Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

Mitochondrial malate dehydrogenase （mMDH） and citrate synthase （CS） are sequential enzymes in Krebs cycle. mMDH, CS and the complex between mMDH and CS （mMDH＋CS） were treated with nitric oxide solution. The roles of notric oxide （NO） on the secondary structures and the interactions between mMDH and CS were studied using circular diehroism （CD） and Fourier transform surface plasmon resonance （FT-SPR）, respectivley. The effects of NO on the activities of mMDH, CS and mMDH＋CS were also studied. And the regulations by NO on mMDH and CS were simulated by PyMOL software. The results of SPR conifrmed that strong interaction between mMDH and CS existed and NO could signiifcantly regulate the interaction between the two enzymes. NO reduced the mass percents ofα-helix and increased that of random in mMDH, CS and mMDH＋CS. NO increased the activities of CS and mMDH＋CS, and inhibited the activity of mMDH. Graphic simulation indicated that covalent bond was formed between NO and Asn242 in active site of CS. However, there was no direct bond between NO and mMDH. The increase in activity of mMDH＋CS complex depended mostly on the interaction between NO and CS. All the results suggested that the regulations by NO on the activity and interaction between mMDH and CS were accord with the changes in mMDH, CS and mMDH＋CS caused by NO.

Malate and Lactate Dehydrogenases of a Freshwater Catfish: Impact of Endosulfan

A sublethal concentration of technical grade endosulfan (END) inhibited 35 to 55% of the activities of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH), and lactate dehydrogenase (LDH) in the liver and the skeletal muscle of a freshwater catfish, Clarias halrachus, after 7 days of exposure. The activity remained in the inhibited state up to 28 days. The withdrawal of END from the medium after 1 week of exposure gradually restored the activities to control levels within 21 days in the skeletal muscle and 28 days in the liver. The administration of actinomycin D or cycloheximide between the 14th and the 21st day of the withdrawal of END almost completely inhibited the withdrawal-dependent recovery in the activities of all the three enzymes. This indicates de novo synthesis of the enzymes during the recovery period. A conjoint treatment of END and triiodothyronine (T_3) raised the activities of cMDH, mMDH, and LDH in the liver and the skeletal muscle up to the control levels. This shows that the inhibitory effect of END may be relieved in presence of T_3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed few changes in the pattern of cytoplasmic proteins of the liver and the skeletal muscle in response to exposure to END. 1990 Academic Press. Inc.

Tear Malate Dehydrogenase,Lactate Dehydrogenase and Their Isoenzymes in Normal Chinese Subjects and Patients of Ocular Surface Disorders

Purose:To determine levels of malate dehydrogenase(MDH),lactate dehydroge-nase(LDH)and their isoenzymes in tears of normal Chinese subjects and patients with ocular surface disorders.Methods:The age range of normal subjects was10-88,with136mal and 128fe-male subjects.123patients suffered from ocular surface disorders.Tears were col-lected from lower fornix on Xinghua filter disc(0.1mm thick,5mm in diameter).The values of tearMDHand LDHwere determined by MONARCH-2000Ana-lyzer(U.S.A)Their isoenzymes were separated by acetate cellulose elec-trophoresis and were determined by Model CDS-200light densitometer.Results:The normal values of tear LDH and MDH were 45.51+23.00-81.35+37.84umol·s^-1/Land11.00+5.33-19.50+9.17umol·s^-1/Lrespectively,dis-regarding sex or eye distriction(P>0.05).The values of tear LDHandMDH in the group aged10-19were significantly lower than in another groups(P<0.05),95%normal ranges of tearMDHaged below19and above20were3.63-19.90umol·s^-1/L.THe MDH isoenzymes comprised MDHs and MDHm,the former accounting for80.0-89.1%.The LDH isoenzymes comprised 5varieties.of which the ratioH/Mof subunit H tosubunit Mwas0.196+0.02.Levels of tear LDH,MDHand their isoenzymes in different diseases were various.Conclusions;Tear LDH/MDHratio reflected sensitively the matabolism of corneae and conjunetival epithelium.The changes in LDH isoenzymes were hel-ful to the differential diagnosis of external eye diseases,and the increase of MDHm reflected sensitively the degree of injury to the corneal epithelium.

