荞麦与大豆叶片乙醇酸氧化酶的纯化和性质比较研究

分别从荞麦与大豆叶片中部分纯化了乙醇酸氧化酶 (GO ,EC1 .1 .3 .1 ) ,并研究其部分性质。结果显示荞麦与大豆叶片中GO的催化特性有明显差异 :大豆叶片中GO对乙醇酸Km值为 0 .3 1mmol/L ,对乙醛酸Km值为 1 .98mmol/L。外源草酸对GO氧化乙醇酸活性影响很小 ,但对其氧化乙醛酸活性抑制明显 ,5mmol/L草酸可抑制 44%。而荞麦叶片中GO性质有所不同 :GO对乙醇酸Km为 0 .46mmol/L ,对乙醛酸Km为 0 .85mmol/L。草酸对荞麦GO氧化乙醇酸活性影响也很小 ,对其氧化乙醛酸活性的抑制作用明显小于大豆 ,5mmol/L草酸只抑制 2 4%。上述研究结果表明 ,荞麦GO对乙醛酸的亲和力明显强于大豆 ,并且草酸对其GO氧化乙醛酸活性影响较小。因此相对于大豆而言 ,GO可能在荞麦叶片草酸合成中起重要作用。     

植物中草酸积累与光呼吸乙醇酸代谢的关系

对几种C3 和C4 植物中草酸含量及相应的乙醇酸氧化酶活性测定结果表明 :叶片光呼吸强度及其关键酶活性大小与草酸积累量没有相关性 ;植物根中均能积累草酸 ,但未测出乙醇酸氧化酶活性。烟草根、叶中的草酸含量在不同生长时期差异明显 ,且二者呈极显著正相关 (y =2 .5 6 5lnx 2 .137,r =0 .749,P <0 .0 0 1) ,说明根中草酸可能来自叶片。氧化乙醇酸的酶的活性与氧化乙醛酸的酶的活性呈极显著线性正相关 (y =0 .2 41x 0 .0 0 6 ,r=0 .96 7,P <0 .0 0 0 1) ,进一步证实是乙醇酸氧化酶催化了两种底物的反应。烟草在不同生长期叶片中草酸总含量变化与相应的乙醇酸氧化酶活性变化亦没有相关性 ;低磷胁迫可显著诱导烟草根叶中的草酸形成和分泌 ,但并未影响乙醇酸氧化酶活性 ,进一步证明草酸积累与该酶活性大小无关     

菜心乙醇酸氧化酶二种催化功能的初步研究(简报)

乙醇酸氧化酶（glyCOlldtCOXid3SC,ECI．1．3．l,GO）是植物光呼吸代谢的关键酶,催化乙醇酸生成乙醛酸,但又有研究者报道其可能还具有进一步氧化乙醛酸生成草酸的能力。Richardson和Tolbert发现甜菜等一些植物的GO均能氧化乙醛酸生成草酸,并且两种催化活性的比值在酶...     

植物中草酸积累与光呼吸惭醇酸代谢的关系

对几种Ｃ３和Ｃ４植物中草酸含量及相应的乙醇酸氧化酶活性测定结果表明,叶片光呼吸强度及其着急酶活性大小与草酸积累量没有相关性;植物根中均能积累草酸,但未测出乙醇酸氧化酶活性。烟草根、叶中的草酸含量在不同生长时期差异明显,且二者呈显著正相关（ｙ＝２．５６５ｌｎｘ＋２．１３７,ｒ＝０．７４９,Ｐ〈０．００１）,说明振中到可能来自叶片。氧化乙醇酸的瓣活性与氧化乙醛酸的酶的活性呈极显著线性正相关（ｙ＝０．２     

水稻叶片中的草酸积累及其与抗白叶枯病的关系(简报)1

测定不同生长时期及感染白叶枯病菌前后,水稻叶片中的草酸含量、乙醇酸氧化酶活性变化的结果,进一步证实乙醇酸氧化酶同时具有氧化乙醛酸的活性,但叶片中的内源草酸含量变化与乙醇酸氧化酶活性变化无关。高感品种玉梅153和高抗品种中二占在染病前后内源草酸含量变化之间并无显著差异。     

不同因子对荞麦中草酸含量的影响

用不同化合物从根部喂养麦幼苗,测定其根叶中草酸含量的变化。结果表明：异柠檬酸、抗坏血酸及其前体物均可不同程度地降低荞麦根叶中草酸含量;而乙醇酸与乙醛酸则显著提高其草酸含量,表明荞麦叶片草酸合成主要来自乙醇酸途径,而非来自抗坏血酸等途径。水培条件下,以铵态氮或尿素等作唯一氮源时,荞麦中草酸含量远低于以硝态氮培养的;将谷氨酸或丝氨酸加到含硝态氮培养液中也能显著降低其草酸含量,不同氮素影响荞麦草酸含量可能与乙醇酸途径有关。     

