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1.
A cytosolic form of dihydroxyacetone phosphate (DHAP) reductase was purified 200,000-fold from spinach ( Spinacia oleracea L.) leaves to apparent electrophoretic homogeneity. The purification procedure included anion-exchange chromatography, gel filtration, hydrophobic chromatography, and dye-ligand chromatography on Green-A and Red-A agaroses. The enzyme, prepared in an overall yield of 14%, had a final specific activity of about 500 μmol of DHAP reduced min −1 mg −1 protein, a subunit molecular mass of 38 kD, and a native molecular mass of 75 kD. A chloroplastic isoform of DHAP reductase was separated from the cytosolic form by anion-exchange chromatography and partially purified 56,000-fold to a specific activity of 135 μmol min −1 mg −1 protein. Antibodies generated in rabbits against the cytosolic form did not cross-react with the chloroplastic isoform. The two reductases were specific for NADH and DHAP. Although they exhibited some dissimilarities, both isoforms were severely inhibited by higher molecular weight fatty acyl coenzyme A esters and phosphohydroxypyruvate and moderately inhibited by nucleotides. In contrast to previous reports, the partially purified chloroplastic enzyme was not stimulated by dithiothreitol or thioredoxin, nor was the purified cytosolic enzyme stimulated by fructose 2,6-bisphosphate. A third DHAP reductase isoform was isolated from spinach leaf peroxisomes that had been prepared by isopycnic sucrose density gradient centrifugation. The peroxisomal DHAP reductase was sensitive to antibodies raised against the cytosolic enzyme and had a slightly smaller subunit molecular weight than the cytosolic isoform. 相似文献
2.
The chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase (PGK; EC 2.7.2.3) of leaves from 18 of a broad range of 21 vascular plant species were separated by either standard or modified anion-exchange Chromatographic procedures. Immunoprecipitation of the isoenzymes with antisera raised against barley chloroplastic and cytosolic PGK isoenzymes showed that the chloroplastic isoenzymes resemble the chloroplastic isoenzymes of other species more closely than the cytosolic isoenzyme of the same species and vice versa for the cytosolic isoenzymes. Each of the two cyanobacterial species tested, yielded only a single PGK fraction on anion-exchange chromatography and gave no reaction with antisera raised against the barley isoenzymes. The cyanobacteria are presumed to contain only a single PGK which is not closely related to either of the barley PGK isoenzymes. In all of the investigated leaf extracts the catalytic activity of the cytosolic PGK was exceeded by that of the chloroplastic PGK with the ratio for many of the C 3 plants falling within the range 595 to 1585 (cytosolic: chloroplastic). The relative amounts of cytosolic PGK activity appeared to be greater in older leaves, in C 4 and CAM plants and in ferns.Abbreviations CAM
crassulacean acid metabolism
- pgk
phosphoglycerate kinase
This work was supported by the Science and Engineering Research Council (grant no. GR/E54504) and also the King's College London Research Strategy Fund. 相似文献
3.
Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves ( Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg 2+. The free Mg 2+ concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg 2+ was independent of the MgATP 2− concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg 2+. Free ATP (ATP 4−) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP 2− concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP 2−, respectively. It was proposed that free ATP competes for the MgATP 2− binding site, whereas free Mg 2+ does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP 2− requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP 2− was required at 0.007 and 0.8 millimolar free ATP in the absence of Pi. At 5 and 25 millimolar Pi, half-maximum saturation was achieved at 9 and 14 micromolar MgATP 2−. PFK activity was inhibited by Ca 2+. The inhibition by Ca 2+ depends upon the total Mg 2+ concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca 2+ at 0.1 and 0.2 millimolar Mg 2+, respectively. At physiological concentrations of about 0.5 millimolar free Mg 2+, Ca 2+ would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5′-triphosphate substrate. Besides MgATP 2−, MgUTP 2−, MgCTP 2−, and MgGTP 2− could be used as a substrate. All four free nucleotides inhibit PFK activity. The physiological consequences of the regulatory properties of cytosolic PFK from spinach leaves will be discussed. A model will be introduced, in an attempt to describe the complex interaction of PFK with substrates and the effectors Mg 2+ and Pi. 相似文献
4.
