dextran-Sepharose chromatography. Incorporation of radioactivity from labeled arginine into nitrate reductase was measured in the presence and absence of cycloheximide. Conditions were found under which the inhibitor completely blocked the incorporation of labeled amino acid, but only slightly decreased the increase in nitrate reductase activity. The results indicate that synthesis of nitrate reductase from amino acids proceeds by way of a protein precursor which is inactive enzymically.     

The role of chloride ion on the photoreactivation of the oxygen-evolving center of tris-washed, 2,6-dichlorophenol indophenol-treated grana

Chloride ion is found to be an essential factor in photoreactivation of the oxygen-evolving center. Tris-washed, 2,6-dichlorophenol indophenol-treated grana are low in both Mn content and oxygen-evolving activity. These grana can restore high oxygen-evolving activity, however, by incorporating Mn2+ ion under weak light in the presence of chloride and calcium ions with dithiothreitol. This restoration is called photoreactivation. When chloride ion is omitted from the medium for the photoreactivation, the recovery of oxygen-evolving activity is inhibited. Other anions, such as bromide and nitrate anions, could also mediate the reactivation; but, anions of weak acids or polyvalent strong acids were not effective. Chloride ion is also required in the light-induced H+ and Mn2+ uptake of these grana, which are essential partial reactions for the reactivation. It is therefore concluded that chloride ion plays an important role in the photoreactivation.     

Photoreactivation inChlamydomonas reinhardi Dangeard

The existence of photoreactivation in the green unicellular algaChlamydomonas reinhardi Dangeard was demonstrated. The dose reduction factor was constant throughout practically the whole of the dose range and was approximately 0.5. Photoreactivation was not found in cells irradiated with X-rays. The maximum photoreactivation after ultraviolet irradiation was reached after about 30 minutes’ illumination with 2,300Lx. No difference was found between the rate of photoreactivation carried out immediately and 30 minutes after ultraviolet irradiation. The rate of photoreactivation, under the given conditions, seems to be limited chiefly by the supply of light energy. The photoreactivation enzyme is probably a stable component of the cell.     

Blue light photoreactivation of nitrate reductase from green algae and higher plants

The inactive form of NADH-nitrate reductase from spinach and $\text{Chlorella fusca}$ is fully reactivated in short periods of time when the enzyme-complex is illuminated with white or blue light but not with red light. Flavin nucleotides greatly accelerate the photoreactivation process. The results suggest that blue light might act as a modulating agent in the assimilation of nitrate in green algae and higher plants.     

Isolation and Characterization of Ultraviolet Light-Sensitive Mutants of the Blue-Green Alga Anacystis nidulans

Three independently isolated ultraviolet light-sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 was most sensitive to UV in the absence of photoreactivation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, indicating the presence of excision enzymes involved in dark repair. Under "black" and "white" illumination, strain uvs-1 displays photoreactivation properties nearly comparable to wild-type culture. Mutants uvs-35 and uvs-88 appeared to have partial photorecovery capacities. Upon pretreatment with chloramphenicol, photoreactivation properties of strains uvs-1 and uvs-88 were not evident although the partial photoreactivation characteristics of strain uvs-35 remained the same. Data indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.     

Photosensitized inhibition of nitrate reductase induction by 4,5',8-trimethylpsoralen and near ultraviolet light

4,5' ,8-trimethylpsoralen photosensitizes the inhibition of the induction of nitrate reductase in XD cells of tobacco, by near ultraviolet light. The photosensitization depends on the concentration of 4,5' ,8-trimethylpsoralen and the near ultraviolet light dose. Concomitant with the inhibition of nitrate reductase induction there is a severe inhibition of DNA and RNA synthesis. Oxygen uptake and protein synthesis are not affected. Translation of preexisting mRNA coding for nitrate reductase is insesitive to 4,5' ,8-trimethylpsoralen and near ultraviolet light. It is concluded that the DNA of these cells is the target for the photochemical reaction of 4,5' ,8-trimethylpsoralen. The template activity of the DNA is inhibited, and the expression of the genome thereby prevented. Translation of preexisting nRNA is not affected.     

Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.     

Anaerobic nitrite production by plant cells and tissues: evidence for two nitrate pools

Tobacco (Nicotiana tabacum L. cv. Xanthi) XD cells containing nitrate and nitrate reductase stopped producing nitrite after approximately 1 hour when incubated under anaerobic conditions. The cessation of nitrite production was not due to an inactivation of the nitrate reducing system. This was shown by the ability of the cells to resume anaerobic nitrite production at a rate similar to the initial rate of nitrite production upon exposure to nitrate, monohydroxy alcohols or pyrazole. Cessation of nitrite production also could not be attributed to leakage of nitrate from the cells. Although some nitrate did leak from the cells, most of the nitrate was still in the cells by the time anaerobic nitrite production ceased. We infer the existence of a small metabolic pool and a large storage pool of nitrate, such that nitrite production ceases when the metabolic pool is depleted of nitrate. The metabolic pool of nitrate in tobacco cells decreased 170-fold as the culture aged from 3 to 5 days. However, total cellular nitrate during this period remained relatively constant.     

