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1.
The activity of enzyme carbonic anhydrase (CA) was investigated in two diazotrophic cyanobacteria, Anabaena sp. (ARM 629) and Nostoc calcicola, in the presence of CO2/NaHCO3 and different inhibitors. The CA activity increased when the cells were pretreated with a high concentration of CO2/NaHCO3 and then transferred to ambient level CO2. Maximum activity of CA was observed after 8 h of incubation in light on transfer of cells from high Ci to ambient level CO2, and was low when incubated in dark. Addition of the photosynthetic inhibitor DCMU brought about a differential reduction in CA activity, depending on the carbon source (NaHCO3/CO2). CA inhibitors--ethoxyzolamide (EZ) and acetazolamide (AZ)--inhibited the enzyme activity in both the genera, but the extent of inhibition was greater in Anabaena sp. than in N. calcicola. Such a variation in extent of inhibition/stimulation of CA activity being different in the two genera reflects differences in their inherent potential and genetic background. The relevance of such cyanobacterial strains as CO2 sinks is also discussed. 相似文献
2.
The activities of antioxidant enzymes viz. glutathione reductase, GR; superoxide dismutase, SOD; peroxidase, POD; catalase, CAT and glutathione-S-transferase, GST and alkaloid accumulation were investigated in leaf pairs (apical, middle, basal) and in roots of Catharanthus roseus seedlings under the conditions of different nitrogen sources (20 mM KNO(3) and 2 mM NH(4)Cl) and salinity, in the absence (non-saline control) and in the presence of 100 mM NaCl in the nutrient solution. Salinity caused a reduction in plant biomass. The biomass production of ammonium-fed plants was lower than that of nitrate-fed plants. The antioxidant enzymes exhibited higher activity in saline-treated plants. Changes in antioxidant enzyme activity caused by different nitrogen sources differed in all leaf pairs, as well as in roots of C. roseus. Ammonium-fed plants showed higher CAT, GR and GST activity in leaf pairs as well as in roots, while POD and SOD activity were higher in nitrate-fed plants. Higher peroxidase activity concomitant with the increased accumulation of alkaloid was found in all leaf pairs, as well as in roots of C. roseus of NO(3)(-) fed plants as compared to NH(4)(+) fed plants. 相似文献
3.
用奎吖咽(quinacrine)作荧光指标剂,测定玉米(ZeamaysL.)根尖微粒体(MIC)膜囊泡的H~+-泵活性,结果表明1mmol/LNaN_3仅抑制该泵活性约8%,而0.8mmol/L钒酸盐(Van)则可抑制其活性达80%,说明MIC制剂中H~+-泵活性主要由质膜(PM)H~+-ATPase产生。此泵活性严格需要Mg~(2+),二价阳离子作用大小的顺序为Mg~(2+)>Mn~(2+)>Zn~(2+)>Ca~(2+)=0;阴离子作用大小的1顺序为Br~->Cl~->NO_3~->SO_4~(2-),并初步证实当质膜同侧发生电子传递时,没有跨膜H~+梯度(△μH~+)生成。 相似文献
4.
Expression of a carbonic anhydrase gene is induced by environmental stresses in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) 总被引:2,自引:0,他引:2
Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO3 and Na2CO3), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses.
Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO3, 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in
rice. 相似文献
5.
Dominique Rumeau Stephan Cuiné Laurent Fina Nathalie Gault Michel Nicole Gilles Peltier 《Planta》1996,199(1):79-88
The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO2 to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M
r of 30000. The chloroplastic isoenzyme (M
r 220000) is also an octamer composed of two different subunits with M
r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M
r-27 000 subunit was three amino acids shorter than that of the M
r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl–, I–, N
3
-
and NO
3
-
) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA
bovine serum albumin
- CA
carbonic anhydrase
- PMSF
phenylmethylsulphonyl fluoride
- BAM
benzamidine
- DTT
dithiothreitol
- 2-ME
2-mercaptoethanol
- PVDF
polyvinylidene difluoride
The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements. 相似文献
6.
Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined 总被引:1,自引:0,他引:1
The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol. 相似文献
7.
Ekinci D Ceyhun SB Sentürk M Erdem D Küfrevioğlu Oİ Supuran CT 《Bioorganic & medicinal chemistry》2011,19(2):744-748
Carbonic anhydrase (CA; EC 4.2.1.1) was purified from the gill of the teleost fish Dicentrarchus labrax (European seabass). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 84.9-fold with a yield of 58%, and a specific activity of 838.9 U/mg proteins. It has an optimum pH at 8.0; an optimum temperature at 10°C. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate. The following anions, H?NSO??, I?, SCN?, NO??, NO??, N??, Br?, Cl?, SO?2?, and F? showed inhibitory effects on the enzyme. Sulfamic acid, iodide, and thiocyanate exhibited the strongest inhibitory action, in the micromolar range (K(i)s of 87-187 μM). NO??, NO?? and N?? were moderate inhibitors, whereas other anions showed only weak actions. All tested anions inhibited the enzyme in a competitive manner. Our findings indicate that these anions inhibit the fish enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various anions differs significantly between the fish and mammalian CAs. 相似文献
8.
Summary Plasma membrane vesicles prepared from the bag re gion of the somatic muscle cell of the parasiteAscaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct
a variety of anions has been investigated using the patch-clamp technique on isolated inside-out patches of muscle membrane.
Symmetrical Cl solutions (140 mm) produced single-channel I/V plots with reversal potentials of 0 mV, substitution of bath
Cl by 140 mM NO3, Br and I caused depolarizing shifts in the reversal potentials. Replacement of the internal Cl by F (140 mM) caused a large
hyperpolarizing shift in the reversal potential. The channel dis played a permeability sequence of I > Br = NO3> Cl > F which differed from the corresponding conductance sequence Cl > NO3 = Br = I > F. The ionic environment within the channel pore has been investigated using Reuter and Stevens (1980) plots to
describe the selectivity and “fluidity” of the channel pore. In addition, the approach of Wright and Diamond (1977) was employed
to estimate the number of cationic binding sites within the channel pore. The channel is relatively fluid but the number of
cationic binding sites varies inversely with the ionic radius of the anion from 2.15 for F to 0.89 for the large planar anion
NO3 相似文献
9.
Charney AN Alexander-Chacko J Gummaconda R Egnor RW 《Biochimica et biophysica acta》2002,1573(2):141-148
Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO(2)-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na(+) and Cl(-), and total HCO(3)(-) generation (estimated as the sum of radiolabeled HCO(3)(-) and CO(2) produced from glucose) were measured in Ussing chambers in nominally CO(2), HCO(3)(-)-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na(+) and Cl(-) absorption. In the ileum, methazolamide reduced J(ms)(Na) and J(ms)(Cl) and caused net Cl(-) secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na(+) and Cl(-) absorption at pH 7.6. Total HCO(3)(-) generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl(-) absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function. 相似文献
10.
We investigated whether changes in sucrose-phosphate synthase (EC 2.4.1.14, SPS) activity could alter N remobilization during leaf senescence. Transgenic rice (Oryza sativa L. cv. Nipponbare) with low SPS activities and wild-type rice plants were grown with basal N (1.0 mM NH4NO3) until the late vegetative stage. Subsequently, half of the plants were transferred to a low N (0.1 mM NH4NO3) condition to accelerate leaf senescence, and the others were continuously grown with basal N. With low N supply, the amounts of chlorophyll and soluble protein in flag leaf blades decreased after anthesis in both the low SPS plants and wild-type plants, although the decrease was less in the low SPS plants. Panicle weights were significantly lower in the low SPS plant than in the wild-type plant. These results suggest that the remobilization of N from flag leaves was diminished by suppressing the development of reproductive sinks in the low SPS plant. 相似文献
11.
