Expression of lactate dehydrogenase is induced during hypoxia via HIF-1 in the mud crab Scylla paramamosain.

Lactate dehydrogenase (LDH) is a key enzyme involved in anaerobic metabolism in most organisms. In the present study, we determined the structure and function of LDH sequence in Scylla paramamosain (SpLDH) by gene cloning, expression and RNA interference techniques in order to explore the genetic characteristics of LDH and its relationship with HIF-1 during hypoxia. The full-length cDNA was 1453 bp with an open reading frame (ORF) of 996 bp, and encoded a polypeptide of 332 amino acids. Homology analysis showed that the SpLDH gene is highly similar to arthropods. The SpLDH transcript increased after hypoxia in all tested tissues. The silencing of HIF-1 blocked the increase in LDH mRNA and activity, which were induced by hypoxia in gill and muscle tissues. Our results indicated that SpLDH expression was regulated transcriptionally by HIF-1.

Circadian rhythm of metabolic oscillation in suprachiasmatic nucleus depends on the mitochondrial oxidation state, reflected by cytochrome C oxidase and lactate dehydrogenase.

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at night. The rhythmic oscillation in the SCN is feedback controlled by the Clock/Bmal1 heterodimer binding to the E-box in target genes (e.g., Arg-vasopressin). Similar transcriptional regulation by Npas2/Bmal1 heterodimer formation operates in the brain, which is dependent on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state and glycolysis levels, we measured glucose, lactate dehydrogenase (LDH), LDH mRNA, and cytochrome C oxidase, energy-producing biochemical materials from mitochondria/cytosol, in rats kept under a light-dark cycle. Mitochondrial cytochrome C oxidase activity, measured by the changes in absorption at 550 nm, was higher during the light period than during the dark period. Glucose concentration was higher during the light period. In contrast, LDH and its coding mRNA were higher during the dark period. Mitochondrial aggregation, which is reflected by mitochondrial membrane potential, indexed by JC-1 fluorescence, was higher during the light period. The results indicate that the glycolysis energy pathway in the SCN, which exhits higher metabolic activity during the day than at night, might be involved in the generation of circadian rhythm.

[Bioinformatics analysis for the structure and function of lactate dehydrogenase from Schistosoma japonicum].

OBJECTIVE: To predict the structure and function of SjLDH using bioinformatics method. METHODS: By online analysis at bioinformatics websites such as NCBI (http://www.ncbi.nlm.nih.gov/) and Expasy (http://cn.expasy. org/), and employing software packages such as Vector NTI suite and PCgene to do multi-sequence homological alignment, phylogenetic analysis, secondary structure and topological prediction, homology modeling of tertiary structure, antigenic epitope analysis, etc. RESULTS: Same conservative sites and key catalytic sites existed among SjLDH and LDHs from other species. Similarity of SjLDH compared to CsLDH, TvLDH and HsLDH was 75%, 17%, 58%-60% respectively. Phylogenetic analysis demonstrated that the evolution relation between SjLDH and DmLDH was closer than the relation between SjLDH and CeLDH, the relationship between SjLDH and HsLDH-B, -C was closer than HsLDH-A. Three transmembrane regions were found, the region of 98aa-106aa in three hydrophilic regions located outside of membrane was inferred as the major antigen epitope. This antigen epitope had significant difference with LDHs from protozoon (Pf., Tg., Tv.) and had 1-3 amino acid residue difference with MmLDH, HsLDH-A, -B, -C, and was the same with CsLDH. One of the key catalytic residues and substrate (pyruvate) binding loop were located in this region. Tertiary structure demonstrated that 98aa-106aa was on the surface of the protein and formed a substrate binding loop, other two key catalytic sites were at the position near the loop. CONCLUSION: The prediction implied that LDH was an ideal molecule for phylogenetic analysis; SjLDH might be a potential molecular target for immunodiagnosis, anti-schistosome drug and vaccine development.

An alpha-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum. Cloning and biochemical characterization of the enzyme.

Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites. The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli. The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD(+)(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate. Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD. The enzyme exhibited strict substrate and cofactor specificity. The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts. A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets. The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in alpha-proteobacterial MDHs. Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also. Treatment of a synchronized culture of P. falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged. Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites. Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs.

Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes.

Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.

Reappraisal of the regulation of lactococcal L-lactate dehydrogenase.

Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed.

Regulating function of coenzymization and decoenzymization of the lactate dehydrogenase isozymes in the mouse tissues during hypoxia.

OBJECTIVE: To study the characteristics of changes of LDH enzyme patterns of mice under slight hypoxia. METHODS: Mice treated with artificial hypoxia, various tissues were made for the test of LDH enzymatic activity by the specific staining technique. LDH (1-5) relative percentage enzymatic activity (RPEA) were measured with CS-910 dual-wavelength thin layer chromatography scanner. RESULTS: The RPEA of LDH isozymes of various tissues after slight hypoxia shifted to the isozymes LDH1 and LDH2, whose principal subunits are H subunits, and the RPEA of LDH1 (H4), LDH2 (H3M) increased, while RPEA of LDH5 (M4) in various tissues decreased prominently except the cardiac muscle, and that of LDH4 (HM3) decreased as well. After polyacrylamide gel electrophoresis (PAGE) of the hypoxia treated cardiac muscle specimen was made, activity subbands originated regularly in the isoyme patterns of LDH, with the regularity of LDH1 (0 subband), LDH2 (0-1 subbands), LDH3 (0-2 subbands), LDH4 (1-3 subbands), LDH5 (2-4 subbands). After adding appropriate amount of NAD+ to the hypoxia treated cardiac muscle specimen, PAGE showed the subbands of four isoymes (LDH2-LDH5) reduced or even totally disappeared in the isozyme patterns. CONCLUSIONS: The negative feedback regulation of coenzymization and decoenzymization of LDH isozymes is one of the mouse stress responses to slight hypoxia.

Classification of lactate dehydrogenase of different origin by liquid chromatography-mass spectrometry and multivariate analysis.

A method intended to serve as a multivariate quality control tool in the production of pharmaceutical proteins is presented. The method is based on multivariate analysis of peptide maps generated with liquid chromatography-mass spectrometry (LC-MS). Lactate dehydrogenase (LDH) from different species and tissues were used as model compounds in the study. The proteins were digested with Endoproteinase Lys-C before the LC-MS analysis. After data pretreatment of the peptide maps, successful classification of the LDHs were obtained by discriminant analysis with partial least squares regression and artificial neural networks. Further, principal component analysis was applied to visualize the relationships between the samples.

Molecular evolution within the L-malate and L-lactate dehydrogenase super-family.

The NAD(P)-dependent malate (L-MalDH) and NAD-dependent lactate (L-LDH) form a large super-family that has been characterized in organisms belonging to the three domains of life. In the first part of this study, the group of [LDH-like] L-MalDH, which are malate dehydrogenases resembling lactate dehydrogenase, were analyzed and clearly defined with respect to the other enzymes. In the second part, the phylogenetic relationships of the whole super-family were presented by taking into account the [LDH-like] L-MalDH. The inferred tree unambiguously shows that two ancestral genes duplications, and not one as generally thought, are needed to explain both the distribution into two enzymatic functions and the observation of three main groups within the super-family: L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. In addition, various cases of functional changes within each group were observed and analyzed. The direction of evolution was found to always be polarized: from enzymes with a high stringency of substrate recognition to enzymes with a broad substrate specificity. A specific phyletic distribution of the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH over the Archaeal, Bacterial, and Eukaryal domains was observed. This was analyzed in the light of biochemical, structural, and genomic data available for the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. This analysis led to the elaboration of a refined evolutionary scenario of the super-family, in which the selection of L-LDH and the fate of L-MalDH during mitochrondrial genesis are presented.

Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases.

The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.

The cDNA sequence of the lactate dehydrogenase-A of the spiny dogfish (Squalus acanthias): corrections to the amino acid sequence and an analysis of the phylogeny of vertebrate lactate dehydrogenases.

