大熊猫乳酸脱氢酶同工酶H_4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５＇－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出了乳酸脱氢酶同工酶Ｈ４．纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＰＡＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分子量为３６０００,等电点为５．４５．经测定大熊猫ＬＤＨ－Ｈ亚基Ｎ端被封闭,Ｃ端氨基酸残基经测定为Ｌｅｕ．氨基酸组成分析表明每个亚基含有５个Ｃｙｓ,９个Ｍｅｔ．     

大熊猫乳酸脱氢酶同工酶M_4的纯化与某些性质的研究

利用8-(6-氨已基)-氨基-5’-AMP Sepharose亲和层析和DEAE-Sephadex A50离子交换层析纯化了大熊猫LDH-M_4。纯化的大熊猫LDH-M_4呈针状晶体,比活为412单位/毫克。聚丙烯酰胺凝胶电泳鉴定为一条区带。SDS凝胶电泳测得其亚基分子量为35,900;等电聚焦电泳测得其等电点为8.05。经氨基酸组成分析,得出每个大熊猫LDH-M亚基含有5个Cys,26个Lys和10个Arg。其N-末端氨基酸残基可能为封闭的,C末端氨基酸残基经测定为Phe。大熊猫LDH-M_4的TPCK-胰蛋白酶水解物在纤维素膜指纹图谱上呈现35个肽斑,与已知序列的猪LDH-M_4的指纹图谱相比较,多数肽斑位置相同,约有10个肽斑在两者指纹图谱上有差异。     

原鸡不同组织乳酸脱氢酶同工酶研究

本文对中国原鸡的血浆,心脏肌肉和肝脏等不同组织,用薄层等电聚焦电泳进行乳酸脱氢酶同工酶试验比较。结果表明,原鸡不同组织在ｐＨ３．５－９．５时,电泳图和相应的扫描图有显著差异。     

细菌乳酸脱氢酶的纯化及其性质研究

从乳酸杆菌发酵液经过两次柱层析,可以得到纯度较高的乳酸脱氢酶,酶的比活力高达６７８．９ｕ／ｍｇ,纯度提高８５．７倍。酶的热稳定性好,ｐＨ稳定范围较宽,在临床上可用于雨氨酸氨基较移酶活力的测定。     

东方田鼠乳酸脱氢酶同工酶的研究

目的研究乳酸脱氢同工酶在3种东方田鼠不同器官的分布.方法以大鼠和小鼠作对照,用聚丙烯酰胺凝胶电泳对3个不同地区的东方田鼠(Microtusfortis)血清、红细胞、肝脏、肾脏、肺中乳酸脱氢酶(LDH)同工酶进行分析.结果东方田鼠血浆中只有LDH5,血清中湖南洞庭湖地区、宁夏青铜峡地区的东方田鼠也只有LDH5,而东北地区的东方田鼠则含有LDH1和LDH5,3个地区的东方田鼠肾脏和肺中都有5种LDH同工酶,肝脏中除东北地区的东方田鼠含5种LDH同工酶外,其余两种东方田鼠均以LDH5为主(LDH5占95%以上).结论东北地区东方田鼠乳酸脱氢酶同工酶谱与其他两地的东方田鼠不相同.     

大熊猫乳酸脱氢酶同工酶H4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层分析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出乳酸脱氢酶同工酶Ｈ４,纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＡＰＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分了量为３６０００,等电点为５．４５。经测定大熊猫ＬＤＨ－Ｈ亚基Ｎ端被封闭,Ｃ端氨基酸残基经测定为Ｌｅｕ。氨基酸组成分析表明     

树鼩组织乳酸脱氢酶同工酶的研究

对云南树鼩(Tupaia belangeri chinensis)心肌、肺、颌下腺、脾脏、肾脏、肾上腺、骨骼肌和大脑8种组织乳酸脱氢酶(LDH)同工酶进行琼脂糖凝胶电泳分析。结果表明,8种组织均呈现为LDH-M型和LDH-H型两种亚基组合而成的5种不同分子形式同工酶。采用分光光度比色定量法测得5种同工酶组分的相对酶活性。对其H亚基和M亚基百分率以及H/M亚基比率进行了统计分析。并就不同酶谱特征与基因表达状况进行了分析讨论。     

华南兔组织乳酸脱氢酶同工酶的研究

本文以聚丙烯酰胺凝胶电泳法分析华南兔(Lepus sinensis sinensis)11种组织乳酸脱氢酶同工酶的分布待征。分析结果:骨骼肌和肝组织5条同工酶带俱全;脑、肺、卵巢和盲肠等组织各含有4条谱带(LDH-1,-2,-3和-4);胃和肾组织含有3条谱带(LDH-1,-2,和-3);眼晶状体和睾丸组织也含有3条谱带,但前者是LDH-3,-4和-5,后者是LDH-1,-2和-5;谱带最少的是心肌组织,只有2条(LDH-1和-2)。此外,还对各组织中的亚基活性分布及电泳图谱特征进行了分析。     

贵州北部四种两栖类乳酸脱氢酶同工酶的比较研究

本文采用聚丙烯酰胺凝胶电泳方法,研究了贵州北部的四种两栖类动物:细痣疣螈、棘指角蟾、绿臭蛙和小弧斑姬蛙的心、肝、肾等组织的乳酸脱氢酶(LDH)同工酶,发现LDH具有明显的种族特异性和组织特异性,不同种的动物和同种动物的不同组织都有各自的LDH同工酶谱型,可以用它们作为识别物种的附加指标。本文初步探讨了生化分类学与系统分类学的关系。     