Evaluation of Tear Malate Dehydrogenase 2 in Mild Dry Eye Disease

Purpose: To evaluate the effect of tear malate dehydrogenase2 on monitoring ocular surface injury in mild dry eye(DE)disease.Methods: A total of 15 DE patients(30 eyes) with mild subjective symptoms but no ocular surface fluorescein staining signs were enrolled in this study.(DE group)..The control group was 15 healthy age- and sex-matched volunteers(30eyes)..All subjects were asked to fill out a DE symptoms questionnaire and take different tests including tear MDH and MDH2 activities evaluation,..tear breakup time.(TBUT),Schirmer I,.and slit-lamp examination of the ocular surface.We investigated different changes in tear MDH and MDH2 activities in the DE group and control group,.discussed the association between tear MDH2 activity and DE symptoms, and the relationship between tear MDH2 activity and diagnostic tests(Schirmer I and TBUT). We also analyzed the changes in tear MDH2 activities after the treatment with artificial tears.Results:.Tear MDH activities in the DE group and control group were 288 ±102 U / L and 259 ±112 U / L,.respectively,and this difference was not statistically significant(P >0.05).The tear MDH2 activities in DE group were significantly increased compared with control group. Tear MDH2 was significantly and negatively correlated with the Schirmer's value(r=-0.733,P<0.01) and the TBUT value(r=-0.841,P<0.01)..MDH2 also had a significant positive correlation with soreness symptoms(r=0.687, P < 0.01). Treatment with artificial tears relieved or eliminated all discomfort symptoms, together with a considerable decrease in MDH2 activities(P <0.01),.but no significant changes in the Schirmer and the TBUT tests were observed.Conclusion: Tear MDH2 activity can indicate ocular surfaceinjury in mild DE patients and may be used to monitor the response to therapy.

Duplication of Locus Coding of Malate Dehydrogenase in Populus tomentosa Carr

Horizontal starch-gel electrophoresis was used to study crude enzyme extraction from young leaves of 234 clones of Populus tomentosa Cart. selected from nine provenances in North China. Ten enzyme systems were resolved. One hundred and fifty-six clones showing unusual allozyme band patterns at locus Mdh-Ⅰ were found. Three allozyme bands at locus Mdh-Ⅰ were 9：6：1 in concentration. Further studies on the electrophoretic patterns of ground mixed pollen extraction of 30 male clones selected at random from the 156 clones were conducted and it was found that allozyme bands at locus Mdh-Ⅰ were composed of two dark-stained bands and a weak band. Only one group of the malate dehydrogenase （MDH） zymogram composed of two bands was obtained from the electrophoretic segregation of pollen leachate of the same clones. A comparison of the electrophoretic patterns one another suggested that the locus Mdh-Ⅰ coding malate dehydrogenase in diploid species of P. tomentosa was duplicated. The duplicate gene locus possessed three same alleles and was located in mitochondria. The locus duplication of alleles coding malate dehydrogenase in P.tomentosa was discovered and reported for the first time.

