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1.
Initial rate studies of spinach ( Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the Vmax and Km for NO 3− yielding Vmax values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and Km values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single Km of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a Ki of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO 3− complex. Binding of Cl − to the enzyme-NO 3− complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase. 相似文献
2.
Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO 3− for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO 3− likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO 3−, the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO 3− occurs during uptake and reduction of exogenous NO 3−. Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO 3−. It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO 3−, (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO 3− is taken up by cells of the cortex and stele, and/or (c) NO 3− is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO 3− reduction. 相似文献
3.
Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley ( Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO 3− uptake by more than 90% but had no effect on NO 2− uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO 3− uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO 3− uptake. The results present the possibility that NO 3− uptake and NO 3− reduction in the PM of barley roots may be related. 相似文献
4.
Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD +, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO 3− with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO 3− reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD +, and NO 3− in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain. 相似文献
5.
Growth chamber studies with soybeans ( Glycine max [L.] Merr.) were designed to determine the relative limitations of NO 3−, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO 3−in vivo, −NO 3−in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO 3− to the in vivo NR assay medium increased activity (over that of the −NO 3−in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO 3− was rate-limiting. The stimulation of in vivo NR activity by NO 3− was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO 3− was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO 2− [g fresh weight, hr] −1) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting. 相似文献
6.
Experiments were designed to study the importance of organic acids as counterions for K + translocation in the xylem during excess cation uptake. A comparison was made of xylem exudate from wheat seedlings treated 72 hours with either 1.0 millimolar KNO 3 or 0.5 millimolar K 2SO 4, both in the presence of 0.2 millimolar CaSO 4. Exudation from KNO 3 plants had twice the volume and twice the K + and Ca 2+ fluxes or rate of delivery to shoots, as K 2SO 4 plants. Malate flux was 25% higher in K 2SO 4 than in KNO 3 exudate. Malate was the principal anion accompanying K + or Ca 2+ in K 2SO 4 treatment, while in the KNO 3 treatment, NO 3− was the principal anion. The contribution of SO 42− was negligible in both treatments. In a second experiment, exudate was collected every 4 hours during the daytime throughout a 72-hour treatment with KNO 3. Malate was the only anion present in exudate at first, just after the CaSO 4 pretreatment had ended. Malate concentration decreased and NO 3− concentration increased with time and these concentrations were negatively correlated. By 62 hours, NO 3− represented 80% of exudate anions. K + and NO 3− concentrations in exudate were strongly correlated with K + and NO 3− uptake, respectively. The first 36 hours of absorption from KNO 3 solution resembled the continuous absorption of K 2SO 4, in that malate was the principal counterion for translocation of K +. 相似文献
7.
Dark-grown, detopped corn seedlings (cv. Pioneer 3369A) were exposed to treatment solutions containing Ca(NO 3) 2, NaNO 3, or KNO 3; KNO 3 plus 50 or 100 millimolar sorbitol; and KNO 3 at root temperatures of 30, 22, or 16 C. In all experiments, the accelerated phase of NO 3− transport had previously been induced by prior exposure to NO 3− for 10 hours. The experimental system allowed direct measurements of net NO 3− uptake and translocation, and calculation of NO 3− reduction in the root. The presence of K + resulted in small increases in NO 3− uptake, but appreciably stimulated NO 3− translocation out of the root. Enhanced translocation was associated with a marked decrease in the proportion of absorbed NO 3− that was reduced in the root. When translocation was slowed by osmoticum or by low root temperatures, a greater proportion of absorbed NO 3− was reduced in the presence of K +. Results support the proposition that NO 3− reduction in the root is reciprocally related to the rate of NO 3− transport through the root symplasm. 相似文献
8.
A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach ( Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg 2+ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl 2 and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl 2, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent Km (NADH) was 74 micromolar and Km (NADPH) was 81 micromolar. Apparent Vmax was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide. 相似文献
9.
The effects of NO 3− and assay temperature on proton translocating ATPases in membranes of barley ( Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H +-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO 3 when assayed at 24°C or above and was tentatively identified as the tonoplast H +-ATPase. A smaller peak of H +-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO 3 and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H +-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO 3 inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO 3. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO 3 caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO 3 was explained by the temperature-dependent delay of the inhibition by KNO 3. The plasma membrane H +-ATPase maintained a pH gradient in the presence of KNO 3 for up to 30 minutes at 24°C. 相似文献
10.
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
11.
