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1.
Summary Nitrate reductase can be induced in isolated embryos of Agrostemma githago by treatment with cytokinin. By labeling proteins with H 2
18O and subsequent isopycnic density gradient centrifugation it was proved that the benzyladenine induction of nitrate reductase is caused by a de-novo synthesis of the enzyme. 相似文献
2.
Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO 3− and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO 3−, either by amplifying the effect of low endogenous NO 3− levels, or by making NO 3− available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO 3− and benzyladenine, additive responses were obtained. The effects of NO 3− and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO 3−, as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO 3− was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO 3− and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increasd again as a result of a second treatment with benzyladenine but not with NO 3−. At the time of the second exposure to benzyladenine, no NO 3− was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO 3−. 相似文献
3.
1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase. 相似文献
4.
Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein M
r150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein M
r150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wilde type. Autoradiography of preparations from cells incubated with 55Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with 99Mo, incorporation of molybdenum into the mutant protein was not detectable.We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.Non-Standard Abbreviations DOC
deoxycholate
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate 相似文献
5.
Induction of nitrate reductase (EC 1.6.6.1) activity was measured in Paul's Scarlet rose cell suspensions cultured in media containing nitrate (NO
3
-
) or urea (U) as nitrogen source, and with (+Mo) or without molybdenum (-Mo). There was a lag of 30 min during induction by NO
3
-
in +Mo cultures but no lag occurred during induction after adding Mo to NO
3
-
-Mo or to U-Mo cultures preincubated with NO
3
-
. Actinomycin D, cycloheximide, and puromycin completely blocked induction by NO
3
-
, but had no effect on the initial rate of induction by Mo. Cycloheximide and puromycin blocked induction by NO
3
-
more quickly than actinomycin D. Induction by NO
3
-
appeared to involve mRNA-dependent synthesis of apoprotein followed by rapid activation with molybdenum in intact cells independently of protein synthesis. Nitrate-induced apoprotein appeared less stable than the holoenzyme. When induced by NO
3
-
in the absence of Mo, apoprotein concentration was about half the amount of maximally induced nitrate reductase. Cycloheximide stabilised preformed nitrate reductase which disappeared steadily in the presence of puromycin. Apoprotein was not stabilised by either antimetabolite.Abbreviations Mo
molybdenum
- NO
3
-
+Mo
standard, MX 1 culture medium
- NO
3
-
-Mo
MX 1 medium purified of Mo
- NR
nitrate reductase
- PSR
Paul's Scarlet rose
- U
urea
- U+Mo
MX 1 medium with NO
3
-
replaced by urea
- U-Mo
MX 1 medium with NO
3
-
replaced by urea and also purified of Mo 相似文献
6.
NADH-nitrate reductase activity in excised embryos of Agrostemma githago develops in response to nitrate as well as benzyladenine. Induction of nitrate reductase by benzyladenine was much more susceptible to inhibition by a mixture of amino acid analogues and by cordycepin than induction by nitrate. In contrast, only induction of nitrate-nitrate reductase was decreased by chloramphenicol. 相似文献
7.
In shoot apices of 7-day-old dark-grown peas the addition of ammonium along with the inducer nitrate resulted in a more than two-fold increase in nitrate reductase activity. Individual amino acids, amides and amino-acid mixture could not replace the ammonium effect. Ammonium also stimulated NADH-glutamate dehydrogenase but not glucose-6-phosphate dehydrogenase. Sucrose caused a marked stimulation of nitrate reductase induction and showed synergistic effect with light. In presence of cordycepin and cycloheximide, induction of nitrate reductase was inhibited more if ammonium or sucrose was supplied along with the inducer. With actinomycin D, α-amanitin or chloramphenicol, no differential inhibition took place in presence of ammonium. The inhibition of enzyme activity by chloramphenicol and 3-(3,4-dichlorophenyl)-l,dimethyl urea was completely relieved by sucrose. Incorporation of 14C-lysine was markedly stimulated by sucrose, but was not affected by ammonium. The effect of sucrose and light on 14C-lysine incorporation was additive. Cordycepin and cycloheximide did not have any differential effect on 14C-lysine incorporation in the presence of ammonium as well as sucrose. The inhibition of 14C-lysine incorporation caused by chloramphenicol was relieved by sucrose. Sucrose also caused a marked increase in 3H-uridine incorporation but ammonium had no effect. Actinomycin D and cordycepin blocked the sucrose dependent increase in 3H-uridine incorporation. The results suggest that ammonium mediated stimulation may depend on a regulatory protein(s) synthesized in response to ammonium, whereas sucrose acts mainly by an overall increase in RNA and protein synthesis. The effect of light does not seem to be dependent on photosynthetic light reactions. 相似文献
8.
