Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate.

Soluble lactate dehydrogenase (EC 1.1.1.27) extracted from brain, skeletal and cardiac muscle and liver of rats, and purified isoenzymes LDH-1 and LDH-5, were incubated with sodium deoxycholate. Deoxycholate almost totally inactivated isoenzyme LDH-5 (A4), whereas it left isoenzyme LDH-1 (B4) unaffected. Tissue lactate dehydrogenase was inactivated to different degrees depending on the origin of the enzyme. Electrophoretic isoenzyme studies of tissue lactate dehydrogenase showed the loss of activity to be quantitatively related to the overall percentage of subunit A distributed among the homotetramer LDH-5 and the heterotetramers LDH-2, LDH-3 and LDH-4. It was concluded that subunit A of lactate dehydrogenase interacts selectively with deoxycholate, irrespective of its association with subunit B. Distinct changes in electrophoretic mobilities of deoxycholate-treated isoenzymes strongly indicated an indiscriminate binding of deoxycholate by all LDH isoenzymes, probably through hydrophobic interactions. The results suggest that the inactivation of the enzyme is non-competitive, but the basis of the selectivity of deoxycholate towards subunit A is not known at present.     

Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein.     

Glycolytic enzymes in polyamine-treated bovine retina

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.     

Separation of rat lactate dehydrogenase isoenzyme C4 from other isoenzymes by affinity and ion-exchange chromatography.

Lactate dehydrogenase C, an isoenzyme composed of C polypeptide subunits and found only in mature testes and spermatozoa, differs kinetically, chemically and immunologically from the five common isoenzymes of lactate dehydrogenase, each of which is a tetramer of A and/or B subunits. In the rat lactate dehydrogenase C exists in two molecular forms, isoenzymes C4 and A1C3. In addition to these two forms of lactate dehydrogenase C, rat testicular homogenate contains all the five isoenzymes of A and B type. Purification of isoenzyme C4 requires its separation from the other six isoenzymes, of which isoenzymes A1C3 and A3B1 are the most difficult ones to separate. In the present study isoenzyme A3B1, along with other enzymes, was separated from isoenzyme C4 by AMP-Sepharose chromatography by using a gradient of increasing concentration of NAD+-pyruvate adduct. In the next step, isoenzyme A1C3 was separated from isoenzyme C4 by DEAD-cellulose chromatography, resulting in a pure lactate dehydrogenase isoenzyme C4 preparation.     

The physiological role of the isoenzymes of lactate dehydrogenase in potatoes

Four of the five isoenzymes of lactate dehydrogenase present in potato tubers have been isolated and their kinetic properties examined. The pyruvate-reductase activity of isoenzyme-4 is greatly reduced at low pH, the affinity for both pyruvate and NADH is reduced and ATP has a stronger inhibitory effect. If the design properties of an enzyme dictate a high affinity for substrates, then the Km values for lactate, glyoxylate and NAD are consistent with an oxidative role for isoenzyme-4. The same considerations do not permit a conclusion about the physiological role of isoenzymes-1 to-3. However, an overview of the kinetic properties of these isoenzymes indicates that isoenzyme-1 is best adapted for the role of pyruvate reductase. Consideration of the relationships between kinetic constants and electrophoretic mobilities of the isoenzymes, leads us to predict that isoenzyme-5 is well adapted for a role in the oxidation of lactate or glyoxylate. The lactate dehydrogenase of potato leaves appears to consist prodominantly of an isoenzyme with the same mobility as isoenzyme-2 of the tubers and the two isoenzymes are probably identical. The kinetic properties of this isoenzyme are consistent with roles in either oxidation or reduction.Abbreviation Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Ultrathin-layer microelectrophoretic determination of lactate dehydrogenase isoenzymes in corneal and conjunctival epithelium of the cow

In order to evaluate the impact of tissue oxygenation on the distribution pattern of lactate dehydrogenase isoenzymes, activities of the isoenzymes were measured in microdissected samples of bovine tissue. A highly sensitive ultrathin-layer electrophoretic technique was used to determine the distribution pattern of lactate dehydrogenase isoenzymes in basal, intermediate and superficial layers of the epithelium of central and peripheral cornea and in the epithelium of the bulbar conjunctiva. Measurements revealed almost homogeneous intraepithelial distribution patterns of lactate dehydrogenase isoenzymes in both tissues. In the cornea the lactate dehydrogenase isoenzymes 4 and 5, which are regarded to be specialized for anaerobic glucose metabolism, were found to predominate. In the well-oxygenated conjunctival epithelium most of the activity could be ascribed to the lactate dehydrogenase isoenzyme 3. In contrast to the isoenzymatic activities, total activity of lactate dehydrogenase was inhomogeneously distributed; maximum activities were found in the basal layer of corneal epithelium and in the intermediate layer of conjunctival epithelium. The results indicate that oxygen supply is relevant rather for the intraepithelial distribution of total enzyme activity than for the expression of lactate dehydrogenase isoenzymes.Parts of this study were presented as an inaugural dissertation to the Medical Faculty of the University of Basel by K. Krieger     

