植物中磷酸甘油醛-3-磷酸脱氢酶（GAPDH）在氧化胁迫下的生理功能

文章介绍植物中持家蛋白磷酸甘油醛-3-磷酸脱氢酶（GAPDH）在氧化胁迫下抑制活性氧生成、诱发磷酸化过程从而激活MAPK信号级联反应、诱导聚合体形成、参与谷胱甘肽修饰和控制电子转运中的生理功能研究进展。     

美洲大蠊3-磷酸甘油醛脱氢酶基因的克隆及表达

旨在通过5’-RACE获得美洲大蠊3-磷酸甘油醛脱氢酶(GAPDH)基因的全长cDNA序列,进行生物信息学分析,构建原核表达载体,诱导重组蛋白表达,为进一步研究其功能奠定基础.通过3’-RACE技术,PCR扩增获取编码美洲大蠊GAPDH蛋白的全长cDNA序列;采用生物信息学方法推导出该序列编码的氨基酸序列及其理化性质;预测信号肽、蛋白疏水性、可溶性、跨膜区结构、二级结构、三级结构,并构建系统发育树;构建原核表达载体pET28a-GAPDH,IPTG诱导重组蛋白表达,并用Histag抗体Western blotting验证.结果显示,美洲大蠊GAPDH基因,其完整阅读框含999个碱基,编码332个氨基酸.序列分析显示,该蛋白与家蚕GAPDH相似性为89％,具有GAPDH保守功能域,经IPTG诱导获得重组蛋白.     

酿酒酵母3-磷酸甘油脱氢酶的诱导、提纯和性质

在YEPD培养基中添加NaCl,可以诱导酿酒酵母(Saccharomyces cerevisiae)细胞内3-磷酸甘油脱氢酶的形成,当NaCl浓度达5％时,酶比活从0.05U/mg提高0.5U/mg;若再限制培养墓中葡萄糖浓度在100mg/L以下,酶比活可达到0.89U/mg。酶比活与培养基中的NaCl浓度的函数关系式为:Sa=0.129C~3-0.038C~2+0.034C+0.063(0≤C≤5％)。粗酶液经Sephadex G-25凝胶过滤,Blue Sepharose亲和层析以及Mono Q离子交换等步骤,提纯123.6倍,得纯酶液。经SDS-凝胶电泳测得分子量为45000±2000。酶的最适温度为51℃,最适pH值为6.8。保温30分钟的半失活温度(t_(1/2))为41℃。NADH和DHAP的Km(mmol/L)值分别为:0.017和0.134。     

Na2CO3胁迫下星星草（Puccinellia tenuiflora）幼苗叶表皮和叶肉细胞中K、Na的相对含量

对不同强度Na2CO3胁迫处理下星星草幼苗叶片表皮和叶肉细胞中K、Na的透射电镜X-射线电子探针显微分析和叶片表面扫描电镜X-射线电子探针显微分析,结果表明：在相同胁迫强度下,无论是表皮细胞还是叶肉细胞的细胞壁和液泡中的Na相对含量均明显高于细胞质中的Na相对含量,并且K的相对含量均明显比相应部位Na的相对含量高,细胞壁与液泡中的Na相对含量变化范围非常接近。在Na2CO3胁迫浓度低于0.1molL-1时,在相同胁迫强度下,K的相对含量高于Na的相对含量,使细胞质保持相对高的K/ Na比。而尽管向细胞壁和液泡分流了大量的Na,但是细胞质中的Na相对含量仍然随着Na2CO3胁迫强度的增加而增加,一方面证明星星草在Na2CO3胁迫下维持相对高的K/ Na比的能力是有一定限度的,另一方面暗示星星草作为盐生植物在盐碱环境中一定程度上Na可以部分地代替K而行使部分K的生理功能。     

Involvement of Glyceraldehyde-3-phosphate Dehydrogenase in the X-Ray Resistance of HeLa Cells

We investigated changes in the sub-cellular distribution of glycelaldehyde-3-phosphate dehydrogenase (GAPDH) after X-ray irradiation in HeLa cells. Twenty-four h after irradiation at 5 Gy, nuclear GAPDH levels increased 2.6-fold, whereas total GAPDH levels increased only 1.2-fold. Knockdown of GAPDH using specific small interfering RNA (siRNA) led to sensitization to X-ray-induced cell death. These results suggest that GAPDH plays a role in the radioresponse.     

