共查询到20条相似文献,搜索用时 15 毫秒
1.
Purification, characterization, and immunological properties of fumarase from Euglena gracilis var. bacillaris. 总被引:3,自引:1,他引:2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A rapid three-step procedure utilizing heat treatment, ammonium sulfate fractionation, and affinity chromatography on Matrex gel Orange A purified fumarase (EC 4.2.1.2) 632-fold with an 18% yield from crude extracts of Euglena gracilis var. bacillaris. The apparent molecular weight of the native enzyme was 120,000 as determined by gel filtration on Sephacryl S-300. The preparation was over 95% pure, and the subunit molecular weight was 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. The pH optimum for E. gracilis fumarase was 8.4. The Km values for malate and fumarate were 1.4 and 0.031 mM, respectively. Preparative two-dimensional gel electrophoresis was used to further purify the enzyme for antibody production. On Ouchterlony double-immunodiffusion gels, the antifumarase serum gave a sharp precipitin line against total E. gracilis protein and purified E. gracilis fumarase. It did not cross-react with purified pig heart fumarase. On immunoblots of purified E. gracilis fumarase and crude cell extracts of E. gracilis, the antibody recognized a single polypeptide with a molecular weight of approximately 60,000, indicating that the antibody is monospecific. This polypeptide was found in E. gracilis mitochondria. The antibody cross-reacted with an Escherichia coli protein whose molecular weight was approximately 60,000, the reported molecular weight of the fumA gene product of E. coli, but it failed to cross-react with proteins found in crude mouse cell extracts, Bacillus subtilis extracts, or purified pig heart fumarase. 相似文献
2.
Coustou V Biran M Besteiro S Rivière L Baltz T Franconi JM Bringaud F 《The Journal of biological chemistry》2006,281(37):26832-26846
The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200). 相似文献
3.
4.
The Bradyrhizobium japonicum fumarase gene (fumC-like) was cloned and sequenced, and a fumC deletion mutant was constructed. This mutant had a Nod+ Fix+ phenotype in symbiosis with the host plant, soybean, and growth in minimal medium with fumarate as sole carbon source was also not affected. The cloned B. japonicum fumC gene fully complemented an Escherichia coli Fum- mutant, strain JH400, for growth in minimal medium with fumarate. The predicted amino acid sequence of the FumC protein showed strong similarity to the E. coli FumC protein, Bacillus subtilis CitG protein, Saccharomyces cerevisiae Fum1 protein, and the mammalian fumarases. The B. japonicum FumC protein accounted for about 40% of the total fumarase activity in aerobically grown cells. The remaining 60% was ascribed to a temperature-labile fumarase. These data suggest that B. japonicum possesses two different fumarase isoenzymes, one of which is encoded by fumC. Besides E. coli, which has three fumarases, B. japonicum is thus the second bacterium for which there is genetic evidence for the existence of more than one fumarase. 相似文献
5.
J S Keruchenko I D Keruchenko K L Gladilin V N Zaitsev N Y Chirgadze 《Biochimica et biophysica acta》1992,1122(1):85-92
Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography. The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities. The native protein consists of four subunits with a approximately 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3. The equilibrium constant for fumarate hydration is about 4.3 (25 degrees C, pH 7.5), the Michaelis constants for fumarate and 1-malate are approximately 30 microM and approximately 250 microM, respectively. The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type. The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method. The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 A, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis. 相似文献
6.
The cDNA encoding fumarase, an enzyme catalyzing reversible hydration of fumarate to L-malate, from the parasitic roundworm Ascaris suum, has been cloned, sequenced, over-expressed in Escherichia coli, and purified. The single open reading frame translates into a protein of 50,502Da containing 467 amino acids. It shows 82, 77, and 58% identity with Caenorhabditis elegans, human, and E. coli fumC fumarases, respectively. The A. suum fumarase shows the signature sequence motif (GSSIMPGKVNPTQCE), which defines not only the class II fumarase family but also a much broader superfamily of proteins containing GSSxMPxKxNPxxxE motif. The coding region was cloned into pET101D-directional TOPO expression vector and transformed into E. coli BL21 Star (DE3). The protein after induction was expressed at high levels, almost 10% of the soluble protein, purified to near homogeneity, and appears identical to the enzyme purified from Ascaris suum. 相似文献
7.