Changes in the lactate dehydrogenase isoenzyme spectrum during T-lymphocyte differentiation

The pattern of lactate dehydrogenase isoenzyme spectrum changes on different stages of T-lymphocyte differentiation was studied An enriched population of stem cells has LDH-5, 4 and 3 isoenzymes, and much less LDH-2 activity. The isoenzyme pattern of thymic cell precursors consists of LDH-5, 4, 3 and 2. All the five LDH isoenzymes were found in cortical thymocytes. Medullary thymocytes reveal LDH-5, 4 and 3 isoenzymes. T-lymphocytes of peripheral lymphoid organs contain mainly LDH-5 and in a lesser degree LDH-4 activity.     

Factors affecting the lactate dehydrogenase isoenzyme pattern of cultured kidney-cortex cells

1. The lactate dehydrogenase isoenzyme pattern of cultured calf kidney-cortex cells was correlated to growth phase, changes in oxygen supply, mean generation time and changes in nutritional supply. 2. During culture of free cells and intact explants the lactate dehydrogenase isoenzyme pattern changed towards a dominance of isoenzymes containing the M subunit. 3. Of the shift in monomer proportion, 58% occurred during the lag phase and 42% during the initial part of the exponential growth phase. During the stationary phase the shift in monomer proportion reversed slightly. It was possible to relate the observed shift in monomer proportion to the glycolytic rate. 4. Factors that depressed glycolysis decreased the shift in monomer proportion. Oxygen was found to limit the decrease in the H subunit/M subunit ratio caused by anaerobic culture in vitro. 5. The results obtained support the view that the altered lactate dehydrogenase isoenzyme pattern of urine in renal ischaemia may be explained by anaerobic changes in the lactate dehydrogenase isoenzyme pattern of cortical tubule cells.     

The excretion of lactate dehydrogenase in human urine after ingestion of aspirin

1. Cells present in normal human urine contain 5-10% of the total lactate dehydrogenase excreted. The enzyme released from these cells by ultrasonication contained a distribution of isoenzymes similar to that found in the bulk of the urine and it is suggested that these cells are the main source of urinary lactate dehydrogenase. 2. Cells were thoroughly washed before examination so it is unlikely that the enzyme found in urinary sediment was simply adsorbed. In addition, full recoveries of added lactate dehydrogenase isoenzymes LDH(1) and LDH(5) showed that adsorption did not occur. 3. Most of the cells in normal urine are of the non-squamous epithelial type and their excretion is greatly increased after the ingestion by the subject of 3g. of aspirin. The possible origin of these non-squamous cells from the kidney is discussed. 4. Starch-block electrophoresis and relative activity measurements of lactate dehydrogenase excreted after the subject had taken aspirin show that the enzymes present in urine and cells are very similar, confirming the conclusion reached above (point 1). They have slightly more M subunits than the normal, shown particularly as an increase in isoenzyme LDH(2). The isoenzyme pattern is like that of the kidney medulla and the possible reasons for this are discussed in terms of the concentration of salicylic acid in various parts of the kidney. 5. The results confirm the previous suggestion that the kidney is the main source of urinary lactate dehydrogenase.     

Lactate dehydrogenase isoenzymes of human semen

1. A lactate dehydrogenase isoenzyme present in human spermatozoa and semen was isolated and characterized biochemically in term of its pH for optimum activity and by means of K(m) values for lactate, NAD(+) and NAD analogues. The results were compared with those obtained with the human heart-type and the liver-type lactate dehydrogenase isoenzymes. 2. The enzyme was characterized by its resistance to digestion with different proteolytic enzymes. The time for 50% digestion in terms of residual dehydrogenase activity was compared with times obtained for the H(4)- and M(4)-types.     

Changes in the lactate dehydrogenase isoenzyme pattern during differentiation of rabbit bone-marrow erythroid cells.

1. Differentiation and maturation of rabbit bone-marrow erythroid cells was accompanied by a 15-fold decrease in lactate dehydrogenase activity from approx. 0.1pmol of NADH utilized/min per cell in basophilic cells to 0.007 pmol of NADH/min per cell in reticulocytes. 2. In early cells, cell division takes place with a corresponding decrease in cell volume, but the concentration of lactate dehydrogenase remains almost constant. 3. When cell division ceases, qualitative as well as quantitative changes in the lactate dehydrogenase isoenzyme pattern become apparent and reticulocytes were found to contain almost exclusively the H4 isoenzyme, whereas early erythroblasts contained also the M4 and hybrid isoenzymes. 4. Extracts from a lysosome-enriched subcellular fraction of bone-marrow erythroid cells specifically degraded the M4 isoenzyme in vitro, but the H4 form was stable. It is suggested that lysosomal enzymes are involved in bringing about the observed changes in lactate dehydrogenase isoenzyme patterns in vivo.     