Cotyledons were excised from 7-day-old dark-grown cucumber seedlings and treated with water, benzyladenine (BA), kinetin (K), zeatin (Z), or zeatin riboside (ZR) in dark after endogenous cytokinin depletion. We have compared changes in chitinase (EC. 3.2.1.14) activity induced by these cytokinins. We find that the activities of chitinase and its isoforms increase by approximately 3- to 6-fold following BA, Z, and ZR treatments. Among these treatments, Z was more effective. K was totally ineffective in inducing chitinase activity. Immunoblot analysis suggests that the cytokinin Z-induction of enzyme activity is due to the induction of higher chitinase protein levels and not the activation of existing enzyme. Furthermore, the Z-induced chitinase activity and its protein accumulation were completely inhibited by the protein kinase inhibitor staurosporine, whereas the protein phosphatase inhibitor sodium fluoride was not effective in such inhibitions. Treatment of cotyledons with extemal CaCl 2 and calcium ionophore increased the basal chitinase activity by 6- and 5-fold, respectively. Moreover, the effects of staurosporine, sodium fluoride, and Ca 2+ on Z-induced chitinase activity correlate with their effects on chitinase protein levels. Taken together, our data suggests Ca 2+ and staurosporine-sensitive protein kinase(s) as components of the cytokinin transduction machinery involving induction of chitinase in cucumber. 相似文献
9.
Comparison of two-dimensional polyacrylamide gel electrophoretic maps of proteins isolated from benzyladenine-treated and untreated pumpkin ( Cucurbita pepo L. cv Halloween) cotyledons showed that the expression of certain proteins is enhanced, induced, or suppressed by the cytokinin treatment. The amount of poly(A) + mRNA isolated from cotyledons incubated with 10 −4 molar benzyladenine for five days was about four-fold over the water-incubated control. The activity of hydroxypyruvate reductase prepared from purified cotyledonous microbodies and analyzed by native gel electrophoresis is proportionally enhanced by sequentially higher concentrations (10 −9 to 10 −4 molar) of benzyladenine. Ethidium bromide (1 microgram per milliliter) did not inhibit hydroxypyruvate reductase activity; thus, the enzyme synthesis does not appear to be controlled by organelle genes. Hydroxypyruvate reductase synthesis is inhibited by cycloheximide, cordycepin, and to a certain degree by actinomycin D. These data support the view of a close association between cytokinin action and gene expression. 相似文献
10.
In order to compare the effects of excess pedospheric and atmospheric nitrogen supply on nitrate reductase activity (NR. EC 1.6.6.1) excised spruce branches were exposed to nitrate solutions or were fumigated with NO 2. Immersion of spruce branches in 6 mM nitrate caused an increase in NR activity by a factor of 14 or 19 in current-year and in one-year-old needles, respectively, as compared to controls incubated in tap water. Exposure to 65 nl I ?1 NO 2 increased NR activity by a factor of 1.5 in current-year needles and by a factor of 2.5 in one-year-old needles as compared to non-fumigated controls. Addition of cycloheximide (0.17 μM) or puromycin (200 μM) to the incubation solution prevented the induction of NR activity from both nitrate and NO 2 exposure. This finding indicates that induction of NR activity by both atmospheric NO 2 or increased nitrate supply of the needles is both caused by de-novo synthesis of NR protein. The increase in NR activity in needles of branches still attached to the tree as a consequence of exposure to 65 nl I ?1 NO 2 was found to be a transient phenomenon. The increase persisted for several days only and was no longer observed after one week of sustained NO 2 exposure. An interruption of phloem transport by girdling, applied subsequent to the induction of NR activity by atmospheric NO 2, prevented the decrease in NR activity. Apparently, export out of the exposed needles and phloem transport within the stem are involved in the regulation of NR activity upon NO 2 exposure. 相似文献
11.