Carrot suspension cells were found to be unable to transport malate directly into the cell but utilized it as a single carbon source in a unique manner -they converted malate extracellularly to fumarate and subsequently used it instead. The uptake of fumarate proved to be inducible and sensitive to pH and protonophore. Immuno-blot experiments using an antibody raised against Arabidopsis fumarase showed that fumarase polypeptide appeared in the medium. Fumarase was not detected in medium when fumarate or glucose was used as a carbon source. The activity of fumarase, which catalyzes the reversible hydration reactions, was induced both in the medium (malate into fumarate, releasing protons) and in the cells (fumarate into malate, requiring protons) and resulted in an increase in the pH gradient across the plasma membrane. The reason for the participation of fumarase in the utilization of malate is discussed. 相似文献
8.
Zimmermann S Hartje S Ehrhardt T Plesch G Mueller-Roeber B 《The Plant journal : for cell and molecular biology》2001,28(5):517-527
An appreciable number of potassium channels mediating K+ uptake have been identified in higher plants. Promoter-beta-glucuronidase reporter gene studies were used here to demonstrate that SKT1, encoding a potato K+ inwardly rectifying channel, is expressed in guard cells in addition to KST1 previously reported. However, whereas KST1 was found to be expressed in essentially all mature guard cells, SKT1 expression was almost exclusively restricted to guard cells of the abaxial leaf epidermis. This suggests that different types of K+ channel subunits contribute to channel formation in potato guard cells and therefore differential regulation of stomatal movements in the two leaf surfaces. The overlapping expression pattern of SKT1 and KST1 in abaxial guard cells indicates that K+in channels of different sub-families contribute to ionic currents in this cell type, thus explaining the different properties of channels expressed solely in heterologous systems and those endogenous to guard cells. Interaction studies had previously suggested that plant K+ inward rectifiers form clusters via their conserved C-terminal domain, KT/HA. K+ channels co-expressed in one cell type may therefore form heteromers, which increase functional variability of K+ currents, a phenomenon well described for animal voltage-gated K+ channels. Co-expression of KST1 and SKT1 in Xenopus oocytes resulted in currents with an intermediate sensitivity towards Cs+, suggesting the presence of heteromers, and a sensitivity towards external Ca2+, which reflected the property of the endogenous K+in current in guard cells. Modulation of KST1 currents in oocytes by co-expressing KST1 with a SKT1 pore-mutant, which by itself was not able to confer activating K+ currents, demonstrated the possibility that KST1 and SKT1 co-assemble to hetero-oligomers. Furthermore, various C-terminal deletions of the mutated SKT1 channel restored KST1 currents, showing that the C-terminal KT motif is essential for heteromeric channel formation. 相似文献
9.
10.
Kozo Yamamoto Tetsuya Tosa Kiyokazu Yamashita Ichiro Chibata 《Biotechnology and bioengineering》1977,19(8):1101-1114
The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells. 相似文献
11.
Larsen K 《Journal of plant physiology》2005,162(11):1263-1269
A cDNA, StEN1, encoding a potato (Solanum tuberosum) endonuclease was cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 906 nucleotides encoding a protein of 302 amino acids, and with a calculated molecular mass of 34.4kDa and a Pi of 5.6. The deduced StEN1 protein contains a putative signal sequence of 25 amino acid residues. The StEN1 encoded protein shows substantial homology to both plant and fungal endonucleases isolated and cloned from other sources. The highest identity (73%) was observed with AgCEL I from celery, Apium graveolens, ZEN1 from Zinnia elegans (69%) and DSA6 from daylily, Hemerocallis (68%). RT-PCR expression analysis demonstrated that the potato StEN1 gene is constitutively expressed in potato, although minor differences in expression level in different tissues were observed. 相似文献
12.