Isozyme Patterns of Lactate Dehydrogenase from Tissues of Mink and Arctic Fox during Postnatal Development

Isozymes of lactate dehydrogenase extracted from heart, kidney, and liver of mink (Mustela vison Briss.) and Arctic fox (Alopex lagopus L.) during postnatal development were separated by agarose gel electrophoresis. Tissue-specific isozyme pattern of lactate dehydrogenase can be revealed at the age of one month, while the definitive pattern is formed at the age of four months. The isozyme patterns of lactate dehydrogenase in the studied tissues of mink and Arctic fox share the properties specific for animal species of various ecogenesis.     

Effects of N-Terminal Deletion Mutation on Rabbit Muscle Lactate Dehydrogenase

Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.     

Modulation of Lactate Dehydrogenase Isozymes by Modified Base Queuine

The modified base queuine is a nutrient factor for lower and higher eukaryotes except yeast. It is synthesized in eubacteria and inserted into the wobble position of specific tRNAs (tRNA_GUN) in exchange of guanine at position 34. The tRNAs of Q family are completely modified in terminally differentiated somatic cells. However, mainly free queuine is present in embryonic and fast proliferating cells, tRNA remains Q deficient. Lactate dehydrogenase (LDH) A mRNA and LDH A protein is known to increase when cells are grown in hypoxic conditions. In the present study, the level of LDH isozymes is analyzed in different tissues of normal and cancerous (DLA) mice and the effect of queuine treatment on LDH isozyme is observed. LDH A isozyme is shown to increase in serum and liver of DLA mice. The level and activity of LDH A decreases on queuine treatment. In skeletal muscle and heart, LDH A isozyme decreases while LDH B increases in DLA mice. Queuine administration leads to change back towards normal. In case of brain, LDH A increases but LDH B decreases in DLA mice. Queuine treatment leads to decrease in A4 anaerobic isozymes of LDH. The results suggest that queuine suppresses anaerobic glycolytic pathway, which leads to tumor suppression of DLA mice.     

Kinetic Formulations for the Reduction of Ketomalonate by Lactate Dehydrogenase

Abstract

Initial rate kinetic studies of lactate dehydrogenase with ketomalonate and NADH as substrates suggest that this enzymatic system is adapted to a rapid equilibrium ordered bi-bi ternary complex mechanism. The application of the reaction product inhibition method reveals the existence of the enzyme-NADH-hydroxy-malonate and enzyme-NAD⁺-ketomalonate abortive complexes. This kinetic behaviour is confirmed by the differential inhibition induced by several alternate products on the pyruvate-lactate dehydrogenase-NADH and ketomalonate-lactate dehydrogenase-NADH systems.     

神经tau聚集物诱导乳酸脱氢酶的失活与构象变化

在37℃,pH 7.2条件下,人类神经tau经过保温形成自聚集物,从而丧失对微管蛋白组装的功能.进一步的实验表明,天然tau具有促进乳酸脱氢酶活性的作用,而tau聚集物却诱导乳酸脱氢酶活性的降低.     

枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究

枯草芽孢杆菌ＢＳ１２的乳酸脱氢酶经硫酸铵分级沉淀、ＣＭ－纤维素、ＤＥＡＥ－纤维素离子交换柱层析、ＳｅｐｈａｄｅｘＧ—２００柱层析,得到了凝胶电脉均一的样品。用ＳＤＳ—ＰＡＧＥ测得其亚基分子量为２８０００Ｄａ。酶反应的最适ｐＨ为７．０,最适温度为３５℃。     

Functional Properties of Lactate Dehydrogenase from Dunaliella salina and Its Role in Glycerol Synthesis

The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) V _max of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle.     

Alteration of Coenzyme Specificity of Lactate Dehydrogenase from Thermus thermophilus by Introducing the Loop Region of NADP(H)-Dependent Malate Dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k _cat?K _m ^coenzyme was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.     

恶性疟原虫乳酸脱氢酶基因的克隆表达及表达产物免疫原性的鉴定

目的：克隆表达恶性疟原虫（Hasmodium falciparum,p．f）海南株（FCC1／HN）乳酸脱氢酶（LDH）,并对其免疫原性进行鉴定．为制备抗LDH抗体用于胶体金法快速检测疟原虫奠定基础。方法：应用PCR技术对恶性疟原虫乳酸脱氢酶（LDHpf）基因进行特异性扩增,将扩增产物克隆入表达载体pET-32a（＋）,重组表达载体经鉴定后诱导表达;重组LDHpf（rLDHpf）经纯化后,免疫家兔制备兔抗rLDHpf免疫血清,间接免疫荧光和Western印迹鉴定表达产物的免疫原性。结果：构建了pET-32a／LDHpf重组表达载体,测序后同源性分析显示p．f不同株间LDH氨基酸序列同源性大于98％,不同种间同源性也在90％以上;间接免疫荧光和Western印迹分析显示rLDHpf具有较好的免疫原性。结论：LDHpf基因高度保守;rLDHpf得到高效表达并具有良好的免疫原性。