It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have i...It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.展开更多
Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycelytic enzyme, glyceraldehyde-3-phosphate dehydrogenase （GA...Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycelytic enzyme, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer （RaGAPDH） has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genornic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligandbinding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coil （EHEC）, belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestiferdisplayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism＇s virulence.展开更多
Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. ...Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.展开更多
Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterizati...Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.展开更多
Glycerol-3-phosphate acyltransferase(GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid(LPA) synthesis. For understanding its catalytic mech...Glycerol-3-phosphate acyltransferase(GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid(LPA) synthesis. For understanding its catalytic mechanism, the structural biology study is expected, but is always hindered by obtaining crystals for X-ray diffraction analysis. In this study, a progressive strategy to optimize the crystal of microalgae plastidial GPAT was presented. After the expression and purification of GPAT, the crystals were screened by hanging-drop and only clusters were obtained. The crystals were optimized by adjusting temperature, pH, protein concentration, or precipitant, but little improvement. To improve the interaction between protein and precipitant, the isopropanol was applied as co-precipitant. The qualified crystals formed. It's suggested that isopropanol is critical to affect protein crystallization by altering polyethylene glycol(PEG)-water-protein interaction when PEG serves as precipitant. The resulting crystal diffracted to a resolution of 2.75 ? and belonged to space group P1, with unit-cell parameters a = 50.79, b = 80.09, c = 88.21 ?, and α = 62.85, β = 73.04, γ = 80.53?. This work introduced a new strategy to optimize the crystal of the heterogeneous catalysis enzymes like GPAT and provided the fundamental structural information for the oriented synthesis of marine microalgae glycerolipid.展开更多
In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, ...In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.展开更多
The gene sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzymatic target site of the herbicide glyphosate, was determined for several purple nutsedge (Cyperus rotundus L.) accessions from g...The gene sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzymatic target site of the herbicide glyphosate, was determined for several purple nutsedge (Cyperus rotundus L.) accessions from geographically distant locations and these were aligned to generate a consensus sequence. The EPSPS sequences each had single nucleotide polymorphisms (SNPs) only a few of which were predicted to cause an amino acid change in the EPSP synthase. None had the proline to serine substitution or other substitutions responsible for glyphosate resistance reported in other species. A dendrogram generated from the cluster analysis of the EPSPS gene sequences indicated similarities between accessions from Tanzania, Indonesia, California-2, Greece, Brazil, Argentina and Iran much like cluster analysis previously reported based on RAPD scores and morphological traits possibly indicating a common genetic background or origin. Considering the differences in EPSPS sequences, the response of these purple nutsedge accessions to 0.84 kg·ae·ha-1 of glyphosate was assessed to determine whether differential tolerance was present. At 7 days after the first application control ranged from 9% for the accession from Greece to 73% for the accession from Tanzania. Control of these accessions increased to 45% and 93% respectively by 14 days after the second application. The I50’s for glyphosate inhibition of growth for four accessions from geographically distant countries (Mississippi, Brazil, Indonesia and Tanzania) were 0.21, 0.10, 0.25 and 0.06 kg·ha-1, respectively, which represented a 4-fold difference. The difference in sensitivity to glyphosate may be a result of a non-target site mechanism such as differences in sequestration, translocation or cuticle thickness rather than alterations in EPSPS.展开更多
Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many ...Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.展开更多