Molecular Weight of Cytoplasmic Malate Dehydrogenase,Mitochondrial Malate Dehydrogenase and Lactate Dehydrogenase of a Freshwater Catfish

The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH ) and lactate dehydrogenase (LDH ) were studied in the liver and skeletal muscle of the freshwater catfish, Clarias batrachus. There were two electrophoretically distinguishable bands (AA andBB) of cMDH and mMDH which suggests that they are apparently encoded at two gene loci (A and B) in both the tissues.However, the presence of a single band (LDH-1 ) of LDH in liver and double bands (LDH-1and LDH-2) in skeletal muscle in which LDH-2 was predominant reflects the differential expression of LDH genes in different metabolic tissues to meet the requirement of energy production. The AA isoform (74 kd) of liver cMDH was smaller than those of the AA form (110 kd) of skeletal muscle. In contrast, the BB isoform of liver (42 kd) and skeletal muscle (54 kd) were more or less similar in size. Unlike the case of cMDH, the molecular weight of AA isoform (115 kd) of liver mMDH was higher than those of the AA form (87kd) of skeletal muscle. Whereas the molecular weight of BB isoform (58 kd) of liver was in proximity to the weight of BB form (44 kd) of skeletal muscle mMDH. The size of AA isoform (74 kd) of liver cMDH was smaller, while the AA isoform (110 kd) of skeletal muscle was larger as compared to AA form of mMDH in the liver (115 kd) and skeletal muscle (87 kd). But the size of BB isoform of both the isozymes was almost equal in these metabolic tissues. The molecular weight of liver LDH-1 (96 kd) was close to the weight of LDH-1 (82 kd) in skeletal muscle. The molecular weight of skeletal muscle LDH-2 was deduced as 37 kd which is much more lower than the weight of LDH-1 in liver and skeletal muscle. The smaller size of LDH-2 in skeletal muscle may be of a physiological significance in this anaerobic tissue

A Review on Molecular Physiology of Malate and Lactate Dehydrogenases in Fishes

The malate(EC 1.1.1.37)and lactate(EC 1.1.1.27)dehydrogenases are themetabolic enzymes directly or indirectly involved in energy production,gluconeogenesis and lipogenesis.Malate dehydrogenase(MDH)exists in twoisoenzymic forms,cytoplasmic(cMDH)and mitochondrial(mMDH),composed of Aand/or B subunits(dimeric molecule:MW 40,000-120,000).Lactate dehydrogenase(LDH)has tetrameric(MW 35,000-110,000)structure made up of either A and/or B,orC(C,E,F)subunits.They catalyze an ordered bisubstrate(substrate and coenzyme)

两种泥鳅不同核质关系下EST和MDH同工酶基因表达的研究

采用聚丙烯酰胺凝胶不连续系统电泳方法分析了泥鳅和大鳞副泥鳅不同交配组合、不同核质关系下，胚胎发育阶段（０～１４５ ｈ） 酯酶（ＥＳＴ） 和苹果酸脱氢酶（ＭＤＨ） 同工酶的分化表达谱式。ＥＳＴ 同工酶随细胞质、细胞核不同及胚胎发育各个时期具有不同的个体发育谱式。以泥鳅卵子为细胞质环境的Ⅰ、Ⅱ两组，杂交组（ Ⅱ） 的ＥＳＴ同工酶基因在受精后３ ｍｉｎ 至原肠中期，雄核基因参与表达与调控。以大鳞副泥鳅为细胞质环境的Ⅲ、Ⅳ两组，各酶均以细胞质调控为主。ＭＤＨ 同工酶在各组合及各个发育阶段均未表现出实质性差异，发现ｍＭＤＨ 为特异性酶谱。并具体分析和讨论了不同核质关系下同工酶基因表达和调控的时空顺序。