西伯利亚蓼甘油醛-3-磷酸脱氢酶基因的cDNA克隆与序列分析

根据NaHCO3胁迫下西伯利亚蓼茎部消减库中甘油醛-3-磷酸脱氢酶基因(GAPDH)表达序列标签序列设计引物,采用cDNA末端快速扩增技术,从西伯利亚蓼茎中扩增出GAPDH的全长cDNA序列。该cDNA序列全长1331bp,完整阅读框1014bp,编码337个氨基酸。属于稳定蛋白,具有GAPDH保守功能域。氨基酸组成与其他已知高等植物来自细胞质中的GAPDH基因cDNA序列具有很高的同源性,最高可以达到96%。通过转酿酒酵母INVSC1的NaHCO3和NaCl胁迫试验表明,转基因INVSC1(pYES2-GAPDH)有明显的抗盐胁迫特性。在10%NaHCO3和4mol·L-1 NaCl胁迫下,转基因INVSC1(pYES2-GAPDH)菌株存活率明显比INVSC1(pYES2)高,可以推测GAPDH基因赋予INVSC1(pYES2-GAPDH)抗NaHCO3和NaCl的能力。该基因的cDNA序列在GenBank中登录号为DQ922680。     

Archaeal Aldehyde Dehydrogenase ST0064 from Sulfolobus tokodaii,a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase,Is a Succinate Semialdehyde Dehydrogenase

Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (K _m of 0.0029 mM and k _cat of 30.0 s^?1) with no allosteric regulation. Activity toward GAP was lower (K _m of 4.6 mM and k _cat of 4.77 s^?1), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.     

Glyceraldehyde-3-phosphate dehydrogenase (NADP) from Sinapis alba: Steady state kinetics

Substrate interaction and product inhibition kinetics of the forward reaction of glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba suggest an Uni Uni Uni Bi Ping Pong mechanism (NAD(P)H on, glyceraldehyde-3-phosphate off, 1,3-diphosphoglycerate on, phosphate off, NAD(P)⁺ off) with an apparent Theorell Chance displacement between 1,3-diphosphoglycerate and phosphate. The proposed mechanism predicts the existence of stable enzyme-NAD(P)⁺ and acyl-enzyme complexes as obligatory intermediates. A comparison of the present findings on the NADP-enzyme with an earlier kinetic analysis of the NAD-specific enzyme from plants (EC 1.2.1.12) by other authors shows that the kinetic mechanisms for the two enzymes, although similar in principle (both show Ping Pong kinetics), differ in some details.     

Different Thermostability of Skeletal Muscle Glyceraldehyde-3-phosphate Dehydrogenase from Hibernating and Euthermic Jerboa （Jaculus orientalis）

D Glyceraldehyde 3 phosphatedehydrogenase(GAPDH ,EC 1.2 .1.12 )isakeyenzymeoftheglycolyticpathwaythatispresentinthecytosolofallorganismssofarstudied[1] .TheglycolyticGAPDHhasbeenremarkablyconservedduringevolution ,havingahomotetramericstructurewithsubunitsof 35 - 37kD[1] .GAPDHhasbeenisolatedfromavarietyofspecies[2 ] ,includingmesophilic ,moderatelythermophilicandhyperthermophilicmicroorganisms[3 ] .Theseenzymes ,whichdifferinthermalstability ,havebeenshowntobehighlysimilarinaminoacidse…     

Glyceraldehyde-3-phosphate dehydrogenase as a biochemical marker of cytotoxicity by vinyl sulfones in cultured murine spleen lymphocytes

Recently, vinyl sulfones have been observed to selectively inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an important ATP-generating enzyme in glycolysis. The possibility of using GAPDH as a biochemical parameter of cytotoxicity by vinyl sulfones was investigated using mouse lymphocytes. Incubation of lymphocyte GAPDH with ethylvinyl sulfone resulted in a pseudo-first-order loss of enzyme activity. The exposure of lymphocytes to ethylvinyl sulfone resulted in the decrease of GAPDH activity followed by ATP depletion and cell death, which were both dependent on the concentration of ethylvinyl sulfone. A further study on the time-dependent change indicated that cell death was preceded by ATP loss. Compared to ethylvinyl sulfone, divinyl sulfone was more than 8 times more potent in causing either ATP depletion or cell death.Abbreviations DTT dithiothreitol - GAPDH glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide     