Isoenzyme pattern and subcellular localisation of enzymes involved in fumaric acid accumulation by Rhizopus oryzae

Summary Electrophoretic studies of fumarase and nicotine adenine dinucleotide (NAD)-malate dehydrogenase were carried out in the fumaric acid-accumulating fungus Rhizopus oryzae. The analyses revealed two fumarase isoenzymes, one localised solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytosolic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested that de novo protein synthesis is required for increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation. Three distinct isoenzymes of NAD-malate dehydrogenase could be detected in R. oryzae. No changes were observed in the isoenzyme pattern of malate dehydrogenase during fumaric acid production.     

LDH isoenzymes in the axial muscle of the sharks Galeus melastomus and Etmopterus spinax

The lactate dehydrogenase isoenzyme patterns have been studied in the axial muscles of the sharks Etmopterus and Galeus. Samples from red, intermediate and white muscle fibres were run separately on a polyacrylamide slab-gel. Both sharks have three isoenzymes; all three are present in the red and intermediate fibres, while the white fibres contain only the two slowest-moving isoenzymes. The red fibres of both sharks contain most of the fastest-moving isoenzyme.
The isoenzymes have a high tolerance towards urea; the slow moving isoenzyme is inhibited at about 2 m urea, the next isoenzyme at 4-6 M urea, and some activity of the fast-moving isoenzyme is still present at 10 M urea in the incubation medium. The LDH distribution in the fibre types is studied by histochemistry on frozen sections.     

Lactate dehydrogenase isoenzyme patterns as a method of pregnancy detection in cattle

The objective of this study was to evaluate serum lactate dehydrogenase isoenzyme patterns in open and pregnant Holstein and Hereford cows as a method of detecting pregnancy. Serum samples were collected from 26 Holstein and 13 Hereford cows and lactate dehydrogenase isoenzyme patterns were examined by electrophoresis and quantitated by scanning densitometry. Lactate dehydrogenase isoenzyme(4) and LDH(5) were found in higher concentration (P 相似文献   

中国林蛙早期胚胎发育过程同工酶的研究

本文采用聚丙烯酰胺凝胶电泳方法,对中国林蛙（Rana chensinensis)早期胚胎发育过程中（受精后0－512h)乳酸脱氢酶（LDH）同工酶和酯酶（EST）同工酶进行了研究,结果表明：1．LDH同工酶在受精卵及早期胚胎发育过程中一直存在,并且在胚胎发育的不同时期差异显著,LDH1同工酶在胚胎发育的各个阶段都占绝对优势,而DH5活性从尾芽期开始增强,LDH2,LDH3,LDH4 3种同工酶的活性较;低同时也发现LDH1,LDH3,LDH4同工酶各亚带也发育阶段的特异性,2．EST同工酶在开口期才开始出现。     

外源钙对根际低氧胁迫下黄瓜幼苗ADH、LDH活性和同工酶的影响

以‘新泰密刺’黄瓜为材料,采用营养液栽培,外源使用Ca2+、钙离子通道抑制剂La3+与钙调素拮抗剂三氟拉嗪(TFP),研究了钙对根际低氧胁迫下黄瓜幼苗根系ADH、LDH活性和同工酶的影响。结果表明,低氧胁迫诱导产生了新的ADH和LDH同工酶条带。低氧胁迫下,ADH、LDH同工酶丰度和活性显著高于对照;外源增施Ca2+有利于Ca2+信号的形成和逆境信号的传递,营养液添加CaCl2缓解了低氧胁迫对黄瓜植株的伤害,ADH、LDH同工酶丰度和活性接近对照水平;La3+抑制Ca2+的吸收和体内运输,营养液添加LaCl3显著抑制了ADH和LDH同工酶丰度和酶活性,黄瓜幼苗植株生长受到抑制,生物量显著低于低氧处理,表明La3+加重了低氧胁迫对黄瓜幼苗植株的伤害;TFP抑制了低氧逆境胁迫信号的传递,营养液添加TFP抑制了ADH和LDH同工酶丰度和酶活性,ADH和LDH同工酶丰度和酶活性显著低于低氧处理,黄瓜幼苗植株生长受到抑制,黄瓜植株的低氧耐性降低。暗示外源Ca2+参与了低氧胁迫下黄瓜根系无氧呼吸代谢的调节,增强了Ca2+向植物体内的运输,缓解了低氧胁迫对黄瓜幼苗植株的伤害,增强了植物对低氧的耐性。     