黏类小麦细胞质雄性不育相关基因cMDH的克隆与表达分析

为深入研究黏类小麦雄性不育的分子遗传机制,利用抑制差减杂交技术构建了黏类小麦育性相关基因的二核期SSH文库。经文库筛选后,得到一个在可育文库中表达的与胞质苹果酸脱氢酶基因同源的EST序列。以该EST序列为信息探针,经电子克隆、RT-PCR、PCR克隆与序列分析,获得了小麦胞质苹果酸脱氢酶(cytosolic malate de-hydrogenases,cMDH)基因的cDNA与DNA序列,利用荧光定量PCR技术对该基因在不育株和可育株花药中的表达进行了分析,并比较了MDH在小麦不育株和可育株中的活性变化。结果表明,该基因的cDNA序列长1213bp,编码333个氨基酸;DNA序列长2908bp,含有7个外显子和6个内含子;该基因在不育株和可育株花药发育3个时期(单核、二核和三核)的表达均表现为先升后降的模式,而且该基因在不育株花药发育的二核期和三核期的表达相对于可育株被明显抑制;MDH在小麦不育株和可育株中的活性变化趋势与定量结果一致。推测该基因在花粉发育早期表达,它的下调表达可能影响了小麦雄蕊发育过程中的能量供应而导致雄性不育。

山药多糖对2型糖尿病大鼠HK SDH及MDH活性的影响

目的:探讨山药多糖对2型糖尿病大鼠糖代谢及关键酶己糖激酶(HK)、琥珀酸脱氢酶(SDH)及苹果酸脱氢酶(MDH)活性的影响,为山药多糖对2型糖尿病治疗的研究提供依据。方法:采用高热量饮食并结合小剂量链脲佐菌素(STZ)腹腔注射的方法制备实验性2型糖尿病大鼠模型,分成正常对照组、糖尿病模型组、山药多糖治疗组(100、70、40mg/kg3个剂量组)和阳性对照组(二甲双胍,100mg/kg)共6组,治疗4周;然后检测HK、SDH和MDH活性。结果:与糖尿病模型组比较,山药多糖显示明显的降血糖作用;山药多糖中、高剂量组的2型糖尿病大鼠的HK、SDH、MDH活性与模型组比较有极显著性差异(P<0.001);山药多糖低剂量组除HK的活性与模型组比较有显著差异外(P<0.05),SDH和MDH活性都无显著差异(P>0.05)。结论:山药多糖对2型糖尿病的治疗机制之一可能是山药多糖直接或间接地提高了糖代谢或关键酶的酶活性。

高氧恢复对低氧训练大鼠骨骼肌SDH和MDH活性的影响

探讨在进行低氧训练后,吸高浓度氧对大鼠骨骼肌琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)活性的影响。将雄性SD大鼠38只随机分为常氧运动组(A)、低氧运动组(B)、常氧运动高氧恢复组(C)、低氧运动高氧恢复组(D),分别进行常氧训练、低氧训练和高氧恢复。4周后测试大鼠骨骼肌SDH和MDH的活性。结果发现,经过4周的实验,高氧恢复干预组大鼠的SDH、MDH活性要比单纯运动或低氧运动组大鼠的活性高,且常氧运动高氧恢复组SDH、MDH活性均显著高于对应的常氧运动组;低氧运动高氧恢复组MDH活性显著高于低氧运动组;而低氧运动组SDH、MDH的活性要高于对应的常氧运动组,且低氧运动组MDH活性显著高于常氧运动组。结果表明,4周低氧训练比常氧训练在提高大鼠股四头肌SDH和MDH活性方面有一定优越性,而在低氧训练和常氧训练后进行高浓度氧恢复则更能显著地提高大鼠股四头肌SDH和MDH的活性。

草鱼胞质苹果酸脱氢酶(cMDH)的序列克隆及分析

以实验室构建的草鱼(Ctenopharyngodon idellus)肠道cDNA文库苹果酸脱氢酶(cMDH)EST序列为基础,采用末端快速扩增法(RACE),从健康雌性草鱼(Ctenopharyngodon idellus)肠道细胞RNA中获得1391 bp的草鱼肠道cMDH的cDNA序列(NCBI登录号:EU569765)。结果显示:该序列包含62 bp的5′非编码区(5′-UTR),330 bp的3′非编码区(3′-UTR),典型加尾信号AATAA位于polyA起点上游16 bp。开放阅读框ORF长999 bp,共编码333个氨基酸,预测蛋白等电点为6.67,大小为36 kDa。多序列比对显示草鱼胞质苹果酸脱氢酶具有典型的苹果酸脱氢酶功能区保守序列,该酶与斑马鱼cMDH相似度最高达到95.5%,与模式植物拟兰介的相似度为57.8%,其预测三级结构具有典型cMDH功能区。Southern杂交结果表明草鱼cMDH属于多拷贝基因家族。

