首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Two isoenzymes of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been separated from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules by a procedure involving (NH4)2SO4 gradient fractionation, gel chromatography, chromatofocusing, and affinity chromatography. The isoenzymes, which have been termed glucose 6-phosphate dehydrogenases I and II, were specific for NADP+ and glucose 6-phosphate and had optimum activity at pH 8.5 and pH 8.1, respectively. Both isoenzymes were labile in the absence of NADP+. The apparent molecular weight of glucose 6-phosphate dehydrogenases I and II at pH 8.3 was estimated by gel chromatography to be approximately 110,000 in the absence of NADP+ and double this size in the presence of NADP+. The apparent molecular weight did not increase when glucose 6-phosphate was added with NADP+ at pH 8.3. Both isoenzymes had very similar kinetic properties, displaying positive cooperativity in their interaction with NADP+ and negative cooperativity with glucose 6-phosphate. The isoenzymes had half-maximal activity at approximately 10 micromolar NADP+ and 70 to 100 micromolar glucose 6-phosphate. NADPH was a potent inhibitor of both of the soybean nodule glucose 6-phosphate dehydrogenases.  相似文献   

2.
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD+ or NADP+ as coenzyme. Kinetic studies showed that NAD+ and NADP+ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP+ and 50% for NAD+. The dissociation constant for NADP+, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined Km of 5.7 μm and Ki of 5 μm. The dissociation constant for NAD+ is 2.5 mm, which is 24 times larger than the previously determined Km of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD+ but not for NADP+. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD+ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD+; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined Ki is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP+-competitive and NAD+-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD+ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.  相似文献   

3.
Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP+, and Mg2+ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (Km = 58 micromolar), but also inhibition by high substrate (Ki = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (Km = 120 micromolar). At both pH conditions, activity response to NADP+ exhibited Michaelis-Menten behavior with Km values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg2+ suggested the presence of at least two Mg2+ - binding sites with different affinity. The Ka values for Mg2+ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg2+ levels could be important for the physiological regulation of NADP-malic enzyme.  相似文献   

4.
The subcellular distribution of NADP+ and NAD+-dependent glucose-6-phosphate and galactose-6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose-6-phosphate and galactose-6-phosphate with either NADP+ or NAD+ as cofactor. While all of the tissues examined had NADP+-dependent glucose-6-phosphate dehydrogenase activity, only rat liver and rat brain soluble fractions had NADP+-dependent galactose-6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose-6-phosphate dehydrogenase activities were kinetically different (Km's 0.5 mm and 10 mm, respectively, for galactose-6-phosphate), although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP+ or NAD+ cofactors but phosphatase activities hydrolyzing 6-phosphogalactonate, galactose-6-phosphate and galactose-1-phosphate were found in crude brain homogenates. In addition, galactose-6-phosphate and 6-phosphogalactonate were tested as inhibitors of various enzymes, with largely negative results, except that 6-phosphogalactonate was a competitive inhibitor (Ki = 0.5 mM) of rat brain 6-phosphogluconate dehydrogenase.  相似文献   

5.
Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP+ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP+. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, Km of NADP+ being 2.5-fold higher for pI 4.3 form. Free ATP (Km = 0.64 millimolar) and ADP (Km = 1.13 millimolar) act as competitive inhibitors with respect to NADP+ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.

  相似文献   

6.
The subcellular location of NADP+-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP+-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP+-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP+-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent Km values of 11 micromolar (NADP+), 35 micromolar (isocitrate), 78 micromolar (Mn2+), 0.3 millimolar (Mg2+) and showed optimum activity at pH 8 to 8.5 with Mn2+ and 8.8 to 9.2 with Mg2+. The NADP+-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP+-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.  相似文献   

7.
Gupta VK  Singh R 《Plant physiology》1988,87(3):741-744
NADP+-isocitrate dehydrogenase (threo-DS-isocitrate: NADP+ oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg2+ or Mn2+, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP+, dl-isocitrate, Mn2+, and Mg2+. Their Km values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP+. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP+ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP+ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP+-IDH in situ is likely to be controlled by intracellular NADPH to NADP+ ratio as well as by the concentration of various substrates and products.  相似文献   

8.
Enzymes of glucose metabolism in normal mouse pancreatic islets   总被引:14,自引:14,他引:0       下载免费PDF全文
1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP+-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had Km 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high Km for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with Km 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had Km values of 2.5 and 21μm for NADP+ and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had Km values of 4 and 22μm for NADP+ and glucose 6-phosphate. The Km for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP+ and apparently mixed with respect to glucose 6-phosphate. 6. NADP+–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.  相似文献   

9.
Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP+, with Km's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a Km of 0.31 mM. The NADP+ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent Km and Vmax values with increasing NADP+, denoting negative cooperativity. The two Km's for high and low NADP+ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP+ binding sites.  相似文献   

10.
NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria   总被引:9,自引:4,他引:5       下载免费PDF全文
Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD+- and NADP+-isocitrate dehydrogenases, NAD+- and NADP+-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP+-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP+-malate dehydrogenase activity is probably due to unspecificity of the NAD+-malate dehydrogenase. NADP+-specific isocitrate dehydrogenase had much lower Kms for NADP+ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD+-specific enzyme (101 micromolar for NAD+ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP+-specific isocitrate dehydrogenase whereas the NAD+-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP+ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP+ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP+-reducing activities of malate dehydrogenase and the NADP+-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.  相似文献   