Two different putative precursor polypeptides of rat liver fumarase were synthesized when RNA prepared from rat liver were translated in vitro using the rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P1) was synthesized as a larger molecular mass than the mature subunit of fumarase (45,000 daltons) by about 5,000 daltons and the other (P2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30 degrees C, P1 and the 35S-labeled mature size fumarase were associated with the mitochondria. Of these, the 35S-labeled mature size fumarase was resistant to externally added protease, but P1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P1, which was energy-dependently imported and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased proportionally to the duration of incubation, while the amount of P1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of the incubation. 2) Only P1 could bind with the mitochondrial outer membrane at 0 degrees C even in the presence of an uncoupler of the oxidative phosphorylation but P2 did not. 3) P1 bound to the mitochondrial outer membrane was imported into the matrix, when the mitochondria binding only P1 at 0 degrees C was reisolated and incubated at 30 degrees C in the presence of an energy-generating system. The specific receptor was involved in the binding of P1 to mitochondria, since a high concentration of NaCl did not interfere with the binding of P1 to the membrane and did not discharge P1 bound onto the membrane. It was shown that P1 formed an aggregate composed of 6 to 8 molecules and P2 was a dimer in the cell-free translation mixture and that P1 and P2 were enzymatically inactive. These results suggest that the precursor for the mitochondrial enzyme has a larger molecular weight than that of the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme. 相似文献
13.
Active band sedimentation studies of pig heart fumarase indicate that the enzyme is predominantly tetrameric at enzyme concentrations between 0.0125 and 0.25 mg/ml and at a fumarate concentration of 2.5 mM. At enzyme concentrations of 0.25--1.0 mg/ml and fumarate concentrations known to activate and inhibit the enzyme, the sedimentation band of fumarase becomes disperse and indicates the presence of polymers greater than tetramers. 相似文献
14.
The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site. 相似文献
15.
Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells. 总被引:18,自引:2,他引:16
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
B Müller-R?ber J Ellenberg N Provart L Willmitzer H Busch D Becker P Dietrich S Hoth R Hedrich 《The EMBO journal》1995,14(11):2409-2416
Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells. 相似文献
16.
Profile of Basic Carbon Pathways in Guard Cells and Other Leaf Cells of Vicia faba L 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Guard cells and three other cell types from Vicia faba L. `Longpod' leaflets were assayed for enzymes that catalyze one step in each of five major carbon pathways in green plants: the photosynthetic carbon reduction pathway (ribulose-bisphosphate carboxylase, EC 4.1.1.39), the photosynthetic carbon oxidation pathway (hydroxypyruvate reductase, EC 1.1.1.81), glycolysis ([NAD] glyceraldehyde-P dehydrogenase, EC 1.2.1.12), the oxidative pentose-P pathway (6-P-gluconate dehydrogenase, EC 1.1.1.44), and the tricarboxylic acid pathway (fumarase, EC 4.2.1.2). Neither ribulose-bisphosphate carboxylase nor hydroxypyruvate reductase could be detected in guard cells or epidermal cells; high levels of these activities were present in mesophyll cells. The specific activity of fumarase (protein basis) was about 4-fold higher in guard cells than in epidermal, palisade parenchyma or spongy parenchyma cells. (NAD) glyceraldehyde-P and 6-P-gluconate dehydrogenases also were present at high protein specific activities in guard cells (2- to 4-fold that in meosphyll cells).
It was concluded that the capacity for metabolite flux through the catabolic pathways is high in guard cells. In addition, other support is provided for the view that photoreduction of CO2 by these guard cells is absent.
相似文献17.