异育银鲫、鲫和白鲫的肌浆蛋白及同工酶(Est、MDH)的电泳比较研究

本文采用聚丙烯酰胺凝胶垂直圆盘电泳,研究了异育银鲫、鲫和白鲫的肌浆蛋白,酯酶(Est)同工酶和苹果酸脱氢酶(MDH)同工酶。结果表明,同工酶类不仅具有种间特异性,而且具有器官特异性,为异育银鲫、鲫和白鲫的分类提供了生化依据。还表明异育银鲫和鲫的亲缘关系较近,而白鲫与异育银鲫和鲫的亲缘关系较远。

MDH和GDH活性与水稻杂种优势预测

本文从杂种优势预测所需的三个基本条件入手,研究了 MDH 和 GDH 活性作为水稻杂种优势预测生理参数的可能性。结果表明:MDH 活性在整个生育期中的变化呈现一定的规律性,并且杂种酶活性是由其双亲遗传性所决定的,同时,MDH 活性与多个生长性状及产量性状有密切的相关性。因此,利用 MDH 活性早期预测杂种后期生长和产量优势表现是一个有意义的生理参数。而 GDH 活性变化复杂,不适于作为优势预测的生理参数。

刚地弓形虫LDH-Like MDHcDNA的克隆和序列分析

目的获取弓形虫RH株速殖子苹果酸脱氢酶(MDH)的cDNA全长序列,并对推导翻译出的氨基酸(aa)序列进行分析。方法将NCBIGenBank中登录的弓形虫MDHEST序列进行比对、拼接和电子延伸,发现近5’末端的部分核酸序列缺失。基于分析,设计的上游、下游引物分别包含起始密码子ATG和终止密码子TGA(TGA→UGA),使经RTPCR扩增出的片段对应完整的CDS或者ORF。之后对MDHCDS推导翻译出的aa序列进行了保守功能域和同源性分析。结果本研究克隆的MDH的cDNA全长951bp,推导翻译出的蛋白质有316个aa组成,约35kDa,与预期大小接近。保守功能域分析表明,弓形虫MDH最接近类乳酸脱氢酶型(lactatedehydrogenaselike)MDH,其准确定名缩写为LDHlikeMDH,同时弓形虫MDH还具有其它脱氢酶类的特征。弓形虫MDH除与隐孢子虫和恶性疟原虫高度同源外,还与很多细菌等物种同源,但与人的同源性最低(22%)。弓形虫MDHcDNA序列在NCBIGenBank中登录号为AY650028,对应aa序列的登录号为AAT67462。结论本研究首次获得了弓形虫MDH基因的全长cDNA序列和对应的aa序列。MDH是三羧酸循环中的重要酶类,该酶活性的改变,将直接影响弓形虫的存活。弓形虫MDHaa序列与人MDH蛋白间的同源性很低,提示基于MDH蛋白作为药靶,经高通量技术筛选抗弓形虫药具有可行性。

中国对虾两个种群的F_1的LDH和MDH同功酶初步分析

采用不连续聚丙烯酰胺凝胶垂直平板电泳法，分析了黄、渤海南移和珠江口北移的中国对虾Ｐｅｎａｅｕｓｏｒｉｅｎｔａｌｉｓ及其杂交于一代的乳酸脱氢酶（ＬＤＨ）和苹果酸脱氨酶（ＭＤＨ）同工酶。结果表明：这两种同工酶具明显的组织特异性，均只在尾部肌肉表达。ＬＤＨ同工酶表现为紧密相邻的两条带。ＭＤＨ的表达谱式与其它物种类似，表现为趋向阳极的上清液型和趋向阴极的线粒体型。本研究分析的两种同工酶未见两种来源的中国对虾及其杂交子一代的差异。文中对此进行了讨论。