The reactions of pyridoxal 5′-phosphate with the M4 and H4 isoenzymes of pig lactate dehydrogenase

The H₄ and M₄ isoenzymes of pig lactate dehydrogenase are both inactivated by reaction with pyridoxal 5′-phosphate. In the early stages, inactivation is largely reversible by the addition of lysine in excess, but may be made irreversible by reduction with borohydride. This indicates that modification of lysine residues probably causes the initial inactivation. Both isoenzymes also undergo a slower process of irreversible inactivation which becomes more evident with increasing concentrations of pyridoxal 5′-phosphate and higher temperature. Although coenzymes give only partial protection of enzyme activity, they nevertheless completely prevent irreversible inactivation. Neither pyruvate nor lactate alone gives any protection. With the M₄ isoenzyme, complete protection against inactivation by pyridoxal 5′-phosphate may be achieved in ternary complexes, but no conditions have been found for complete protection of the H₄ isoenzyme. In the course of irreversible inactivation of H₄ lactate dehydrogenase, complete loss of activity can be correlated with the loss of approximately two free thiol groups per subunit. Present findings with regard to the importance of temperature and reagent concentration in determining the outcome of the chemical modification appear to resolve earlier controversy.     

Lactate dehydrogenase from the northern krill Meganyctiphanes norvegica: comparison with LDH from the Antarctic krill Euphausia superba

Electrophoretic polymorphism of lactate dehydrogenase (LDH, EC 1.1.1.27) from abdominal muscle is reported in the northern krill Meganyctiphanes norvegica. In the population, from the Gullmarsfjord (west coast of Sweden), LDH was encoded for by two different Ldh-A* and -B* loci. The isoenzymes were named according to their electrophoretic mobilities. Ldh-A* locus was polymorphic. The allelic frequencies were a=0.99, a'=0.002, a"=0.004, a"'=0.004. The level of LDH polymorphism is low. Most individuals possess the same amount of two LDH homopolymers (LDH-A*(4) and LDH-B*(4)). The Meganyctiphanes norvegica LDH-A*(4) and LDH-B*(4) isoenzymes and the predominant LDH-A*(4) isoenzyme from Euphausia superba were purified to specific activities of 294, 306 and 464 micromol NADH min(-1) mg(-1), respectively. In both species the LDH isoenzymes were separated by chromatofocusing. All three isoenzymes are L-specific tetramers with molecular weight of approximately 160 kDa. Northern krill LDH-A*(4) has higher affinity for pyruvate and lactate and is more thermostable than LDH-B*(4). Both isoenzymes are inhibited significantly by high concentration of pyruvate but not lactate. Antarctic krill isoenzyme exhibits high substrate affinities, high NAD inhibition, high inhibition at 10 mM pyruvate, lack of lactate inhibition, and high heat stability and resembles northern krill LDH-A*(4) isoenzyme.     

The distribution of enzyme and isoenzyme activities between parenchymal and haematopoietic cells in the liver of the foetal guinea pig

The hepatocyte and haematopoietic cell contents of the liver of the foetal guinea pig were measured over the latter half of gestation. Hepatocytes represented about 30% of liver volume at mid-gestation and this increased to 70-80% by term; cell volume remained fairly constant until 5-7 days before term, then more than doubled. Haematopoietic cells represented about 5% of liver volume at mid-gestation and this progressively fell to <1% by term. At 75% of gestation hepatocytes and haematopoietic cells were prepared from perfused foetal livers by collagenase digestion. Enzyme activity of the hepatocyte was, without exception, similar to that of the whole liver. In general, enzyme activity in the haematopoietic cells was similar to that in erythrocytes, with relatively low values for aldolase, glycerol 3-phosphate dehydrogenase, phosphoglycerate mutase, enolase, lactate dehydrogenase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, isocitrate dehydrogenase, ;malic' enzyme, glutamate dehydrogenase and aspartate aminotransferase. The haematopoietic cell contribution to total enzyme activity in the foetal liver was usually much less than 10% and could thus not account for the major changes in hepatic enzyme activity over the latter half of gestation. Hepatocytes contained hexokinase isoenzymes I and III, aldolase isoenzymes A and B and pyruvate kinase isoenzymes 1, 2 and 4. The haematopoietic cells contained hexokinase isoenzyme I and two additional bands of activity with slightly greater mobility, aldolase isoenzyme A and pyruvate kinase isoenzymes 2 and 4.     

Changes in cytosolic Ca2+ levels correspond to fluctuations of lactate levels in crosstalk of astrocyte-neuron cell lines

Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with CSD1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.