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1.
A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [14C]glucose 1-phosphate from 14C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg2+, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed. 相似文献
2.
In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K
m
values for PEP and NaHCO3 were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K
m
for Mn2+, Co2+ and Mg2+ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg2+. Mn2+ and Co2+ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K
a
0.18 mM) or propionyl CoA (K
a
0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP
phosphoenolpyruvate
- OAA
oxaloacetate
- MW
molecular weight
- TEMG buffer
50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2, 1 mM glutathione
- HEPES
N-2-hydroxyethylpiperazine-N-ethanesulfonic acid 相似文献
3.
S.K. Mukerji 《Archives of biochemistry and biophysics》1977,179(1):360-365
Sulfite ion, the hydrated form of SO2 which is an air pollutant, was found to be an inhibitor of phosphoenolpyruvate carboxylase(s) isolated from corn leaves. The inhibition was partial even in the presence of excess SO32?. It inhibited the enzyme competitively with respect to HCO3?, noncompetitively with respect to phosphoenolpyruvate, and uncompetitively with respect to Mg2+. The kinetics of inhibition suggest that an alternate pathway is operative in the presence of SO32?. The enzyme(s) were activated by glucose 6-phosphate which affected primarily the affinity of the enzyme for phosphoenolpyruvate. The binding site of glucose 6-phosphate was apparently distinct from the catalytic site of the enzyme since partial destruction of the catalytic site by heat had no effect on the inhibition by SO32?, but glucose 6-phosphate lost its activating effect. The inhibition due to SO32? was relieved by glucose 6-phosphate. 相似文献
4.
The allosteric inhibition of Ml-type pyruvate kinase from rabbit skeletal muscle by phenylalanine is reciprocally dependent on Mg2+ and phosphoenolpyruvate concentrations . At pH 8, phenylalanine acts as a competitive inhibitor with respect to Mg2+ and phosphoenolpyruvate, and vice versa. Phenylalanine introduces sigmoidicity into the dependence of the reaction velocity on [Mg2+]. In vitro kinetic analysis indicates that phenylalanine inhibition of muscle pyruvate kinase is unlikely to have regulatory significance in vivo. 相似文献
5.
Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria
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Graesser RJ 《Plant physiology》1977,59(2):126-128
Phosphoenolpyruvate partially inhibits the accumulation of Ca2+ in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca2+ uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca2+ uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca2+ uptake completely while partially inhibiting NADH-supported Ca2+ uptake. The oxaloacetate inhibition of NADH-supported Ca2+ uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca2+ uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca2+ uptake. 相似文献
6.
Prakorn Chudapongse 《BBA》1976,423(2):196-202
Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca2+-induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca2+ in the presence of glutamate and inorganic phosphate causes the release of Ca2+ from mitochondria. The phosphoenolpyruvate-stimulated Ca2+ efflux can be observed with mitochondria loaded with low initial Ca2+ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H+ translocation produced by addition of Ca2+ to respiring mitochondria show that phosphoenolpyruvate depresses H+ ejection and enhances H+ uptake by mitochondria. The Ca2+-releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca2+ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca2+ transport. 相似文献
7.