The effects of Cl(-), Mn(2+), Ca(2+), and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl(-), and strongly inhibited above this concentration. This enzyme is activated by Mn(2+) and stimulated somewhat by Ca(2+). The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl(-) up to 0.4 M. Ca(2+) appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn(4) complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent. 相似文献
12.
Diazotrophic heterocystous cyanobacteria Nostoc calcicola and Anabaena sp. ARM 629 were investigated for their ability to grow in presence of sodium bicarbonate (NaHCO3) or carbon dioxide (CO2) under cultural conditions. Maximum growth was observed in 75 mM NaHCO3 and 5% CO2 in N. calcicola and Anabaena ARM 629, respectively. Although their growth rate declined, N. calcicola and Anabaena sp. could tolerate upto 250 mM NaHCO3 and 20% CO2, respectively. N-methyl-N'-nitro N nitrosoguanidine induced mutants of these cyanobacteria were isolated which showed growth upto 1 M NaHCO3 (N. calcicola) or 50% CO2 (Anabaena sp.) in comparison to their wild types. The mutants also showed cross-resistance to either of the inorganic carbon compounds, which was not observed for wild type. It was concluded that mutants were altered in multiple properties enabling them to grow at elevated levels of inorganic carbon compounds. 相似文献
13.
Nitric oxide counteracts the senescence of detached rice leaves induced by dehydration and polyethylene glycol but not by sorbitol 总被引:12,自引:0,他引:12
In the present study, we evaluated the protective effect of nitric oxide(NO) against senescence of rice leaves enhanced by water deficit. Dehydration(DH), polyethylene glycol (PEG) and sorbitol (ST) treatments were used toinducewater deficit. Senescence of rice leaves was determined by the decrease ofprotein content. NO donors[N-tert-butyl--phenylnitrone (PBN), sodiumnitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and ascorbic acid +NaNO2] were effective in inhibiting senescence of dehydrated andPEG-treated rice leaves, but had no effect on senescence of ST-treated riceleaves. PEG or DH resulted in a marked increase in malondialdehyde (MDA)contentand decrease in superoxide dismutase (SOD) activity, but ST had no effect onMDAcontent and SOD activity. Treatment with NO donors caused a reduction of PEG-and DH-induced increase in MDA content and decrease in SOD activity. Theprotective effect of NO donors on promotion of senescence, increase in lipidperoxidation and decrease in SOD activity induced by PEG and DH was reversed by2-(4-carboxy-2-phenyl)-4,4,5,5- tetra-methylinmidazoline-1-oxyl-3-oxide, a NOspecific scavenger, suggesting that the protective effect of NO donors isattributed to NO released. The inhibition of PEG- and DH- enhanced senescenceofrice leaves by NO is most likely mediated through increasing SOD activity anddecrease in lipid peroxidation. 相似文献
14.
Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore. 相似文献
15.
K Nakajima N Niisato Y Marunaka 《Biochemical and biophysical research communications》2012,425(2):225-229
In growing neurite of neuronal cells, it is suggested that α/β-tubulin heterodimers assemble to form microtubule, and assembly of microtubule promotes neurite elongation. On the other hand, recent studies reveal importance of intracellular Cl(-) in regulation of various cellular functions such as cell cycle progression, differentiation, cell migration, and elongation of neurite in neuronal cells. In this study, we investigated effects of Cl(-) on in vitro tubulin polymerization. We found that efficiency of in vitro tubulin polymerization (the number of microtubule) was higher (3 to 5-fold) in Cl(-)-containing solutions than that in Cl(-)-free solutions containing Br(-) or NO(3)(-). On the other hand, GTPase activity of tubulin was lower (2/3-fold) in Cl(-)-containing solutions than that in Cl(-)-free solutions containing Br(-) or NO(3)(-). Efficiency of in vitro tubulin polymerization in solutions containing a non-hydrolyzable analogue of GTP (GpCpp) instead of GTP was much higher than that in the presence of GTP. Effects of replacement of GTP with GpCpp on in vitro tubulin polymerization was weaker in Cl(-) solutions (10-fold increases) than that in Br(-) or NO(3)(-) solutions (20-fold increases), although the efficiency of in vitro tubulin polymerization in Cl(-) solutions containing GpCpp was still higher than that in Br(-) or NO(3)(-) solutions containing GpCpp. Our results suggest that a part of stimulatory effects of Cl(-) on in vitro tubulin polymerization is mediated via an inhibitory effect on GTPase activity of tubulin, although Cl(-) would also regulate in vitro tubulin polymerization by factors other than an inhibitory effect on GTPase activity. 相似文献
16.