The cDNA sequence of the lactate dehydrogenase-A (LDH-A) of the spiny dogfish was determined. The deduced amino acid sequence differed from a previously determined protein sequence by 5%. Separate maximum parsimony analyses of the two sequences along with LDHs of other vertebrates resulted in shorter trees with the sequence presented here, as well as fewer equally parsimonious trees. The new sequence also indicates a greater conservation of length among vertebrate LDHs than was previously suspected. Analyses of the phylogeny of vertebrate LDHs resulted in a monophyletic grouping of LDH-As, from within which mammalian LDH-C is derived. The phylogeny of LDH-As did not exactly match the phylogeny of the organisms, raising the possibility of multiple origins and losses of a muscle-predominant gene. LDH-Bs appear to have shared a single origin.

Allozyme differentiation among nine populations of the corn borer (Ostrinia) in China.

To test the hypothesis of the migration of the corn borer, the allozymes of nine populations of the corn borer (Ostrinia) in China were checked using vertical-slab polyacrylamide gel electrophoresis. Eight loci of six allozymes were analyzed. The mean of the genetic identities among the nine populations calculated from the allele frequencies was 0.99068, much closer than that of other species and geographical populations. The mean (0.97955) of the genetic identities between the XJYN population (Ostrinia nubilalis Hübner, collected from Yining, Xinjiang Autonomous Region) and each of the other eight populations (O. furnacalis Guenée) was significantly smaller than that between the pairs of the eight populations (0.99386; t test, P < 0.01). Although the population XJYN clearly deviates from the other eight populations in the dendrogram, the relationship of the two species of corn borer was very close. It is possible that the speciation of corn borer may have resulted from single-gene substitutions.

Diversity of lactate metabolism in halophilic archaea.

D-Lactate is readily used as a substrate for the growth of species of halophilic archaea belonging to the genera Haloferax and Haloarcula. L-Lactate was used by Haloferax species (Haloferax volcanii, Haloferax mediterranei) only when a substantial concentration of the D-isomer was also present in the medium. On the enzymatic level, considerable diversity was found in the lactate metabolism of the different representatives of the Halobacteriaceae. At least three types of lactate dehydrogenases were detected in halophilic archaea. A high level of activity of an NAD-linked enzyme was present constitutively in Haloarcula species, and a low level of activity was also detected in Haloferax mediterranei. NAD-independent lactate dehydrogenases, oxidizing L-lactate and D-lactate with 2,6-dichlorophenol-indophenol as electron acceptor, were detected in all nine species tested, but L-lactate dehydrogenase activity in Halobacterium species was very low, and Haloarcula species, which possess a high level of activity of NAD-linked lactate dehydrogenase, showed very low activities of both NAD-independent D- and L-lactate dehydrogenase. An inducible lactate racemase, displaying an unusually high pH optimum, was found in Haloferax volcanii. Lactate racemase activity was found constitutively in Haloarcula species, but no activity was detected in Halobacterium species and in Haloferax mediterranei.

Multiple lactate dehydrogenase activities of the rumen bacterium Selenomonas ruminantium.

The lactate utilizing strain of Selenomonas ruminantium 5934e was found to contain three lactate dehydrogenase (LDH) activities in sonicated cell extracts. One activity, an NAD dependent L-LDH (L-nLDH) was measured at 15-fold greater levels in extracts of cells grown to mid-exponential phase on glucose compared to cells grown to the equivalent growth stage on DL-lactate. A second nLDH activity specific for D-lactate (D-nLDH) was detected at similar levels in both lactate-grown cell extracts and glucose-grown cell extracts. The third activity, an NAD independent DLDH (D-iLDH) was very low in cells grown on glucose but was induced more than 10-fold when DL-lactate was used as the carbon source. The three LDH activities could be separated by gel filtration. Recovery of the activities was low due to the apparent instability of the enzymes at 4 degrees C, which was most pronounced in the case of the D-iLDH. A Km for lactate of 0.5 mM was estimated for the D-iLDH and this was considerably lower than the values of 45 mM and 70 mM measured for L-nLDH and D-nLDH respectively. It is proposed that the D-iLDH may be largely responsible for the formation of pyruvate in lactate-grown cells of S. ruminantium strain 5934e. Three other lactate utilizing strains of S. ruminantium, HD4, 5521C1 and JW13 exhibited a similar profile of LDH activities to strain 5934e when grown on glucose and DL-lactate.

Breast cancer diagnosis by lactate dehydrogenase isozymes in nipple discharge.