Detached first leaves of 3-day-old corn seedlings ( Zea mays L. W64AxW183E) were incubated with nitrate in air or 100% O 2 in the light. Nitrate accumulation in the leaves was not depressed by O 2. NADH:nitrate reductase activity and enzyme protein, as measured with an enzyme-linked immunosorbent assay, increased in parallel during the 8 h nitrate treatment in air, but in O 2 the levels of enzyme activity and protein were depressed. NADH:nitrate reductase mRNA levels were the same in the air-and O 2-treated leaves. Total soluble protein levels in leaves were slightly depressed by O 2 and shifting from O 2 to an air environment increased the protein level. Incorporation of [ 35S]methionine during nitrate treatment revealed that total soluble protein and nitrate reductase protein synthesis were both depressed by the O 2 environment relative to air, but both recovered when leaves were shifted from O 2 to air. Although O 2 accelerated inactivation of nitrate reductase in vitro, the in vivo inactivation rate appeared to be too low to account for the depressed level of nitrate reductase activity in O 2-treated leaves. We concluded that O 2 inhibition of nitrate reductase biosynthesis in detached corn leaves was largely due to inhibition of total soluble protein synthesis at the level of translation. 相似文献
12.
After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO 3− as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH 4+-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein. 相似文献
13.
Nitrate and light are known to modulate barley ( Hordeum vulgare L.) nitrate reductase activity. The objective of this investigation was to determine whether barley nitrate reductase is regulated by enzyme synthesis and degradation or by an activation-inactivation mechanism. Barley seedling nitrate reductase protein (cross-reacting material) was determined by rocket immunoelectrophoresis and a qualitative immunochemical technique (western blot) during the induction and decay of nitrate reductase activity. Nitrate reductase cross-reacting material was not detected in root or shoot extracts from seedlings grown without nitrate. Low levels of nitrate reductase activity and cross-reacting material were observed in leaf extracts from plants grown on nitrate in the dark. Upon nitrate induction or transfer of nitrate-grown etiolated plants to the light, increases in nitrate reductase activity were positively correlated with increases in immunological cross-reactivity. Root and shoot nitrate reductase activity and cross-reacting material decreased when nitrate-induced seedlings were transferred to a nitrate-free nutrient solution or from light to darkness. These results indicate that barley nitrate reductase levels are regulated by de novo synthesis and protein degradation. 相似文献
14.
Light was required for induction of nitrate reductase (NR, E.C. 1.6.6.1) in intact cotyledons of 2-day old seedlings of Lactuca sativa L. Molybdate strongly enhanced efficiency of induction. Benzyladenine (BA), gibberellin, and succinic acid-2,2-dimethylhydrazide
reduced the enzyme activity. BA thrice enhanced incorporation of labelled leucine to the protein fraction. (2-chloroethyl)trimethylammonium
chloride did not affect NR activity and markedly inhibited greening and protein synthesis. KNO 3 stimulated protein synthesis as well as growth of the cotyledons. 相似文献
15.
Supply of 100 μM spermidine (Spd) in the nutrient solution containing 10 mM nitrate as the sole nitrogen source, increased growth of roots and shoots, total nitrogen content and in vivo or in vitro nitrate reductase (NR) activity of leaves of 10-d old Leucaena leucocephala seedlings. Spd and the cytokinins benzyladenine or kinetin also increased growth, total nitrogen and in vivo NR activity of isolated cotyledons. The synergistic effects of nitrate, kinetin and Spd in increasing NR activity, indicate that the Spd acted at different level than the nitrate or cytokinin. 相似文献
16.
The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH 2OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH 2OH. To demonstrate that the terminal activity was readily inactivted by NH 2OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH 2OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH 2OH appears to be competitive versus NO 3−. Since NO 3−, as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO 3−), protect the enzyme from NH 2OH inactivation, it is suggested that NH 2OH binds to the nitrate active site. The NH 2OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN −. NH 2OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance. 相似文献
17.
With respect to cofactor requirements, NADH, and FMNH 2 were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH 2 required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH 2 was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH 2). All attempts to separate the NADH2- and FMNH2-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH2 activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH2-dependent activities were not additive. A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35° in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted. In those instances where NADH activity but not FMNH2 activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine. Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH2 as cofactor. However the high concentration of FMNH2 required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1). 相似文献
18.