Cytosolic ATP-dependent phosphofructokinase (PFK) from spinach leaves ( Spinacia oleracea L.) was enriched 2600-fold by (NH 4) 2SO 4 fractionation, DEAE anion exchange chromatography, Blue Sepharose CL-6B, and ATP agarose type 3-affinity chromatography. The final preparation had a specific activity of 417 nkat per milligram protein and exhibited four bands between 50 and 70 kilodaltons following denaturing electrophoresis. Only one band of ATP- and fructose 6-phosphate (F-6-P)-dependent, Pistimulated activity was detected following isoelectric focusing PAGE and nondenaturing discontinuous PAGE of the final preparation. Crude extracts contained, in addition to the band observed in the final preparation, a second band that was inhibited by Pi. The latter band is presumably chloroplastic PFK. PFK was stimulated by the anions Pi 2−, Cl −, SO 42−, NO 3−, HAsO 42−, and HCO 3− but was not affected by NH 4+. Pi and Mg 2+ changed the response of PFK toward pH and affected the saturation kinetics of F-6-P. In general, activity was highest when Pi was high and (or) Mg 2+ was low. Phosphoenolpyruvate (PEP), 2-PGA, and PPi, but not 3-PGA, inhibited PFK. Although the inhibition by PEP and 2-PGA was reduced or relieved by Pi, the inhibition by PPi was not affected by Pi. F-2, 6-P 2 had no effect upon the activity of PFK. It is proposed that, in the cytosol of spinach leaves, PFK is likely to be more active during the dark, when cytosolic Pi levels are high, than in the light. 相似文献
5.
During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS 2) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS 2. The synthesis of GS 2 was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS 2 protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH
heavy chain of antibodies
- AbL
light chain of antibodies
- AP
acid phosphatase
- CH
cycloheximide
- G6PDH
glucose-6-phosphate dehydrogenase
- GS
glutamine synthetase
- GS 1
cytosolic glutamine synthetase
- GS 2
chloroplastic glutamine synthetase
- LC
lincomycin
- NAD-MDH
NAD malate dehydrogenase
- NADP-G3PDH
NADP glyceraldehyde-3-phosphate dehydrogenase 相似文献
6.
Two distinct membrane fractions containing H +-ATPase activity were prepared from red beet. One fraction contained a H +-ATPase activity that was inhibited by NO 3− while the other contained a H +-ATPase inhibited by vanadate. We have previously proposed that these H +-ATPases are associated with tonoplast (NO 3−-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP 2−, and Mg 2+ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP ( i.e. ATP concentrations in excess of Mg 2+) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP. Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP2− in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner. Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP2− complex. The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H+-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg2+ could modulate ATPase activity at both the tonoplast and plasma membrane. 相似文献
7.
5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP + oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy- d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP
3-deoxy- d-arabino-heptulosonate 7-phosphate
- DEAE
diethylaminoethyl
- DHQase
3-dehydroquinate dehydratase
- DTT
dithiothreitol
- EPSP
5-enolpyruvylshikimate 3-phosphate
- SORase
shikimate:NADP + oxidoreductase 相似文献
8.
During the germination of pumpkin ( Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH 3 are discussed. 相似文献
9.
(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of Q A (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O 2 evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O 2 evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149–1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.Abbreviations and symbols Chl
chlorophyll
- Fru6P
fructose-6-phosphate
- Frul,6bisP
fructose-1,6-bisphosphate
- Fru-1,6Pase
fructose-1,6-bisphosphatase
- Fru2,6bisP
fructose-2,6-bisphosphate
- Fru2,6Pase
fructose-2,6-bisphosphatase
- Glc6P
glucose-6-phosphate
- PGI
phosphoglucose isomerase
- Pi
inorganic phosphate
- Q A
acceptor for photosystem II
- Ru1,5bisP
ributose-1,5-bisphosphate
- SPS
sucrose-phosphate synthase 相似文献
10.
In leaves of spinach plants ( Spinacia oleracea L.) grown in ambient CO 2 the subcellular contents of adenylates, pyridine nucleotides, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, glutamate, 2-oxoglutarate, and aspartate were assayed in the light and in the dark by nonaqueous fractionation technique. From the concentrations of NADP and NADPH determined in the chloroplast fraction of illuminated leaves the stromal NADPH to NADP ratio is calculated to be 0.5. For the cytosol a NADH to NAD ratio of 10 −3 is calculated from the assay of the concentrations of NAD, malate, glutamate, aspartate, and 2-oxoglutarate on the assumption that the reactions catalyzed by the cytosolic glutamate oxaloacetate transaminase and malate dehydrogenase are not far away from equilibrium. For the transfer of redox equivalents from the chloroplastic NADPH to the cytosolic NAD two metabolite shuttles are operating across the inner envelope membrane: the triosephosphate-3-phosphoglycerate shuttle and the malate-oxaloacetate shuttle. Although both shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, this apparently does not occur. To gain an insight into the regulatory processes we calculated the free energy of the enzymic reactions and of the translocation steps involved. From the results it is concluded that the triosephosphate-3-phosphoglycerate shuttle is mainly controlled by the chloroplastic reaction of 3-phosphoglycerate reduction and of the cytosolic reaction of triosephosphate oxidation. The malate-oxaloacetate shuttle is found to be regulated by the chloroplastic NADP-malate dehydrogenase and also by the translocating step across the envelope membrane. 相似文献
11.