M J MacDonald 《Biochemical and biophysical research communications》1979,90(3):741-749
3-Mercaptopicolinic acid is known to be an inhibitor of phosphoenolpyruvate carboxykinase and 3-aminopicolinic acid permits Fe2+ to activate the enzyme. The potency of mercaptopicolinate is increased by incubating the enzyme with Fe2+ prior to assaying for activity. In the present work, the average combining ratio of either pyridine carboxylate with Fe2+ at pH 7.5 was determined to be 2:1 when measured by the method of continuous variation of Job or by elemental analysis of the isolated pyridine carboxylate-Fe2+ complexes. The ratio of 3-mercaptopicolinate or 3-aminopicolinate to Fe2+ that caused the greatest inhibition or activation of purified phosphoenolpyruvate carboxykinase was 2:1. In the absence of Fe2+, neither pyridine carboxylate altered the activity of the enzyme. These results indicate that the two pyridine carboxylates can interact with phosphoenolpyruvate carboxykinase as Fe2+ coordination complexes. 相似文献
8.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(1):68-73
Glutathione reductase (GR; E.C. 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). In this study we tested the effects of Al3+, Ba2+, Ca2+, Li+, Mn2+, Mo6+, Cd2+, Ni2+, and Zn2+ on purified bovine liver GR. In a range of 10?μM–10?mM concentrations, Al3+, Ba2+, Li+, Mn2+, and Mo6+, and Ca2+ at 5?μM–1.25?mM, had no effect on bovine liver GR. Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effects on this enzyme. The obtained IC50 values of Cd2+, Ni2+, and Zn2+ were 0.08, 0.8, and 1?mM, respectively. Cd2+ inhibition was non-competitive with respect to both GSSG (KiGSSG 0.221?±?0.02?mM) and NADPH (KiNADPH 0.113?±?0.008?mM). Ni2+ inhibition was non-competitive with respect to GSSG (KiGSSG 0.313?±?0.01?mM) and uncompetitive with respect to NADPH (KiNADPH 0.932?±?0.03?mM). The effect of Zn2+ on GR activity was consistent with a non-competitive inhibition pattern when the varied substrates were GSSG (KiGSSG 0.320?±?0.018?mM) and NADPH (KiNADPH 0.761?±?0.04?mM), respectively. 相似文献
9.
Vladimir Ossipov Andrei Chernov Galina Zrazhevskaya Igor Shein 《Trees - Structure and Function》1995,10(1):46-51
Quinate:NAP(P)+-oxidoreductase (QORase, EC 1.1.1.24), which catalyzes the interconversion of quinic and 3-dehydroquinic acids, was purified from the needles and developing xylem cells of Larix sibirica. The enzymes from these two tissues were partially characterized and compared. QORase from needles had optimum pH at 9.0 and apparent Km values of 1.84 mM for quinic acid and 0.19 mM for NADP+. The enzyme was activated by phosphoenolpyruvate. Gallic and protocatechuic acids were formed in a reaction mixture of purified enzyme from needles as final products of quinic acid transformation. QORase from developing xylem cells showed pH optimum at 10.0 and had apparent Km values of 0.70 mM for quinic acid and 0.05 mM for NADP+. The enzyme was not affected by PEP. The divalent cations Co2+ and Mn2+ at least doubled activity of QORase from both sources but Mg2+ affected the enzyme from needles only. The spatial organization and regulation of quinic acid metabolism in the autotrophic and heterotrophic cells of conifers and the role of QORase in this process are discussed. 相似文献
10.
Kinetic analysis of the Mg2+-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP2- is the substrate of ATPase: MgATP2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg2ATP is formed. No evidence for a direct role of Mg2+ as activator or inhibitor was found. These data corroborate previous findings that MgATP2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes
2-(N-morpholino)ethane+Sulfonic acid 相似文献
11.
Chick brain microsomal ATPase was strongly inhibited by Cu2+. (Na+ + K+)-ATPase was more susceptible to low levels of Cu2+ than Mg2+-ATPase. The inhibition of (Na+ + K+)-ATPase could be partially protected from Cu2+ in the presence of ATP in the preincubation period. When Cu2+ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu2+ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu2+ (1.0 μM) on (Na+ + K+)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K+ (5 mM), V with Na+ was markedly decreased in the presence of Cu2+ and Km was about twice that of the control. However, at high K+ concentration (20 mM), the Km for Na+ was not affected. At both low (25 mM) and high (100 mM) Na+, Cu2+ displayed non-competitive inhibition of the enzyme with respect to K+.On the basis of these data, we suggest that Cu2+ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu2+ interacts reversibly with the enzyme. 相似文献
12.
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)-1·min-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. Km values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (Ka) for Mg2+ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW
molecular weight
- PEP (Case)
phosphoenolpyruvate (carboxylase)
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis 相似文献
13.