1. Possible mechanisms regulating the activities of three enzymes involved in nitrate assimilation, nitrate reductase, nitrite reductase and glutamate dehydrogenase, were studied in radish cotyledons. 2. Nitrate-reductase and nitrite-reductase activities are low in nitrogen-deficient cotyledons, and are induced by their substrates. 3. Glutamate dehydrogenase is present regardless of the nitrogen status, and the enzyme can be increased only slightly by long-term growth on ammonia. 4. Although nitrate is the best inducer of nitrate reductase, lower levels of induction are also obtained with nitrite and ammonia. The experiments did not distinguish between direct or indirect induction by these two molecules. 5. Nitrite reductase is induced by nitrite and only indirectly by nitrate. 6. The induction of both nitrate reductase and nitrite reductase is prevented by the inhibitors actinomycin D, puromycin and cycloheximide, indicating a requirement for the synthesis of RNA and protein. 7. The decay of nitrate reductase, determined after inhibition of protein synthesis, is slower than the synthesis of the enzyme. Nitrite reductase is much more stable than nitrate reductase. 8. The synthesis of nitrate reductase is not repressed by ammonia, but is repressed by growth on a nitrite medium. 9. There is no inhibition of nitrate reductase, nitrite reductase or glutamate dehydrogenase by the normal end products of assimilation, but cyanate is a fairly specific inhibitor of nitrate reductase. 相似文献
19.
The nitrate reductase in the mature root extract of 3-day maize ( Zea mays) seedlings was relatively labile in vitro. Insoluble polyvinylpyrrolidone used in the extraction medium produced only a slight increase in the stability of the enzyme. Mixing the mature root extract with that of the root tip promoted the inactivation of nitrate reductase in the latter. The inactivating factor in the mature root was separated from nitrate reductase by (NH 4) 2SO 4 precipitation. Nitrate reductase was found in the 40% (NH 4) 2SO 4 precipitate, while the inactivating factor was largely precipitated by 40 to 55% (NH 4) 2SO 4. The latter fraction of the mature root inactivated the nitrate reductase isolated from the root tip, mature root, and scutellum. The inactivating factor, which has a Q 10 15 to 25 C of 2.2, was heat labile, and hence has been designated as a nitrate reductase inactivating enzyme. The reduced flavin mononucleotide nitrate reductase was also inactivated, while an NADH cytochrome c reductase in nitrate-grown seedlings was inactivated but at a slower rate. The inactivating enzyme had no influence on the activity of nitrite reductase, glutamate dehydrogenase, xanthine oxidase, and isocitrate lyase. The activity of the nitrate reductase inactivating enzyme was not influenced by nitrate and was also found in the mature root of minus nitrate-grown seedlings. 相似文献
20.
Bromphenol blue, which was reduced with dithionite, was found to support nitrate reduction catalyzed by squash NADH:nitrate reductase at a rate about 5 times greater than NADH with freshly prepared enzyme and 10 times or more with enzyme having been frozen and thawed. Kinetic analysis of bromphenol blue as a substrate for squash nitrate reductase yielded apparent Km values of 60 micromolar for bromphenol blue at 10 millimolar nitrate and 500 micromolar for nitrate at 0.2 millimolar bromphenol blue. With the same preparation of enzyme the apparent Km values were 9 micromolar for NADH at 10 millimolar nitrate and 50 micromolar nitrate at 0.1 millimolar NADH. Bromphenol blue was found to be a noncompetitive inhibitor versus NADH with a Ki of 0.3 millimolar. When squash NADH:nitrate reductase activity was inactivated with p-hydroxymercuribenzoate or denatured by heating at 40°C, the bromphenol blue nitrate reductase activity was not lost. These results were taken to indicate that bromphenol blue and NADH donated electrons to nitrate reductase at different sites. When monoclonal antibodies prepared against corn and squash nitrate reductases were used to inhibit the nitrate reductase activities supported by NADH, bromphenol blue, and methyl viologen, differential inhibition was found which tended to indicate that the three electron donors were interacting with the enzyme at different sites. One monoclonal antibody prepared against squash nitrate reductase inhibited all three activities of both corn and squash nitrate reductase. It appears this antibody may bind to a highly conserved antigenic site in the nitrate binding region of the enzyme. 相似文献
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