The isotherm for isocitrate oxidation by potato ( Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate Km for the membrane enzyme was calculated to be approximately 5.9 × 10 −4 molar, while the S 0.5 for the matrix was 6.9 × 10 −4 molar. The NAD Km for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms. 相似文献
12.
The comparative induction of nitrate reductase (NR) by ambient NO 3− and NO 2− as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley ( Hordeum valgare L.) seedlings was determined. The dynamic interaction of NO 3− influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO 3−. As the ambient concentration of NO 3− increased, the relative influences imposed by influx and reduction on NO 3− accumulation changed with influx becoming a more predominant regulant. Significant levels of NO 3− accumulated in NO 2−-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO 2−, about 60% more NO 3− accumulated in the leaves than in the absence of the inhibitors. In NO 3−-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 m m. No NR induction occurred in leaves supplied with NO 2− until the ambient NO 2− concentration was 0.5 m m. In fact, NR induction from NO 2− solutions was not seen until NO 3− was detected in the leaves. The amount of NO 3− accumulating in NO 2−-fed leaves induced similar levels of NR as did equivalent amounts of NO 3− accumulating from NO 3−-fed leaves. In all cases the internal concentration of NO 3−, but not NO 2−, was highly correlated with the amount of NR induced. The evidence indicated that NO 3− was a more likely inducer of NR than was NO 2−. 相似文献
13.
The role of NO 3− and NO 2− in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley ( Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO 2−/gram fresh weight × hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO 2− and NO 3− induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO 2− and NO 3− over a 24 hour period). In NO 3−-supplied leaves, NiR induction occurred at an ambient NO 3− concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO 2− until the ambient NO 2− concentration was 0.5 millimolar. Nitrate accumulated in NO 2−-fed leaves. The amount of NO 3− accumulating in NO 2−-fed leaves induced similar levels of NiR as did equivalent amounts of NO 3− accumulating in NO 3−-fed leaves. Induction of NiR in NO 2−-fed leaves was not seen until NO 3− was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO 3−, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO 3− to NO 2− was inhibited by WO 42−, the induction of NiR was inhibited only partially. The results indicate that in barley leaves NiR is induced by NO 3− directly, i.e. without being reduced to NO 2−, and that absorbed NO 2− induces the enzyme activity indirectly after being oxidized to NO 3− within the leaf. 相似文献
14.
Corn seedlings ( Zea mays cv W64A × W182E) were grown hydroponically, in the presence or absence of NO 3−, with or without light and with NH 4Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO 3−. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO 3− was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO 3−. Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO 3− was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH 4Cl − was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO 3− or by some other unidentified factor, and that higher levels of NO 3− are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO 3− to NO 2−. 相似文献
15.
The influence of NH 4+, in the external medium, on fluxes of NO 3− and K + were investigated using barley ( Hordeum vulgare cv Betzes) plants. NH 4+ was without effect on NO 3− ( 36ClO 3−) influx whereas inhibition of net uptake appeared to be a function of previous NO 3− provision. Plants grown at 10 micromolar NO 3− were sensitive to external NH 4+ when uptake was measured in 100 micromolar NO 3−. By contrast, NO 3− uptake (from 100 micromolar NO 3−) by plants previously grown at this concentration was not reduced by NH 4+ treatment. Plants pretreated for 2 days with 5 millimolar NO 3− showed net efflux of NO 3− when roots were transferred to 100 micromolar NO 3−. This efflux was stimulated in the presence of NH 4+. NH 4+ also stimulated NO 3− efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO 3− occur via effects upon efflux. By contrast to the situation for NO 3−, net K + uptake and influx of 36Rb +-labeled K + was inhibited by NH 4+ regardless of the nutrient history of the plants. Inhibition of net K + uptake reached its maximum value within 2 minutes of NH 4+ addition. It is concluded that the latter ion exerts a direct effect upon K + influx. 相似文献
16.