BACKGROUND: The diagnosis of breast cancer based on nipple discharge, often the only clinical manifestation of early breast cancer, is currently unsatisfactory. Because M subunits of lactate dehydrogenase (LDH) have been noted to increase in cancer tissue, the author assessed the value of using LDH isozyme patterns in nipple discharge for the diagnosis of breast cancer. METHODS: LDH isozyme levels in (1) nipple discharge of patients diagnosed as having breast cancer, intraductal papilloma, mastopathy, drug-induced nipple discharge, mastitis, or benign nipple discharge; (2) control samples of normal nipple discharge (milk) 6 days, 1-5 months, and 6 months to 2 years postpartum; (3) the serum of patients presenting with nipple discharge; and (4) normal and cancerous breast tissue extracts were measured using a Ciba-Corning LDH isozyme system (Ciba Corning Diagnostic Corp., Tokyo, Japan). RESULTS: LDH isozyme levels in the nipple discharge of patients with benign breast diseases displayed various patterns. Levels in the nipple discharge of patients with breast cancer, including noninvasive carcinoma, tended to increase in ascending order from LDH1 to LDH5. This pattern was similar to that in breast cancer tissue and was unrelated to the pattern in serum. CONCLUSION: LDH isozyme assay of nipple discharge may be a useful technique for providing a supporting diagnosis of breast cancer.

Neisseria gonorrhoeae possesses two nicotinamide adenine dinucleotide-independent lactate dehydrogenases.

An important metabolic capability of Neisseria gonorrhoeae is the utilization of host-derived lactate. Two isoenzymes of the membrane-associated, pyridine dinucleotide-independent type of lactate dehydrogenase (iLDH) participate in lactate assimilation, but exhibit distinctive properties. Isoenzyme iLDH-I utilized lactate exclusively as substrate, exhibiting a preference for the D-isomer. In contrast, isoenzyme iLDH-II exhibited broad substrate specificity (lactate, phenyllactate, and 4-hydroxyphenyllactate), but was stereospecific for the L-isomers. These results explain the difficulty in isolating mutants unable to utilize lactate.

Prognostic value of the determination of serum lactic dehydrogenase and its isoenzymes in children with acute lymphoblastic leukaemia.

Serum total lactic dehydrogenase (LDH) levels have been found to be significantly higher in 21 children with acute lymphoblastic leukaemia (ALL) at initial diagnosis than the values of 12 children who achieved remission. The mean value of serum LDH levels in patients with high-risk factors was 2347 +/- 1490 U/ml (range 430-5460 U/ml), while in patients at standard risk it was 652 +/- 385 U/ml (range 110-1320 U/ml). The serum LDH values were above 1320 U/ml in 70% of the 13 children with high-risk factors. The serum isoenzyme patterns were analyzed in 17 of these children at the initial diagnosis. Although LDH-3 and LDH-2 were prominent at the time of diagnosis; LDH-2 and LDH-1 were the predominant isoenzymes in remission. The highest concentrations of LDH-3 were observed in the high-risk group at diagnosis and the ratio of LDH-3 to LDH-2 exceeding 1.0 was found in children who had high-risk factors, but not in any patients in the standard risk group.

Stability of solutions of human granulocyte lactate dehydrogenase stored at different temperatures.

Granulocyte suspensions from eight healthy individuals were lysed ultrasonically in 0.15 M NaCl or serum. The leukolysates were tested for lactate dehydrogenase after being stored at 22 C, 5 C, -20 C and -196 C for one, two, three, four and seven days. The LDH content of granulocytes was 291 IU/10(10) cells (mean) and the enzyme had a characteristic isoenzyme pattern rich in LDH-5 with stepwise increases in each fraction from LDH-1 to LDH-5. The enzyme was unstable in saline solutions. This was marked at -20 C and related to the duration of storage. All the isoenzyme fractions showed significant inactivation. A mean 10% loss of activity occurred at -196 C. This loss was independent of the duration of storage and related to the process of freezing and thawing. Moderate inactivation occurred at room temperature while refrigerated samples were stable for four days. Serum protected the enzyme from denaturation and samples stored at -20, -196 and 22 C were stable for the seven days of the experiment.