Relative thermotolerance of the enzyme, L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4), from the chloroplastic and cytosolic sources of Diplopterygium glaucum was studied. The purification involved streptomycin sulphate precipitation, ammonium sulphate fractionation, ion-exchange chromatography, and molecular sieve chromatography. After the final chromatography, 16.62% of chloroplastic and 13.47% of cytosolic MIPS could be recovered. Between 15 degrees C and 55 degrees C, the two forms of MIPS exhibited differential thermal stability, which is related to the presence of the MIPS co-factor, NAD+. Added NAD+ increased the lower thermotolerance of the chloroplastic MIPS and the removal of 'built-in' NAD+ decreased the higher thermal stability of the cytosolic MIPS. 相似文献
12.
Soybean plants ( Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N 2 fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N 2-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO 3− and NH 4+ (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N 2-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N 2-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N 2-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the Km values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N 2-fixing as well as N-supplemented plants, and G1,6BP levels in N 2-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N 2-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N 2-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO 3− and NH 4+, the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N 2-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N 2-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis. 相似文献
13.
Bisphosphoglycerate synthase from horse red cells has been purified to apparent homogeneity by a simple and efficient new procedure incorporating chromatography on a column of Sepharose 4B derivatized with blue dextran. The enzyme is similar to the human red cell synthase in subunit size. It is phosphorylated by either glycerate-1,3-P 2 or glycerate-2,3-P 2 to form a phosphoenzyme with the acid-lability of a histidyl phosphate. In addition to the synthase activity (glycerate-1,3-P 2 → glycerate-2,3-P 2), kcat 12.5 s ?1, the enzyme has bisphosphoglycerate phosphatase activity in the presence of glycolate-2-P (glycerate-2,3-P 2 → glycerate-P + P i), kcat 2.6 s ?1 and phosphoglycerate mutase activity (3-PGA ? 2-PGA), kcat 1.7 s ?1. The energy of activation for the synthase reaction is 9.38 kcal/mol. Lineweaver-Burk plots of the kinetic data are parallel lines. In contrast intersecting patterns were obtained from similar experiments done with the human red cell enzyme. Further investigation is required to explain these differences. This enzyme may function as both synthase and phosphatase for bisphosphoglycerate in the red blood cell. 相似文献
14.
1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for the brain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves for MgATP 2− based upon Bachelard's (1971) studies can be corrected to hyperbolic curves by use of a stability constant for MgATP 2− complex formation. 3. A number of other effects related to the concentration-dependent stability of the MgATP 2− complex and to the presence of the inhibitory free uncomplexed ATP 4− concentration are also explained in terms of a non-allosteric role for either Mg 2+ or MgATP 2− fully consistent with a number of previous reports on this enzyme. 4. A brief discussion of the validity of Hill plots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model is presented that demonstrates how enzymes obeying Michaelis–Menten kinetics can demonstrate sigmoidal velocity responses if the true substrate of the reaction is the metal–substrate complex. 相似文献
15.
Pea ( Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase. 相似文献
16.
The relationship between the gas-exchange characteristics of attached leaves of Zea mays L. and the contents of photosynthetic intermediates was examined at different intercellular partial pressure of CO 2 and at different irradiances at a constant intercellular partial pressure of CO 2. (i) The behaviour of the pools of the C 4-cycle intermediates, phosphoenolpyruvate and pyruvate, provides evidence for light regulation of their consumption. However, light regulation of phosphoenolpyruvate carboxylase does not influence the assimilation rate at limiting intercellular partial pressures of CO 2. (ii) A close correlation between the pools of phosphoenolpyruvate and glycerate-3-phosphate exists under many different flux conditions, consistent with the notion that the pools of C 4 and C 3 cycles are connected via the interconversion of glycerate-3-phosphate and phosphoenolpyruvate. (iii) The ratio of triose-phosphate to glycerate-3-phosphate is used as an indicator of the availability of ATP and NADPH. Changes of this ratio with CO 2 and with irradiance are compared with results obtained in C 3 leaves and indicate that the mechanism of regulation of carbon assimilation by light in leaves of C 4 plants may differ from that in C 3 plants. (iv) The behaviour of the ribulose-1,5-bisphosphate pool with CO 2 and irradiance is contrasted with the behaviour of these pools measured in leaves of C 3 plants.Abbreviations
P
i
intercellular CO 2 pressure
- RuBP
ribulose-1,5-bisphosphate
- PEP
phosphoenolpyruvate
- triose-P
triose phosphates
- PGA
glycerate-3-phosphate 相似文献
18.
