鱼类乳酸脱氢酶同工酶聚丙烯酰胺凝胶电泳技术研究

本文对用聚丙烯酰胺凝胶电泳（ＰＡＧＥ）分离鱼类乳酸脱氢酶（ＬＤＨ）同工酶的技术进行了研究。结果表明：该方法优于淀粉凝胶电泳及琼脂糖凝胶电泳分析鱼类ＬＤＨ同工酶,具有较高的分辩率。     

Lactate dehydrogenase in the cyanobacterium Microcystis PCC7806

Abstract The cyanobacterium Microcystis PCC7806 was found to possess an NAD-dependent lactate dehydrogenase (EC 1.1.1.27) which catalyzes the reduction of pyruvate to l-lactate. The enzyme required fructose 1,6-bisphosphate for activity and displayed positive cooperativity towards pyruvate. Lactate was not formed during fermentation by cell suspensions, possibly due to low intracellular concentrations of fructose 1,6-bisphosphate and/or pyruvate.     

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     

Trypanosoma cruzi: Lactate dehydrogenase isoenzymes and infections in mice

Total plasma LDH isoenzyme (EC 1.1.1.27) levels increased significantly over the normal level in mice infected with strains of Trypanosoma cruzi from three different geographic locations, but some strain differences were observed. The most rapid increase was exhibited by the blood-induced Tulahuen strain, but this strain, unlike the House 510 or House 11, did not elicit an increase during the early period of infection. Overall increases in LDH-1 and LDH-2, heart isoenzymes, were most marked in vector-derived House 510 infections, but, as in the Tulahuen strain, a considerable increase was also observed in blood-induced infections. The House 510 strain also elicited significant increases in LDH-4; these were particularly high during the early period of the blood-induced infection. By contrast, the vector-derived Tulahuen strain elicited a higher increase in LDH-4 during the early period than the House 510 or House 11 strains. Comparable similarites and differences were also observed in regard to LDH-3, 5, and “X.” The most marked isoenzyme increases were those of “LDH-X” exhibited by the blood-induced House 510 and vector-derived Tulahuen strains.Parallel histopathologic studies of liver, heart, and skeletal muscle disclosed significant pathology in all the infections. Animals with blood-induced Tulahuen strain infections characteristically showed extensive necrosis with marked multiplication of parasites throughout the liver, but little or no evident damage to the heart and skeleal muscle. Animals infected with House 510 and House 11 strains exhibited minimal pathology in the liver but severe damage to the heart and skeletal muscle. Increases in LDH-4 and LDH-5, isoenzymes which represent both liver and skeletal muscle, in blood-induced Tulahuen infections were attributed largely to liver damage, but in the House 510 and House 11 infections were related more to skeletal muscle.     

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.     

The host antigen phenomenon in experimental murine schistosomiasis: The transfer of 3-week old Schistosoma mansoni between two inbred strains of mice

A surgical technique for the transfer of 3-week old Schistosoma mansoni to the mesenteric veins of mice is described. Parasites grown in a donor C3H/StCrl strain survive on transfer to recipient C57BL/10J mice despite prior immunization with C3H/StCrl cells or skin grafts. The findings indicate that the ‘species-specific’ mouse host antigens associated with adult worms in xenogeneic transfers do not have an allogeneic basis in these two strains.     

Rat lactate dehydrogenase A and B subunit concentrations are not regulated by mRNA abundance in liver and heart

RNA was isolated from rat liver and heart tissues at various times up to 12 weeks after birth, and probed on slot blots with lactate dehydrogenase A and B cDNA probes. Although the relative abundances of LDH A in liver and LDH B in heart increased substantially in the 12 weeks after birth, mRNAs for both isoenzymes remained remarkably stable in both tissues over the same period. The implications of these observations for the regulation of constitutive gene expression are discussed.     