11.
The enzyme catalysing the l-proline-dependent reduction of NAD+has been purified over 600-fold from wheat germ acetone powder extracts. l-Proline, 3,4 dehydro-dl-proline, thiazolidine-4-carboxylate were the only substrates utilized readily. The Km for l-proline was 1·0 mM and for NAD+ 0·8 mM. The enzyme was highly specific for NAD+ with NADP+ and NADPH acting as effective competitive inhibitors with a Ki of 1·8 and 0·4 μM, respectively. All ribonucleoside triphosphates tested were good non-competitive inhibitors, in particular UTP. The purified enzyme could reduce pyrroline-5-carboxylate, either chemically synthesized or generated in a linked assay system from ornithine by a highly-purified ornithine transaminase. In the latter case both NADH and NADPH were utilized equally well as the reductant. With chemically synthesized dl-pyrroline-5-carboxy-late as the substrate. NADPH was used at only 25% the rate of NADH, and NADPH strongly inhibited the oxidation of NADH.  相似文献   

12.
In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0 ± 7.9 μM) and the K0.5 values determined for G6P (57.9 ± 2.5 and 104.5 ± 9.3 μM in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.  相似文献   

13.
The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K m's are for G6PDH: 80M (substrate) and 20M (NADP+); for 6PGDH: 90M (substrate) and 25M (NADP+). In Synechococcus 6716 the apparent K m's are for G6PDH: 550M (substrate) and 30M (NADP+); for 6PGDH: 40M (substrate) and 10M (NADP+). None of the K m's is influenced by the growth temperature and only the K m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine  相似文献   

14.
Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP+-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP+-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (Ki:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.  相似文献   

15.
Human glucose 6-phosphate dehydrogenase (G6PD) has both the “catalytic” NADP+ site and a “structural” NADP+ site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PDWisconsin (R393G) and G6PDNashville (R393H), and G6PDFukaya (G488S) and G6PDCampinas (G488V), in which the mutations are in the vicinity of the “structural” NADP+ site, showed elevated Kd values of the “structural” NADP+, ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP+ and DTT at 25 °C. The refolding yields of the mutants exhibited strong NADP+-dependence and ranged from 1.5% to 59.4% with 1000 μM NADP+, in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of “structural” NADP+ can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state.  相似文献   

16.
All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD+ and NADP+. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH4Cl and 1.5 at 12.5 millimolar NH4Cl. Km values for NH4+ and NAD(P)H are reduced at low salt concentrations. The low Km values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.  相似文献   

17.
《BBA》2020,1861(3):148140
Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches.The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s−1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins.  相似文献   

18.
Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25°C, with a photon flux density of 500 mol m-2s-1. Measurements were made of net CO2 exchange, leaf adenylates (ATP, ADP and AMP), and leaf nicotinamide nucleotides (NAD+, NADP+, NADH, NADPH), over the diurnal period (16h light/8 h dark) and during photosynthetic induction. All the measurements were carried out on recently expanded leaves from 5-week-old plants. When the lights were switched on in the growth chamber, the rate of photosynthetic CO2 uptake, and the levels of leaf ATP and NADPH increased to a maximum in 30 min and remained there throughout the light period. The increase in ATP over the first few minutes of illumination was associated with the phosphorylation of ADP to ATP and the increase in NADPH with the reduction of NADP+; subsequently, the increase in ATP was associated with an increase in total adenylates while the increase in NADPH was associated with an accumulation of NADP+ and NADPH due to the light-driven phosphorylation of NAD+ to NADP+. On return to darkness, ATP and NADPH values decreased much more slowly, requiring 2 to 4 hours to reach minimum values. From these results we suggest that (i) the total adenylate and NADPH and NADP+ (but not NAD+ and NADH) pools increase following exposure to light; (ii) the increase in pool size is not accompanied by any large change in the energy or redox states of the system; and (iii) the measured ratios of ATP/ADP and NADPH/NADP+ for intact leaves are low and constant during steady-state illumination.Abbreviations AEC adenylate energy charge - DHAP dihydroxyacetone phosphate - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide - PES phenazine ethosulfate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - PFD photon flux density - Ru5P ribulose-5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase  相似文献   

19.
Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The Km value for glucose-6-phosphate is 1.6 × 10?4 and 6.3 × 10?4M at low (1.0–6.0 × 10?4M) and high (6.0–30.0 × 10?4M) concentrations of the substrate, respectively. The Km value for NADP+ is 1.4 × 10?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg2+, and Mn2+. In the presence of NADPH, the plot of activity vs. NADP+ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg2+ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.  相似文献   

20.
The (K+,Mg2+)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co2+ > Mg2+ > Mn2+ > Zn2+ > Ca2+) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg2+, the enzyme was further activated by monovalent cations (K+, NH4+, Rb+ Na+, Cs+, Li+). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K+ approached simple Michaelis-Menten kinetics, with a Km of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K+, and preferred Mn2+ to Mg2+. The results demonstrate that the (K+,Mg2+)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号