Kaori Ohki Jonathan P. Zehr Paul G. Falkowski Yoshihiko Fujita 《Archives of microbiology》1991,156(5):335-337
The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N2 as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N2 was not suppressed after 7 h incubation with 2 mM NaNO3 or 0.02 mM NH4Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO3, 0.5 mM urea or 0.02 mM NH4Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo–Fe protein, revealed the following: (1) both the Fe-protein and the Mo–Fe protein were synthesized in cells grown with N2 as well as in cells grown with NaNO3 or low concentration of NH4Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO3 or low concentration of NH4Cl; (4) neither the Fe-protein nor the Mo–Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1. 相似文献
17.
Exogenous nitric oxide enhances cadmium tolerance of rice by increasing pectin and hemicellulose contents in root cell wall 总被引:6,自引:0,他引:6
To study the mechanisms of exogenous NO contribution to alleviate the cadmium (Cd) toxicity in rice (Oryza sativa), rice plantlets subjected to 0.2-mM CdCl2 exposure were treated with different concentrations of sodium nitroprusside (SNP, a NO donor), and Cd toxicity was evaluated
by the decreases in plant length, biomass production and chlorophyll content. The results indicated that 0.1 mM SNP alleviated
Cd toxicity most obviously. Atomic absorption spectrometry and fluorescence localization showed that treatment with 0.1 mM
SNP decreased Cd accumulation in both cell walls and soluble fraction of leaves, although treatment with 0.1 mM SNP increased
Cd accumulation in the cell wall of rice roots obviously. Treatment with 0.1 mM SNP in nutrient solution had little effect
on the transpiration rate of rice leaves, but this treatment increased pectin and hemicellulose content and decreased cellulose
content significantly in the cell walls of rice roots. Based on these results, we conclude that decreased distribution of
Cd in the soluble fraction of leaves and roots and increased distribution of Cd in the cell walls of roots are responsible
for the NO-induced increase of Cd tolerance in rice. It seems that exogenous NO enhances Cd tolerance of rice by increasing
pectin and hemicellulose content in the cell wall of roots, increasing Cd accumulation in root cell wall and decreasing Cd
accumulation in soluble fraction of leaves. 相似文献
18.
Responses of rice cultivars with different nitrogen use efficiency to partial nitrate nutrition 总被引:5,自引:0,他引:5
BACKGROUND AND AIMS: There is increased evidence that partial nitrate (NO3-) nutrition (PNN) improves growth of rice (Oryza sativa), although the crop prefers ammonium (NH4+) to NO3- nutrition. It is not known whether the response to NO3- supply is related to nitrogen (N) use efficiency (NUE) in rice cultivars. Methods Solution culture experiments were carried out to study the response of two rice cultivars, Nanguang (High-NUE) and Elio (Low-NUE), to partial NO3- supply in terms of dry weight, N accumulation, grain yield, NH4+ uptake and ammonium transporter expression [real-time polymerase chain reaction (PCR)]. KEY RESULTS: A ratio of 75/25 NH4+ -N/NO3- -N increased dry weight, N accumulation and grain yield of 'Nanguang' by 30, 36 and 21 %, respectively, but no effect was found in 'Elio' when compared with those of 100/0 NH4+ -N/NO3- -N. Uptake experiments with 15N-NH4+ showed that NO3- increased NH4+ uptake efficiency in 'Nanguang' by increasing Vmax (14 %), but there was no effect on Km. This indicated that partial replacement of NH4+ by NO3- could increase the number of the ammonium transporters but did not affect the affinity of the transporters for NH4+. Real-time PCR showed that expression of OsAMT1s in 'Nanguang' was improved by PNN, while that in 'Elio' did not change, which is in accordance with the differing responses of these two cultivars to PNN. Conclusions Increased NUE by PNN can be attributed to improved N uptake. The rice cultivar with a higher NUE has a more positive response to PNN than that with a low NUE, suggesting that there might be a relationship between PNN and NUE. 相似文献
19.