Increase in the fraction of non-repaired mutations in Escherichia coli cells, incubated in the absence of glucose after UV-irradiation

In E. coli WP2 trpE65 cells irradiated with UV-dose of 11 J/m2, the additional small portion of induced Trp+ mutations became resistant to photoreactivation or "dark" (excision) repair after a short-termed (10-30 min) postirradiation incubation of bacteria in a minimal medium deprived of glucose and tryptophan. Since protein synthesis could not proceed in those cells because of the lack of energy and tryptophan, the data indicate that an unknown mechanism exists which imparts some mutations with the resistance to antimutagenic repair in the absence of the inducible mutagenic system. In the light of this result, one could suggest that the normal process of mutation fixation (that is the loss of sensitivity of mutations to photoreactivation or to excision repair in cells incubated in growth medium after irradiation) should not necessarily be a direct consequence of manifestation of the activity of an inducible mutagenic system.     

UV-B light induces an adaptive response to UV-C exposure via photoreactivation activity in Euglena gracilis.

Phytoplankton such as Euglena are constantly exposed to solar light which is used for photosynthesis. Although the solar ultraviolet (UV) induces DNA damage such as cyclobutane-pyrimidine dimers (CPDs), many kinds of living organisms can repair CPDs by photoreactivation (PR) utilizing the near-UV/blue light component in sunlight. Euglena cells are known to possess such PR activity. In the present paper, the formation of CPDs induced by UV-C exposure and the photoreactivation PR repair of these CPDs by UV-A are demonstrated. To clarify the adaptive responses prior UV-B irradiation on PR activity, cells were cultured in the dark or under UV-B light. When the cells were cultured in the dark for 3 d prior to UV-C exposure, PR activity decreased. When the cells were cultured under UV-B light, however, PR activity increased. These results suggest that exposing the cells to UV-B prior to exposure to UV-C induced an adaptive response towards DNA damage caused by UV-C exposure, and this UV-C induced damage was repaired through PR activity.     

The light-induced development of nitrate reductase in etiolated barley shoots: An inhibitory effect of laevulinic acid

Induction of nitrate reductase EC 1.6.6.1 in etiolated barley (Hordeum vulgare L., var. Proctor) required continuous illumination and showed a lag period of about three hours. During the first 16 h of illumination the ratio NADH/NAD and NADPH/NADP, taken as a measure of internal oxidation reduction potential, declined. The inhibitor DCMU applied to whole leaves at concentrations shown to inhibit the reduction of cytochrome f by Photosystem 2 light did not inhibit the induction of nitrate reductase nor did it diminish the ratio of reduced to oxidised puridine nucleotides in the early hours of greening. It was concluded that light driven electron flow was not necessary for nitrate reductase induction. Chloramphenicol gave a slight inhibition of nitrate reductase induction. Laevulinic acid was added to greening barley leaves to inhibit tetrapyrrole pigment biosynthesis and plastid development. It strongly inhibited chlorophyll synthesis and nitrate reductase induction, with relatively little effect upon Photosystem 1 and 2 activities in isolated plastids. The activities of other inducible enzymes and control enzymes were little affected by laevulinic acid. Laevulinic acid also inhibited nitrate reductase induction by added nitrate in fully-greened illuminated plants grown in nitrate-free medium and so is unlikely to be acting through inhibition of plastid development. This inhibitor lowered the level of protohaem in whole leaves and plastids of greening barley and it is postulated that it may diminish the protohaem available for the assembly of a cytochrome b component of nitrate reductase.Abbreviations DCMU 3-(3:4-Dichlorophenyl)-1:1-dimethylurea - LA laevulinic acid     

Excision-repair capacity in Streptococcus pneumoniae: cloning and expression of a uvr-like gene

Although deficient in photoreactivation and some SOS-like functions, Streptococcus pneumoniae has the capacity to carry out excision repair when exposed to UV light. The repair ability and sensitivity to UV irradiation or treatment with chemical agents in the wild type and a UV-sensitive mutant strain indicate that UV-induced pyrimidine dimers might be repaired in pneumococcus by a system similar to the uvr-dependent system in Escherichia coli. A gene complementing the mutation conferring UV sensitivity of the mutant strain has been cloned. The coding region directs the synthesis of a polypeptide with a molecular weight of 78 kDa. The relationship with uvr-like protein in E. coli is discussed.