Identification of two different glyceraldehyde-3-phosphate dehydrogenases (phosphorylating) in the photosynthetic protist Cyanophora paradoxa

Two different glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) activities, namely NAD- and NADP-dependent, have been found in cell extracts of the cyanelle-bearing photosynthetic protist Cyanophora paradoxa. Whereas the two G3P dehydrogenase activities were detected with similar specific activity levels (0.1 to 0.2 U/mg of protein) in extracts of the photosynthetic organelles (cyanelles), only the NAD-dependent activity was found in the cytosol. Thus, a differential intracellular localization occurred. The perfect overlapping of the two G3P dehydrogenase activity peaks of the cyanelle in both hydrophobic interaction chromatography and subsequent FPLC (fast protein liquid chromatography) gel filtration indicated that the two activities were due in fact to a single NAD(P)-dependent G3P dehydrogenase (EC 1.2.1.-) with a molecular mass of 148,000. SDS-PAGE of active fractions from FPLC gel filtration showed that the intensity of the major protein band (molecular mass, 38,000) of the enzyme preparation clearly paralleled the activity elution profile, thus suggesting a tetrameric structure for the cyanelle dehydrogenase. On the other hand, FPLC gel filtration analysis of the cytoplasmic fraction revealed a NAD-dependent G3P dehydrogenase with a native molecular mass of 142,000, being equivalent to the classical glycolytic enzyme (EC 1.2.1.12) present in the cytosol of all the organisms so far studied. The significance of these results is discussed taking into account that the cyanobacteria, photosynthetic prokaryotes which share many structural and biochemical features with cyanelles and are considered as their ancestors, have a similar NAD(P)-dependent G3P dehydrogenase.Abbreviation FPLC Fast protein liquid chromatography     

Cotransport of Glyceraldehyde-3-Phosphate Dehydrogenase and Actin in Axons of Chicken Motoneurons

1.To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [³⁵S]methionine and, 1–20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I–Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography.2.In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).3.The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods.4.Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3–5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity.5.Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.     

Glyceraldehyde-3-Phosphate Dehydrogenase Activity and F-Actin Associations in Synaptosomes and Postsynaptic Densities of Porcine Cerebral Cortex

1. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) is a glycolytic enzyme that has also been implicated in a wide variety of functions within neurons. Because of the well-documented role of G3PD as an actin-binding protein, we sought evidence for a G3PD–actin complex in synaptosomes and postsynaptic densities (PSDs).2. We have shown G3PD association with 0.5-m synaptosomal particles by immunofluorescence as similarly demonstrated for actin (Toh et al., Nature 264:648–650, 1976). An immunoblot analysis also showed G3PD and actin to be enriched in synaptosomes. Further analysis of subcellular fractions from synaptosomes showed the PSD but not the synaptosomal plasma membranes to be enriched in G3PD and actin.3. Highest levels of G3PD catalytic activity were found in synaptosomes and PSDs. Although synaptosomes showed significant activity for phosphoglyceratekinase (PGK), an enzyme in sequence with G3PD for ATP production in the glycolytic pathway, no such activity was detected in the PSD fraction.4. Our studies indicate that a G3PD–actin complex may exist at the synapse. A physical association of G3PD with endogenous F-actin in synaptosomes and PSDs was demonstrated by combined phalloidin shift velocity sedimentation/immunoblot studies. By this approach, synaptosomal G3PD–actin complexes were also found to be significantly less dense than the PSD G3PD–actin complexes.5. G3PD and PGK catalytic activity in synaptosomes suggests a role in glycolysis, as well as actin binding, in the presynaptic terminals. On the other hand, the high levels of G3PD activity in PSDs but lack of PGK activity suggests that G3PD is involved in nonglycolytic functions, such as actin binding and actin filament network organization.