The human erythrocyte membrane is an efficient enhancer of both high (CA II) and low (CA I) activity isozymes of red blood cell carbonic anhydrase. The presence of membrane increased CO2 hydration catalyzed by bovine CA II 1.6-fold, human CA II 3.5-fold, and human CA I 1.6-fold. With the high activity CA isozymes, maximal stimulation was observed in the presence of 1-3 micrograms membrane protein/ml. The Vmax for bovine CA II (4 nM) rose from 0.302 to 0.839 mM/s, while that for human CA II (6 nM) increased from 0.113 to 0.414 mM/s in the absence and presence of membrane, respectively. The apparent Km for CO2 increased from 13.2 to 51.2 mM for bovine CA II, and from 6.5 to 38.5 mM for human CA II. Mixtures of membrane plus enzyme, upon centrifugation through linear sucrose density gradients, displayed enhanced Ca activity only in membrane-containing gradient fractions, verifying the stimulatory ability of membranes on enzyme activity and indicating tight and stable complex formation. Membrane enhancement of CA activity appears to be a general phenomenon in that mouse hepatocyte membranes also stimulated CA activity, although less efficiently than erythrocyte membranes. Of the many soluble putative effectors assayed, only imidazole enhanced CA II activity to an extent comparable with erythrocyte membranes; imidazole did not, however, stimulate the activity of human CA I. The data are consistent with a model of CA II activation by membrane association that may effect a distortion of the enzyme conformation in such a way as to facilitate intra- and/or intermolecular proton transfer between membrane-bound and enzyme-bound proton shuttling residues (perhaps the imidazole moiety of histidine) and the Zn-bound hydroxide at the catalytic site of the enzyme. 相似文献
20.
Spring wheat (Triticum aestivum cv. Hanno) was grown at ambient (350 micromol mol(-1)) or elevated CO(2) (700 micromol mol(-1)) in charcoal/Purafil-filtered air (CFA <5 nmol mol(-1)) or ozone (CFA +75 nmol mol(-1) 7 h d(-1)) at three levels of N supply (1.5, 4 and 14 mM NO(-3)), to test the hypothesis that the combined impacts of elevated CO(2) and O(3) on plant growth and photosynthetic capacity are affected by nitrogen availability. Shifts in foliar N content reflected the level of N supplied, and the growth stimulation induced by elevated CO(2) was dependent on the level of N supply. At 60 d after transfer (DAT), elevated CO(2) was found to increase total biomass by 44%, 29%, 12% in plants supplied with 14, 4 and 1.5 mM NO(-3), respectively, and there was no evidence of photosynthetic acclimation to elevated CO(2) across N treatments; the maximum in vivo rate of Rubisco carboxylation (V(cmax)) was similar in plants raised at elevated and ambient CO(2). At 60 DAT, ozone exposure was found to suppress plant relative growth rate (RGR) and net photosynthesis (A) in plants supplied with 14 and 4 mM NO(-3). However, O(3) had no effect on the RGR of plants supplied with 1.5 mM NO(-3) and this effect was accompanied by a reduced impact of the pollutant on A. Elevated CO(2) counteracted the detrimental effects of O(3) (i.e. the same ozone concentration that depressed RGR and A at ambient CO(2) resulted in no significant effects when plants were raised at elevated CO(2)) at all levels of N supply and the effect was associated with a decline in O(3) uptake at the leaf level. 相似文献