In order to investigate the relationship between the lipid composition in thylakoid membrane and thermostability of photosynthetic apparatus, tobacco transformed with sweet pepper sense glycerol-3-phosphate acyltransf...In order to investigate the relationship between the lipid composition in thylakoid membrane and thermostability of photosynthetic apparatus, tobacco transformed with sweet pepper sense glycerol-3-phosphate acyltransferase （GPAT） gene were used to analyze the lipid composition in thylakoid membrane, the net photosynthetic rate and chlorophyll fluorescence parameters under high temperature stress. The results showed that the saturated extent of monogalactosyldiacylglycerol （MGDG）, sulfoquinovosyldiacylglycerol, digalactosyldiacylglycerol and phosphatidylglycerol in thylakoid membrane of transgenic tobacco T1 lines increased generally. Particularly, the saturated extent in MGDG increased obviously by 16.2% and 12.6% in T1-2 and T1-1, respectively. With stress temperature elevating, the maximum efficiency of photosystem Ⅱ （PSⅡ） photochemistry （Fv/Fm）, actual photochemical efficiency of PSll in the light （ФPSⅡ） and net photosynthetic rate （Pn） of the two lines and wild type tobacco plants decreased gradually, but those parameters decreased much less in transgenic plants. Even though the recovery process appeared differently in the donor and acceptor side of PSⅡ in transgenic tobacco compared with wild-type plants, the entire capability of PSⅡ recovered faster in transgenic tobacco, which was shown in the parameters of PI, Fv/Fm and ФPSⅡ, as a result, the recovery of Pn was accelerated. Conclusively, we proposed that the increase in saturated extent of thylakoid membrane lipids in transgenic plants enhanced the stability of photosynthetic apparatus under high temperature stress.展开更多
RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in pl...RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（3）P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.展开更多
Substrate selectivity of glycerol-3-phosphate acyltransferase （EC 2. 3. 1. 15） of rice （Oryza sativa L.） was explored in a comparative study of acyltransferases from seven plant species. In vitro labeling of acyl ...Substrate selectivity of glycerol-3-phosphate acyltransferase （EC 2. 3. 1. 15） of rice （Oryza sativa L.） was explored in a comparative study of acyltransferases from seven plant species. In vitro labeling of acyl carrier protein （ACP） with ^14C or 3H showed that acyltransferase from chill-sensitive plants, such as rice that uses either oleic （18：1） or palmitic acid （16：0） as acyl donor at comparable rates, displays lower selectivity than the enzyme from chill-resistant plants, such as spinach, which preferentially uses oleic acid （18：1） rather than palmitic acid （16：0） as an acyl donor. This may be a result of the size and character of the substrate-binding pocket of acyltransferase. Homology modeling and protein structure-based sequence alignment of acyltransferases revealed that proteins from either chill-sensitive or chill-tolerant plants shared a highly conserved domain containing the proposed substrate-binding pocket. However, the aligned residues surrounding the substrate-binding pocket are highly heterogeneous and may have an influence mainly on the size of the substrate binding pockets of acyltransferases. The substrate selectivity of acyltransferase of rice can be improved by enlarging the substrate-binding pocket using molecular biological methods.展开更多
The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to...The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.展开更多
In contrast with the coezyme, two coenzyme analogs, ADP-ribose and SNAD, bind non-cooperatively to D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Palinurus versicolor (PV) GAPDH complexed with ADP-ribose and SNAD...In contrast with the coezyme, two coenzyme analogs, ADP-ribose and SNAD, bind non-cooperatively to D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Palinurus versicolor (PV) GAPDH complexed with ADP-ribose and SNAD has been crystallized by the method of sitting-drop vapor diffusion. X-ray diffraction data analysis reveals that both crystals belong to the same space group (C2), and have similar cell dimensions: a =152.80 A, b =100.35 A, c =128.31 A, β=110.28° and a =153.41 A, b =100.51 A, c =128.44 A, β =110.48°, respectively. It is estimated that the asymmetric unit in each crystal contains 4 subunits. This is a novel crystal form which is quite different from that previously reported for holo- and apo-GAPDH from the same spurce. The result suggests that the binding of the two coenzyme analogs to GAPDH may lead to some significant conformational changes, which are different from those induced by the coenzyme binding. The self-rotation function indicates that the tetramer of these two GAPDH展开更多
D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell param...D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.展开更多
文摘It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.
基金Project supported by the Fundamental Research Funds of the Central Universities of China(No.XDJK2013C009)
文摘Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycelytic enzyme, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer （RaGAPDH） has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genornic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligandbinding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coil （EHEC）, belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestiferdisplayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism＇s virulence.