香蕉果实采后成熟过程中苹果酸脱氢酶(MDH)及苹果酸含量的变化

以巴西香蕉果实为材料,研究果实采后正常成熟过程中乙烯释放速率,苹果酸脱氢酶(MDH)活性,苹果酸、淀粉以及可溶性总糖含量的变化。结果表明:香蕉果实采后成熟过程中乙烯释放速率在采后10d开始增加,到采后14d达到高峰;MDH酶活性在采后10d迅速增强,到采后16d达到峰值;苹果酸含量在果实成熟早期上升,晚期下降,可溶性总糖含量逐渐增加,而淀粉含量持续下降。推测MDH通过改变香蕉品质而参与果实成熟。

籼型两用核不育系水稻培矮64S的育性表达──幼穗发育过程中的NAD^+-MDH和AP同工酶的变化

以籼型两用不育系水稻培矮645和常规稻粳籼89为材料，采用PAGE分析同工酶的方法，比较水稻幼穗发育过程中花粉母细胞形成期、减数分裂期和花粉完熟期NAD＋-MDH和AP同工酶的变化，以期了解不育系的育性转换与表达机理。结果表明，在1993年2月25日、3月10日和3月30日三期播种的水稻培矮645均表现不育，花粉不育度在99.7％以上，结实率在0.3％以下，1994年8月1日播种的培矮645表现可育，结实率在19％以上，而同期播种的常规稻粳籼89均能得到正常结实，结实率在68％以上。同时发现1993年培矮645的不育性在花粉完熟期与颖花及花药的NAD＋-MDH活性的显著降低关系较大。在花粉母细胞形成期一减数分裂期与颖花AP的活性及同工酶组成的变化关系较密切。1994年表现可育的培矮645与粳籼89相比不存在这种差异。由此可认为培矮645育性表达可能与花粉发育前期的脂肪代谢以及花粉发育后期的呼吸代谢有关。

牡丹PsMDH基因的克隆、表达及载体构建

以牡丹品种赵粉为试材,采用RT-PCR和RACE方法从雄蕊中获得了一个牡丹苹果酸脱氢酶基因cDNA全长,命名为PsMDH,GenBank登录号为HQ449567。其cDNA全长1 283 bp,包含80 bp的5'非编码区、203 bp的3'非编码区和一个长度为999 bp编码332个氨基酸的开放阅读框。序列比对和系统进化分析表明,PsMDH与毛果杨的亲缘关系最近,相似性达93.37%以上。相对荧光定量PCR分析表明,PsMDH在心皮中的表达量最高,在雄蕊中表达量最低。成功构建植物表达载体,为牡丹品种改良提供基础。

不同类型意大利蜜蜂MDHⅡ同工酶的研究

运用聚丙烯酰胺凝胶等电聚焦技术(IEF-PAGE),对有代表性的王浆蜂蜜双高产的“浙农大1号”意蜂品种(浙意,Ea)和中国本地意蜂(本意,Eb)、美国意蜂(美意,Em)、澳大利亚意蜂(澳意,Eo)、意大利意蜂(原意,Ee)等5个意大利蜂(ApismelliferaLigustica)品种试飞前的幼龄工蜂进行了苹果酸脱氢酶(MDH)同工酶的基因型、基因型频率、基因频率、杂合度和纯合度的研究。由a、b、c3个等位基因编码的MDH图谱第二区带应有的a/a、a/b、a/c、b/b、b/c和c/c6种基因型,在Ea、Eb、Ee3种意蜂中全部出现;在Eo中出现a/b,a/c,b/c,c/c4种,在Em中出现a/c,b/c,c/c3种。联列表独立性检验结果,5个意蜂品种间的基因型频率、基因频率、杂合度、纯合度存在显著差异。