Theodora R. Devereux Richard M. Philpot James R. Fouts 《Chemico-biological interactions》1977,18(3):277-287
Solubilization and partial purification of the rabbit pulmonary and hepatic N,N-dimethylaniline N-oxidases were carried out in order to study the effect of Hg2+ in vitro observed previously in the microsomal enzymes. Rabbit lung microsomal N,N-dimethylaniline (DMA) N-oxidase activity was stimulated 1.5–2 times by 0.1 mM Hg2+ added in vitro. This concentration of mercury inhibited hepatic microsomal N-oxidase by 50%. Upon solubilization and partial purification of the lung N-oxidase enzyme, stimulation of the N-oxidase activity by 0.1 mM Hg2+ was lost. It was found that the concentration of Hg2+ that would stimulate the partially purified pulmonary N-oxidases was 25 μM or less. Stimulation by 0.1 mM Hg2+ of the partially purified N-oxidase from lung was restored by addition of flavins (FMN or FAD) or a heat-stable (NH4)2SO4 precipitated fraction obtained during the purification of the N-oxidase from solubilized pulmonary or hepatic microsomes. However, addition of the flavins or the solubilized, heat-stable fraction from liver or lung microsomes did not reverse inhibition by 0.1 mM Hg2+ of the N-oxidase in hepatic microsomes or in partially purified preparations from these hepatic microsomes. Kinetic data suggest that flavins and the heatstable factor isolated from microsomes lower the concentration of free Hg2+.The determination of kinetics of Hg2+ inhibition (liver) and activation (lung) with the partially purified N-oxidases showed that the pulmonary and hepatic DMA N-oxidase enzymes are markedly different with respect to their in vitro response to Hg2+. This suggests that the N-oxidases from liver and lung may be different enzymes. 相似文献
14.
NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent K
m for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn2+ and Ca2+ could be substituted for Mg2+. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a K
i of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.This paper is dedicated to Sydney C. Rittenberg on the occassion of his retirement, with respect and much affection, in appreciation for his friendship and years of distinguished service as a teacher and scientist 相似文献
15.
Berivan Tandoğan N. Nuray Ulusu 《Journal of enzyme inhibition and medicinal chemistry》2013,28(4):489-495
Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn2 + (up to 2 mM) and activated above this concentration. Ca2 + inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals. 相似文献
16.
The individual and interactive role of calcium and abscisic acid (ABA) in amelioration of water stress simulated by polyethylene glycol (PEG) 6000 was investigated in two contrasting wheat genotypes. PEG solution (osmotic potential –1.5 MPa) was applied to 10-d-old seedlings growing under controlled conditions and changes in photosynthetic rate, activities of ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase, water potential and stomatal conductance were observed in the presence of 0.1 mM ABA, 5 mM calcium chloride, 1 mM verapamil (Ca2+ channel blocker), and 1 mM fluridone (inhibitor of ABA biosynthesis). ABA and calcium chloride ameliorated the effects of water stress and the combination of the two was more effective. The two genotypes varied for their sensitivity to ABA and Ca2+ under stress. As was evident from application of their inhibitors, ABA caused more alleviation in C 306 (drought tolerant) while HD 2380 (drought susceptible) was more sensitive to Ca2+. 相似文献
17.
18.
Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K+ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K+ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the Km(PEP) and Vmax values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM
Crassulacean acid metabolism
- GCP
guard-cell protoplast
- PEP
phosphoenolpyruvate
Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday 相似文献
19.
Jobst-Heinrich Klemme 《Archives of microbiology》1976,107(2):189-192
The kinetic and regulatory properties of partially purified phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.32) from Rhodospirillum rubrum were studied. The enzyme was active with guanosine-and inosinephosphates and must thus be classified as GTP (ITP): oxaloacetate carboxylyase (transphosphorylating). In the direction of oxaloacetate-formation, the enzyme was strongly inhibited by ATP (Ki=0.03 mM). ITP, UTP, CTP and GTP were less inhibitory. The inhibition was competitive with respect to GDP or IDP, but not with respect to PEP. In the direction of PEP-synthesis, the enzyme was not inhibited, but rather activated by ATP. 相似文献
20.
RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn2+ optimum of 1·25 mM and a Mg2+ optimum at 10mM. The Mn2+/Mg2+ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH4)2SO4. 相似文献