基金This work was supported by the National Natural Science Foundation of China(No.30170736)China National 863'High-tech Program(Grant No.2004AA603220).
文摘Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.
文摘Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.
基金financially supported by the National Natural Science Foundation of China (Nos. 21576253, 31500294 and 31470432)
文摘Glycerol-3-phosphate acyltransferase(GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid(LPA) synthesis. For understanding its catalytic mechanism, the structural biology study is expected, but is always hindered by obtaining crystals for X-ray diffraction analysis. In this study, a progressive strategy to optimize the crystal of microalgae plastidial GPAT was presented. After the expression and purification of GPAT, the crystals were screened by hanging-drop and only clusters were obtained. The crystals were optimized by adjusting temperature, pH, protein concentration, or precipitant, but little improvement. To improve the interaction between protein and precipitant, the isopropanol was applied as co-precipitant. The qualified crystals formed. It's suggested that isopropanol is critical to affect protein crystallization by altering polyethylene glycol(PEG)-water-protein interaction when PEG serves as precipitant. The resulting crystal diffracted to a resolution of 2.75 ? and belonged to space group P1, with unit-cell parameters a = 50.79, b = 80.09, c = 88.21 ?, and α = 62.85, β = 73.04, γ = 80.53?. This work introduced a new strategy to optimize the crystal of the heterogeneous catalysis enzymes like GPAT and provided the fundamental structural information for the oriented synthesis of marine microalgae glycerolipid.
基金Supported by Social Service Project of New Countryside Development Research Institute of Yangtze University(201411)
文摘In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.
文摘The gene sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzymatic target site of the herbicide glyphosate, was determined for several purple nutsedge (Cyperus rotundus L.) accessions from geographically distant locations and these were aligned to generate a consensus sequence. The EPSPS sequences each had single nucleotide polymorphisms (SNPs) only a few of which were predicted to cause an amino acid change in the EPSP synthase. None had the proline to serine substitution or other substitutions responsible for glyphosate resistance reported in other species. A dendrogram generated from the cluster analysis of the EPSPS gene sequences indicated similarities between accessions from Tanzania, Indonesia, California-2, Greece, Brazil, Argentina and Iran much like cluster analysis previously reported based on RAPD scores and morphological traits possibly indicating a common genetic background or origin. Considering the differences in EPSPS sequences, the response of these purple nutsedge accessions to 0.84 kg·ae·ha-1 of glyphosate was assessed to determine whether differential tolerance was present. At 7 days after the first application control ranged from 9% for the accession from Greece to 73% for the accession from Tanzania. Control of these accessions increased to 45% and 93% respectively by 14 days after the second application. The I50’s for glyphosate inhibition of growth for four accessions from geographically distant countries (Mississippi, Brazil, Indonesia and Tanzania) were 0.21, 0.10, 0.25 and 0.06 kg·ha-1, respectively, which represented a 4-fold difference. The difference in sensitivity to glyphosate may be a result of a non-target site mechanism such as differences in sequestration, translocation or cuticle thickness rather than alterations in EPSPS.
文摘Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.
基金the Early Stage of China Key Development Project for BasicResearch (CB116208)the National Natural Science Foundation of China(30471053)the Shandong Provincial Natural Science Foundation ofChina (Y2007D50).