Pyrophosphorylytic kinetic constants (S 0.5, Vmax) of partially purified UDP-glucose- and ADP-glucose pyrophosphorylases from potato tubers were determined in the presence of various intermediary metabolites. The S 0.5 of UDP-glucose pyrophosphorylase for UDP-glucose (0.17 millimolar) or pyrophosphate (0.30 millimolar) and the Vmax were not influenced by high concentrations (2 millimolar) of these substances. The most efficient activator of ADP-glucose pyrophosphorylase was 3-P-glycerate (A 0.5 = 4.5 × 10 −6 molar). The S 0.5 for ADP-glucose and pyrophosphate was increased 3.5-fold (0.83 to 0.24 millimolar) and 1.8-fold (0.18 to 0.10 millimolar), respectively, with 0.1 millimolar 3-P-glycerate while the Vmax was increased nearly 4-fold. The magnitude of 3-P-glycerate stimulation was dependent upon the integrity of key sulfhydryl groups (−SH) and pH. Oxidation or blockage of −SH groups resulted in a marked reduction of enzyme activity. Stimulations of 3.1-, 2.9-, 4.8-, and 9.5-fold were observed at pH 7.5, 8.0, 8.5, and 9.0, respectively, in the presence of 3-P-glycerate (2 millimolar). The most potent inhibitor of ADP-glucose pyrophosphorylase was orthophosphate (I 0.5 = 8.8 × 10 −5. molar). This inhibition was reversed with 3-P-glycerate (1.2 × 10 −4 molar), resulting in an increased I 0.5 value of 1.5 × 10 −3 molar. Likewise, orthophosphate (7.5 × 10 −4 molar) caused a decrease in the activation efficiency of 3-P-glycerate (A 0.5 from 4.5 × 10 −6 molar to 6.7 × 10 −5 molar). The significance of 3-P-glycerate activation and orthophosphate inhibition in the regulation of α-glucan biosynthesis in Solanum tuberosum is discussed. 相似文献
17.
The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms. The enzyme uses either Mg2+ or Mn2+ as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn2+ the Km for malate2− is lower than with Mg2+, but Vmax is lower than with Mg2+. In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C3 plants. In the reverse reaction, run with Mn2+, the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 × 10−3 molar. The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this. The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH. The enzyme is activated by coenzyme A with Ka = 4 micromolar. AMP also shows competitive activation, with Ka = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate2− is a strong activator, with Ka = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with Ki = 4.9 millimolar. 相似文献
18.
The inducibility and kinetics of the NO 3−, NO 2−, and NH 4+ transporters in roots of wheat seedlings ( Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for and the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO 3− and NO 2− transporters. Evidence for substrate inducibility of the NH 4+ transporters requiring 5 hours is presented. The transport of NO 3− was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO 2− and NH 4+. The Km values for NO 3−, NO 2−, and NH 4+ were, respectively, 0.027, 0.054, and 0.05 millimolar. The Km for mechanism II of NO 3− transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar. 相似文献
19.
A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO (x)) evolution activity extracted from soybean ( Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO (x) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent Km for nitrite of 0.49 millimolar, and an apparent Km for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO (x) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH 2) and reduced methyl viologen (MV) can serve as electron donors for NO (x) evolution activity. The NADPH-, FMNH 2-, and reduced MV-NO (x) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH 2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO (x). NADPH eluted both NR and NO (x) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean ( Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean. 相似文献
20.
Nitrate and NO 2− transport by roots of 8-day-old uninduced and induced intact barley ( Hordeum vulgare L. var CM 72) seedlings were compared to kinetic patterns, reciprocal inhibition of the transport systems, and the effect of the inhibitor, p-hydroxymercuribenzoate. Net uptake of NO 3− and NO 2− was measured by following the depletion of the ions from the uptake solutions. The roots of uninduced seedlings possessed a low concentration, saturable, low Km, possibly a constitutive uptake system, and a linear system for both NO 3− and NO 2−. The low Km system followed Michaelis-Menten kinetics and approached saturation between 40 and 100 micromolar, whereas the linear system was detected between 100 and 500 micromolar. In roots of induced seedlings, rates for both NO 3− and NO 2− uptake followed Michaelis-Menten kinetics and approached saturation at about 200 micromolar. In induced roots, two kinetically identifiable transport systems were resolved for each anion. At the lower substrate concentrations, less than 10 micromolar, the apparent low Kms of NO 3− and NO 2− uptake were 7 and 9 micromolar, respectively, and were similar to those of the low Km system in uninduced roots. At substrate concentrations between 10 and 200 micromolar, the apparent high Km values of NO 3− uptake ranged from 34 to 36 micromolar and of NO 2− uptake ranged from 41 to 49 micromolar. A linear system was also found in induced seedlings at concentrations above 500 micromolar. Double reciprocal plots indicated that NO 3− and NO 2− inhibited the uptake of each other competitively in both uninduced and induced seedlings; however, Ki values showed that NO 3− was a more effective inhibitor than NO 2−. Nitrate and NO 2− transport by both the low and high Km systems were greatly inhibited by p-hydroxymercuribenzoate, whereas the linear system was only slightly inhibited. 相似文献
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