Determination of lactate dehydrogenase isoenzymes in single lymphocytes from normal and leukemia cell lines

This work demonstrates that our previously developed technique for single-erythrocyte analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be applied to study individual lymphocytes, with some modification in the cell lysing procedure. A tesla coil was shown to be capable of lysing the lymphocyte cells inside the capillary. The electromagnetic field induced by the tesla coil was believed to be responsible for breaking the cell membrane. The lactate dehydrogenase (LDH) isoenzyme activities and the relative ratios between different LDH isoenzymes were measured for normal lymphocytes as well as B-type and T-type acute lymphoblastic leukemia cells. Both the LDH activity and the isoenzyme ratios show large variations among individual cells. The former is expected due to variations in cell size. The latter implies that single-cell measurements are less useful than the average values over a cell population as markers for leukemia.     

中国林蛙乳酸脱氢酶多基因系统及基因间连锁关系的研究

（１）用聚丙烯酰胺凝胶电泳对山西产中国林蛙４个地理群的３３３只林蛙进行了分析,结果表明：中国林蛙的ＬＤＨ由ＬＤＨ－Ａ,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ３个基因决定。ＬＤＨ－Ａ是单态座位,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ均为多态座位,每个多态座位均有两个等位基因。ＬＤＨ－Ｂ与ＬＤＨ－Ｃ呈紧密连锁关系。认为ＬＤＨ－Ｃ是ＬＤＨ－Ｂ的重复产物。（２）热稳定性、尿素处理稳定性及组织特异性研究表明：ＬＤＨ对温度和尿素处理稳定性顺序为Ａ４＞Ｂ’４＞Ｂ４＞Ｃ４。Ａ４在骨骼肌和肝脏等组织中活力最大,Ｂ４在心肌和卵巢中活力最强,ＬＤＨ－Ｃ主要在眼球和卵巢中表达。（３）Ｌｄｈ－ｂ和Ｌｄｈ－ｂ＇在不同地理群间呈差异分布,随着纬度的增高,Ｌｄｈ－ｂ在种群中的频率增大。     

Activity variation in alcohol dehydrogenase paralogs is associated with adaptation to cactus host use in cactophilic Drosophila

Drosophila mojavensis and Drosophila arizonae are species of cactophilic flies that share a recent duplication of the alcohol dehydrogenase (Adh) locus. One paralog (Adh-2) is expressed in adult tissues and the other (Adh-1) in larvae and ovaries. Enzyme activity measurements of the ADH-2 amino acid polymorphism in D. mojavensis suggest that the Fast allozyme allele has a higher activity on 2-propanol than 1-propanol. The Fast allele was found at highest frequency in populations that utilize hosts with high proportions of 2-propanol, while the Slow allele is most frequent in populations that utilize hosts with high proportions of 1-propanol. This suggests that selection for ADH-2 allozyme alleles with higher activity on the most abundant alcohols is occurring in each D. mojavensis population. In the other paralog, ADH-1, significant differences between D. mojavensis and D. arizonae are associated with a previously shown pattern of adaptive protein evolution in D. mojavensis. Examination of protein sequences showed that a large number of amino acid fixations between the paralogs have occurred in catalytic residues. These changes are potentially responsible for the significant difference in substrate specificity between the paralogs. Both functional and sequence variation within and between paralogs suggests that Adh has played an important role in the adaptation of D. mojavensis and D. arizonae to their cactophilic life.     

Genetic differentiation between two host “races” and two species of cleptoparasitic bees and between their two hosts

In this paper we test the following two hypotheses: (1) that apparently conspecific samples of the cleptoparasitic beeCoelioxys funeraria, differing markedly in size and reared from different host species, do indeed represent one panmictic population; (2) that bees that nest in holes in wood or twigs have higher levels of genetic variation than those nesting in the ground. Based upon 41 loci, the genetic differences between the two samples ofC. funeraria could be explained entirely in terms of sampling error. In contrast, the sympatricC. moesta showed 16 fixed allelic differences from theC. funeraria samples. Similarly, the two hosts ofC. funeraria, Megachile relativa andM. inermis, had 21 fixed allelic differences between them out of 42 presumptive gene loci. Heterozygosities among the wood-nesting bees were not particularly high for Hymenoptera, ranging from 0.045 to 0.054. Comparisons of heterozygosity estimates among bees remain ambiguous as to whether soil nesting confers sufficient environmental buffering effects to reduce possible advantages of heterosis in ground-nesting species.     