文摘In order to investigate the relationship between the lipid composition in thylakoid membrane and thermostability of photosynthetic apparatus, tobacco transformed with sweet pepper sense glycerol-3-phosphate acyltransferase （GPAT） gene were used to analyze the lipid composition in thylakoid membrane, the net photosynthetic rate and chlorophyll fluorescence parameters under high temperature stress. The results showed that the saturated extent of monogalactosyldiacylglycerol （MGDG）, sulfoquinovosyldiacylglycerol, digalactosyldiacylglycerol and phosphatidylglycerol in thylakoid membrane of transgenic tobacco T1 lines increased generally. Particularly, the saturated extent in MGDG increased obviously by 16.2% and 12.6% in T1-2 and T1-1, respectively. With stress temperature elevating, the maximum efficiency of photosystem Ⅱ （PSⅡ） photochemistry （Fv/Fm）, actual photochemical efficiency of PSll in the light （ФPSⅡ） and net photosynthetic rate （Pn） of the two lines and wild type tobacco plants decreased gradually, but those parameters decreased much less in transgenic plants. Even though the recovery process appeared differently in the donor and acceptor side of PSⅡ in transgenic tobacco compared with wild-type plants, the entire capability of PSⅡ recovered faster in transgenic tobacco, which was shown in the parameters of PI, Fv/Fm and ФPSⅡ, as a result, the recovery of Pn was accelerated. Conclusively, we proposed that the increase in saturated extent of thylakoid membrane lipids in transgenic plants enhanced the stability of photosynthetic apparatus under high temperature stress.
基金This work was supported by grants from the National Natural Science Foundation of China （No. 30971889）, National Science and Technology Major Projects （No. 2009ZX08005-003B）, and the Natural Science Foundation of Jiangsu Province （No. BK2012027）.We appreciate Prof. Brett Tyler （Oregon State） for manuscript preparation, and Yuanming Zhang （Nanjing Agricultural University） for suggestions in data analysis. No conflict of interest declared.
文摘RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（3）P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.
基金Supported by the National Natural Science Foundation of China (30270794)the Jiangsu Provincial Science Foundation of China (BK2007063 and BK2005041)the Natural Science Foundation of the Education Bureau of Jiangsu Province (06KJB180087 and 04KJB210107)
文摘Substrate selectivity of glycerol-3-phosphate acyltransferase （EC 2. 3. 1. 15） of rice （Oryza sativa L.） was explored in a comparative study of acyltransferases from seven plant species. In vitro labeling of acyl carrier protein （ACP） with ^14C or 3H showed that acyltransferase from chill-sensitive plants, such as rice that uses either oleic （18：1） or palmitic acid （16：0） as acyl donor at comparable rates, displays lower selectivity than the enzyme from chill-resistant plants, such as spinach, which preferentially uses oleic acid （18：1） rather than palmitic acid （16：0） as an acyl donor. This may be a result of the size and character of the substrate-binding pocket of acyltransferase. Homology modeling and protein structure-based sequence alignment of acyltransferases revealed that proteins from either chill-sensitive or chill-tolerant plants shared a highly conserved domain containing the proposed substrate-binding pocket. However, the aligned residues surrounding the substrate-binding pocket are highly heterogeneous and may have an influence mainly on the size of the substrate binding pockets of acyltransferases. The substrate selectivity of acyltransferase of rice can be improved by enlarging the substrate-binding pocket using molecular biological methods.
基金This work was supported by the National High-Tech Program (863), National Natural Science Foundation of China (Grant Nos. 39989001, 39580012 & 39880023) National Special Program for Research and Industrialization of Transgenic Plants, and Rockefeller F
文摘The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.
文摘In contrast with the coezyme, two coenzyme analogs, ADP-ribose and SNAD, bind non-cooperatively to D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Palinurus versicolor (PV) GAPDH complexed with ADP-ribose and SNAD has been crystallized by the method of sitting-drop vapor diffusion. X-ray diffraction data analysis reveals that both crystals belong to the same space group (C2), and have similar cell dimensions: a =152.80 A, b =100.35 A, c =128.31 A, β=110.28° and a =153.41 A, b =100.51 A, c =128.44 A, β =110.48°, respectively. It is estimated that the asymmetric unit in each crystal contains 4 subunits. This is a novel crystal form which is quite different from that previously reported for holo- and apo-GAPDH from the same spurce. The result suggests that the binding of the two coenzyme analogs to GAPDH may lead to some significant conformational changes, which are different from those induced by the coenzyme binding. The self-rotation function indicates that the tetramer of these two GAPDH
文摘D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.