水稻叶片中的草酸积累及其与抗白叶枯病的关系（简报）

测定不同生长时期及感染白叶枯病菌前后,水稻叶片中的草酸含量、乙醇酸氧化酶活性变化的结果,进一步证实乙醇酸氧化酶同时具有氧化乙本钱酸的活性,但叶片另的内源草酸含量变化与乙醇酸氧化酶活性变化无关,高感品种玉梅１５３和高抗品种中二占在染病前后内源草酸含量变化之间并无显著差异。     

植物的乙醇酸氧化酶

乙醇酸氧化酶存在于植物细胞过氧物酶体中,是一种黄素蛋白,以FMN为辅基,含8个亚基。催化乙醇酸氧化为乙醛酸和乙醛酸氧化为草酸。受光活化。硫化钠和氰化物促进其活性:巯基抑制剂、α-羟基磺酸类、α-羟基丁炔酸抑制其活性。多种代谢物对其活性有调节作用。为光呼吸的关键酶之一,其活性随植物发育、矿质营养及感病而发生变化。     

不同氮素形态培养下荞麦叶片中草酸积累的变化

用1/5浓度Hoagland(pH6.0)营养液培养荞麦幼苗3d后,取其中一部分继续用此营养液(硝态氮);另一部分用硫酸氨和氯化钙取代硝态氮(氨态氮)的营养液,均培养至荞麦第一片真叶完全展开。结果表明,以氨态氮为唯一氮源培养荞麦时,植株叶片中草酸含量显著下降。进一步研究表明,氨态氮培养下荞麦根中及根分泌草酸的速率也显著下降,结果排除了叶片中草酸含量的下降是由于叶片中草酸向其根系转运或是因为根分泌草酸速率的差异造成的,而可能与其草酸代谢改变有关。氨态氮培养下叶片中与草酸代谢相关的有机酸含量以及相关酶活性也显著下降,这可能意味着荞麦叶片草酸形成积累可能与相关有机酸代谢有关。     

光呼吸代谢物乙醇酸、乙醛酸和草酸对烟草叶片硝酸还原的影响

乙醇酸、乙醛酸和草酸能明显促进烟草(Nicotiana rustica)叶片在黑暗中的硝酸还原,光呼吸抑制剂a-羟基吡啶甲烷磺酸能消除前二者的促进作用而不能完全消除草酸的作用。草酸+NAD~+能显著促进离体的硝酸还原。烟叶提取液加入草酸和NAD~+后生成NADH和CO_2认为活体内由乙醛酸氧化生成的草酸是经脱氢生成NADH供硝酸还原之用。未能证明在烟叶内存在乙醇酸脱氨酶,因此排除由乙醇酸直接脱氢以还原硝酸的可能。     

Compartmentation of aluminium in leaves of an Al-accumulator,Fagopyrum esculentum Moench

Buckwheat (Fagopyrum esculentum Moench.) is an Al-accumulating plant, but the internal mechanism(s) of detoxification of Al is not fully understood. We investigated the subcellular localization of Al in the leaves of this plant (cv. Jianxi) by directly isolating protoplasts and vacuoles. Pure protoplasts and vacuoles from the leaves of buckwheat, grown hydroponically in Al solution, were obtained based on light-microscopic observation and the activities of marker enzymes of cytosol and vacuoles. More than 80% of total Al in the leaves was present in the protoplasts, and was identified as an Al-oxalate complex (1:3 ratio) by (27)Al-nuclear magnetic resonance. Oxalate and Al in the protoplasts was localized in the vacuoles. These results suggest that internal detoxification of Al in the buckwheat leaves is achieved by both complexation with oxalate and sequestration into vacuoles.     

Active site and loop 4 movements within human glycolate oxidase: implications for substrate specificity and drug design

Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most alpha-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with alpha-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.     

植物叶片中抗坏血酸含量与草酸积累的关系

不同植物和不同生长期烟草叶片中抗坏血酸含量变化与相应的草酸含量变化之间都无显著的相关性;喂饲外源抗坏血酸后的水稻和荞麦叶中草酸含量提高不明显。据此推测：尽管不能排除抗坏血酸可能是植物草酸合成的前体,但其内源含量高低不一定影响植物中草酸积累。     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

Synthesis of oxalic Acid by enzymes from lettuce leaves

A rapid purification of lactate dehydrogenase and glycolate oxidase from lettuce (Lactuca sativa) leaves is described. The kinetics of both enzymes are reported in relation to their possible roles in the production of oxalate. Lettuce lactate dehydrogenase behaves like mammalian dehydrogenase, catalyzing the dismutation of glyoxylate to glycolate and oxalate. A model is proposed in which glycolate oxidase in the peroxisomes and lactate dehydrogenase in the cytosol are involved in the production of oxalate. The effect of pH on the balance between oxalate and glycolate produced from glyoxylate suggests that in leaves lactate dehydrogenase may function as part of an oxalate-based biochemical, pH-stat.