Glutamine synthetase and glutamate dehydrogenase in cadmium-stressed triticale seedlings

The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO ₃ ⁻ as the nitrogen source, with the addition of 0.5 mM CdCl₂. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS₁) and chloroplastic (GS₂). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions, result from dramatic decrease in GS₂ activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS₁) activity by approx. 96 %. Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic specificity.     

Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae

The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

Enzyme Activities in Relation to pH and Lactate in Postmortem Brain in Alzheimer-Type and Other Dementias

Phosphate-activated glutaminase, glutamic acid decarboxylase, pyruvate dehydrogenase, succinic dehydrogenase, pH, and lactate were measured in frontal cortex and caudate nucleus of postmortem brains from cases of Alzheimer-type dementia (ATD), Down's syndrome, Huntington's disease, and one case of Pick's disease, as well as from sudden death and agonal controls. Lactate levels were higher and pH, phosphate-activated glutaminase, and glutamic acid decarboxylase levels were lower in the agonal controls than in the sudden death controls. Phosphate-activated glutaminase and glutamic acid decarboxylase were correlated with tissue pH and lactate, and also were reduced by in vitro acidification, suggesting that the low activities of these enzymes in agonal controls were related to decreased pH consequent upon lactate accumulation. Compared with control tissues at the same pH, phosphate-activated glutaminase and glutamic acid decarboxylase were unaltered in ATD and Down's frontal cortex and reduced in Huntington's caudate nucleus, and glutamic acid decarboxylase was reduced in Huntington's frontal cortex. These data suggest that GABAergic neurons are not affected in ATD and confirm the GABAergic defect in Huntington's disease. Pyruvate dehydrogenase and succinic dehydrogenase activities were the same in agonal controls and sudden death controls and were unaffected by acid pH and lactate in vitro, and pyruvate dehydrogenase was not correlated with pH or lactate. Reduced pyruvate dehydrogenase in frontal cortex of individual ATD, Down's, and Pick's cases, and in the caudate nucleus of Huntington's and Down's cases, was accompanied by gliosis/neuron loss. We conclude that decreased pyruvate dehydrogenase reflects neuronal loss.     

Parasites on an intertidal Corophium-bed: factors determining the phenology of microphallid trematodes in the intermediate host populations of the mud-snail Hydrobia ulvae and the amphipod Corophium volutator

The phenology of microphallid trematodes within their intermediate hostpopulations has been studied on an intertidal mud flat. The parasites usethe mud snail Hydrobia ulvae and the infaunal amphipod Corophium volutatoras first and secondary intermediate host, respectively. Migratory shorebirdsact as final hosts. Our results show a general trend of decline in thedensity of infected intermediate hosts during both spring and autumn, whichcould mainly be ascribed to shorebird predation. During summer the densityof both infected snails and infected amphipods increased considerably, witha culmination in June within the snail population (1000 infectedm-² and in August within the amphipod population (40 000infected m-². This time lag in parasite occurrence could berelated to (1) the development time of larval trematodes within the snails,(2) higher ambient temperatures in late summer increasing parasitetransmission between snails and amphipods during this period, and (3) ageneral increase in the Corophium population during late summer. Fromsamples collected between 1990 and 1995 it is shown that microphallidtrematodes occasionally may give rise to mass mortality in the amphipodpopulation. The prerequisites for such an event are a high parasiteprevalence within the first intermediate host population and unusually highambient temperatures, facilitating parasite transmission to the secondaryintermediate host, C. volutator.     

A study of lactate dehydrogenase levels and turnover rates during postnatal development in the rat

Specific radioimmunoassays for lactate dehydrogenase A and B subunits have been employed to quantify cellular contents of these proteins more precisely than hitherto possible and to monitor changes during postnatal development. Liver, skeletal muscle, heart muscle and kidney cortex all demonstrated alterations in cellular levels of lactate dehydrogenase subunits over the first 56 days of life, the particular pattern being specific to each tissue. Studies on the turnover of lactate dehydrogenase in vivo and in vitro indicated that the developmental changes in total lactate dehydrogenase content in liver and kidney were regulated at some point(s) during both the biosynthesis and the degradation of the proteins.     

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.