Exposure of dark-grown restingEuglena gracilis Klebs var.bacillaris Cori to light, ethanol, or malate produced an increase in the specific activity of fumarase (EC. 4.2.1.2) and succinate dehydrogenase (EC. 1.3.99.1) during the first 8–12 h of exposure to inducer, followed by a decrease in the specific activity of both mitochondrial enzymes between 12 and 72 h. The increased specific activity represented a net increase in the level of active enzyme, and it was dependent upon cytoplasmic protein synthesis. The photoinduction of fumarase required continuous illumination while the subsequent decrease in fumarase specific activity was independent of light. Light had little effect on the ethanol and malate induction of fumarase and succinate dehydrogenase. In the mutant W3BUL, which has no detectable protochlorophyll(ide) and chloroplast DNA, light induced both mitochondrial enzymes and the kinetics of enzyme induction were similar to the induction kinetics in wild-type cells. The induction of mitochondrial enzymes appears to be controlled by a non-chloroplast photoreceptor. Dark-grown resting cells of the plastidless mutant W10SmL have lost the ability to regulate fumarase levels. In this mutant, the specific activity of fumarase fluctuated and light had little effect on these fluctuations, indicating that fumarase synthesis was uncoupled from the nonchloroplast photoreceptor. Ethanol addition produced transient changes in fumarase specific activity in W10SmL indicating that in this mutant, mitochondrial enzymes are still inductible by metabolites. Fumarase synthesis in wild-type cells was not induced in the dark by levulinic acid, a chemical inducer of the breakdown ofEuglena storage carbohydrates. Taken together, our results indicate that the photoinduction of mitochondrial enzyme synthesis is not a result of the photoinduction of carbohydrate breakdown. The mechanisms by which light and organic carbon induce the synthesis ofEuglena mitochondria may differ. 相似文献
18.
Isolation and characterization of 3-hydroxyacyl coenzyme A dehydrogenase-binding protein from pig heart inner mitochondrial membrane 总被引:1,自引:0,他引:1
3-Hydroxyacyl coenzyme A (CoA) dehydrogenase-binding protein was solubilized from inner mitochondrial membrane by using taurodeoxycholate at high ionic strength. The binding protein was isolated from the suspension using 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The protein eluted from the affinity column had a molecular weight of approximately 150,000, as determined by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protein is a dimer consisting of 69,000 and 71,000 molecular weight subunits. The enzyme binding capacity of this protein was tested with a polyethylene glycol precipitation method: 0.5 mg of enzyme could be precipitated together with 1 mg of binding protein, showing that 1 mol of binding protein binds 1 mol of enzyme. This protein had no affinity toward malic dehydrogenase, citrate synthase, and fumarase. The approximately 2-fold increase in the 3-hydroxyacyl-CoA dehydrogenase activity when it was measured in the presence of the binding protein is additional evidence of enzyme-binding protein interaction. When incorporated into liposomes, the binding protein retained its ability to bind 3-hydroxyacyl-CoA dehydrogenase, but did not bind malic dehydrogenase, citrate synthase, and fumarase. These results suggest that the protein isolated by us has a specific function in anchoring a beta-oxidation enzyme to the matrix surface of the mitochondrial membrane. 相似文献
19.
20.
M.A. Horrum S.D. Schwartzbach 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(3):407-414
Exposure of dark-grown resting (carbon deficient) Euglena to light, ethanol or malate produced a transient increase in the specific activity of fumarase. Fumarase levels decreased 8–12 h after the start of induction and this decrease could not be prevented by additional inducer. During the period of fumarase accumulation, cycloheximide prevented further fumarase synthesis and enzyme levels decreased at a rate comparable to the rate of decline normally observed 8–12 h after the start of induction. Although the addition of ethanol to ethanol-induced cultures or malate to malate-induced cultures 12 or 24 h after the initial induction failed to maintain or induce additional fumarase synthesis, the addition of organic carbon to photoinduced cells 8 or 24 h after light exposure induced additional enzyme synthesis. Additional enzyme synthesis was not induced when ethanol- or malate-induced cells were exposed to light 12 or 24 h after organic carbon addition. Light exposure or ethanol addition failed to induce fumarase synthesis during balanced growth indicating that fumarse inducibility is a property of resting cells. 相似文献