The major metabolites of the phosphatidylinositol cycle from extracts of [ 32PO 4]- and [ 3H]-inositol-labeled Samanea saman pulvini were separated. The membrane localized phosphoinositides were separated by thin layer chromatography, identified by comparison with purified lipid standards, and quantitated based on incorporation of radiolabel. The ratio of radioactivity in phosphatidylinositol:phosphatidylinositol 4-phosphate:phosphatidylinositol 4,5-bisphosphate is about 32:8:1. The aqueous inositol phosphates were separated by anion exchange chromatography using conventional liquid chromatography and by high performance liquid chromatography (HPLC) and were identified by comparison with standards. Analysis by HPLC reveals that 32P-labeled pulvini have inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate that co-migrate with red blood cell inositol phosphates, but 3H-inositol-labeled pulvini appear to have a variant profile. 相似文献
19.
We report the first purification and characterization of a eukaryotic algal phosphoglycerate kinase (PGK), Two forms of PGK (PGK 1 and PGK 2) from the green alga Selenastrum minutum (Naeg.) Collins were purified to electrophoretic homogeneity with specific activities of 1100 and 1069 units · mg ?1 protein, respectively. The portion of PGK 1 and PGK 2 (probably the cytosolic and chloroplastic forms, respectively) in this organism was estimated as 32 and 68%, respectively. PGK 1 was more heat-stable than PGK 2. The M r estimation for PGK 1 and PGK 2 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration indicated that they both were monomeric with a similar M r of approximately 44 kDa. Antibodies raised against S. minutum PGK 1 cross-reacted with PGK 2 as well as PGKs from prokaryotic and eukaryotic sources, suggesting that PGK 1 was structurally and immunologically closely related to PGK 2 and other PGKs, which was consistent with NH 2-terminal sequence analysis. Comparative kinetic and regulatory properties of PGK 1 and PGK 2 from S. minutum were investigated, Both forms exhibited hyperbolic kinetics with respect to both 3-phosphoglycerate (3-PGA) and Mg-adenosine triphosphate 2- (MgATP 2-) under the conditions tested and had similar K m values for each substrate (PGK 1; K m (MgATP 2-) = 0.37 mM, K m(3-PGA) = 0.59 mM; PGK 2; K m(MgATP 2-) = 0.32 mM, K m(3-PGA) = 0.46 mM). PGK 1 and PGK 2, however, differed significantly in several other kinetic properties. PGK 2 had a broad pH optimum between 7.3 and 7.8, as compared to PGK 1, with a pH optimum of 7.3 Mg 2+ was the most efficient cofactor for both forms; it inhibited PGK 1 but not PGK 2 at higher concentrations (>10 mM). Other divalent cations (Mn 2+, Zn 2+, Co 2+, Cd 2+, and Ca 2+) only partially replaced Mg 2+ and were more effective for PGK 1 than for PGK 2, A wide range of metabolites was examined for regulatory properties. Energy charge was the most important factor in regulating the two forms of S. minutum PGK. These results were interpreted in light of the regulation of this kinase in response to the cell energy requirement and the need for glycolytic carbon flow to provide carbon skeletons for amino acid biosynthesis. 相似文献
20.
Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH 1 and NADP-IDH 2 (EC 1.1.1.42) were identified in pea ( Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH 1 while NADP-IDH 2 represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH 2 was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH 1, and as a consequence NADP-IDH 2 does not result from a post-translational modification of NADP-IDH 1. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH 1 is restricted to the cytosol and NADP-IDH 2 to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH 2 was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA
bovine serum albumin
- DEAE
diethylaminoethyl
- NADP-IDH
NADP-isocitrate dehydrogenase
- NADP-IDH 1
cytosolic NADP-IDH
- NADP-IDH 2
chloroplastic NADP-IDH 相似文献
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