Lactate dehydrogenase-B cDNA from the teleost Fundulus heteroclitus: evolutionary implications

A cDNA that encodes the heart-type lactate dehydrogenase (LDH-B) from the teleost fish Fundulus heteroclitus was cloned and sequenced. The protein encoded by the cDNA was analyzed in relation to 13 LDH proteins from a variety of taxa. One of the deductions from this analysis is that LDH-B proteins have residues in the active site that are unique and that may be important in determining the biochemistry of the heart-type isozyme. Phylogenetic analysis of the LDH sequences indicates that the branch lengths are greater in lower vertebrates, suggesting that the amino acid replacement rates vary depending on the evolutionary constraints within each taxon. Furthermore, the analysis suggests that LDH-C arose prior to the divergence of the LDH-A and LDH-B isozymes and thus that it is probably ancestral to these isozymes.     

Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin

Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.     

The cDNA and protein sequences of human lactate dehydrogenase B.

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

Characters of amino acid substitution in LDH-A and LDH-B in the evolutionary line of vertebrates

Structure of lactate dehydrogenase LDH-A (muscle) and LDH-B (heart) subunits is compared in the evolutionary line of vertebrates from Chondrosteous fishes to Mammals. It is revealed persistent differences between them in the amino acid set determining the physical and chemical characteristics of macromolecules. The polypeptide chain of LDH-A is shorter then that of LDH-B but it contains amino acids with higher molecular weight. In LDH-A polarized amino acids are less in number but charged amino acids are more numerous, positive charged amino acids prevail over negative charged ones. The features of polypeptide structure are discussed in connection with differences in the level of intraspecific variability of allozymes in the evolutionary line of vertebrates.     

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.      

Lactate dehydrogenase genes of caiman and Chinese soft-shelled turtle, with emphasis on the molecular phylogenetics and evolution of reptiles.

L-Lactate dehydrogenase (LDH) cDNAs encoding for LDH-A(4) (muscle) and LDH-B(4) (heart) isozymes from caiman (Caiman crocodilus apaporiensis) belonging to the order Crocodilia and Chinese soft-shelled turtle (Pelodiscus sinensis) belonging to the order Chelonia were sequenced. The phylogenetic relationships of the newly determined cDNA and their deduced protein sequences, as well as the previously published sequences of vertebrate LDH isozymes, were analyzed by various phylogenetic tree construction methods. These results indicated that Chelonia is indeed more closely related to Crocodilia. The divergent times between caiman and alligator, turtle and soft-shelled turtle, and Chelonia and Crocodilia were estimated to be approximately 36, 100 and 177 million years, respectively.     

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。     

Relative mRNA expression of the lactate dehydrogenase A and B subunits as determined by simultaneous amplification and single strand conformation polymorphism. Relation with subunit enzyme activity

To explore if it is correlated in human tumor cells that the expression of LDH homologous gene and LDH isoenzymes, we used RT-PCR-SSCP technique to measure the relative expression of genes with homologous sequences. The combination of PCR using common primers designed in the highly conserved regions and single-strand conformation polymorphism analysis of the products is used for quantitative determination of the proportions of LDH-A mRNA in human cancer cell lines. The proportion is compared with that of the activities of isoenzymes. The results indicated that the enzyme activity of LDH-A was consistent with mRNA levels in the human tumor cell. The present procedure using a single pair of primers for two fragments can overcome disadvantages in quantitative analysis using multiplex PCR. Template concentrations and PCR cycles did not affect the proportions of LDH-A and LDH-B in the product.     

Molecular evidence for a clade of turtles.

Although turtles have been generally grouped with the most primitive reptile species, the origin and phylogenetic relationships of turtles have remained unresolved to date. To confirm the phylogenetic position of turtles in amniotes, we have cloned and determined the cDNA sequences encoding for skink lactate dehydrogenase (LDH)-A and LDH-B, snake LDH-A, and African clawed frog LDH-A; four alpha-enolase cDNA sequences from turtle, alligator, skink, and snake were also cloned and determined. All of these eight cDNA sequences, as well as the previously published LDH-A, LDH-B, and alpha-enolase of mammals, birds, reptiles, and African clawed frog, were analyzed by the phylogenetic tree reconstruction methods of neighbor-joining, maximum parsimony, and maximum likelihood. In the phylogenetic analyses, the turtle was found to be closely related to the alligator. Also, we found that the turtle had diverged after the divergence of squamates and birds. This departs from previous hypotheses of turtle evolution and further suggests that turtles are the latest of divergent reptiles, having been derived from an ancestor of crocodilian lineage within the last 200 million years.     

Estimation of the gene frequency of lactate dehydrogenase subunit deficiencies

To detect the frequency of lactate dehydrogenase (LDH) subunit deficiency, screening for LDH subunit deficiency was performed on 3,776 blood samples from healthy individuals in Shizuoka Prefecture by means of electrophoresis. The frequency of heterozygote with LDH-A subunit deficiency was found to be 0.185%, and with LDH-B subunit deficiency, 0.159%. The frequencies of both subunit deficiencies were not significantly different. Gene frequencies of LDH subunit deficiencies were calculated by the simple counting procedure, and the results are as follows: gene frequency of LDH-A subunit deficiency was 11.9 X 10(-4), and that of LDH-B subunit deficiency, 7.9 X 10(-4). In addition, the second case in the world of a homozygous individual with LDH-A subunit deficiency was detected by this screening. This case with regard to the characteristics of LDH-A subunit deficiency are summarized herein.     

Enzyme activities in fish spermatozoa with focus on lactate dehydrogenase isoenzymes from herring Clupea harengus

The activities of NAD- and NADP-dependent dehydrogenases and creatine kinase were compared in extracts of spermatozoa from herring (Clupea harengus), carp (Cyprinus carpio) and catfish (Clarias gariepinus). The activity of malic enzyme in herring spermatozoa was approximately 5 and 36 times higher than in carp and catfish spermatozoa. In contrast, lactate dehydrogenase activity in herring spermatozoa was very low. Herring spermatozoa possess two isoenzymes of lactate dehydrogenase: LDH-A(2)B(2) and LDH-B(4). Both herring spermatozoa isozymes were separated, partly purified and characterized by kinetic and physico-chemical properties. The pH optima and K(m) values for pyruvate reduction were 7.1, 7.25, 7.6 and 0.22, 0.07, 0.09 mM for LDH-A(4), LDH-A(2)B(2) and LDH-B(4), respectively. The isoenzymes also have different thermostabilities. High activity of malic enzyme in herring spermatozoa suggests adaptation to metabolism at high oxygen tension.     

Nucleotide sequences of the cDNA and an intronless pseudogene for human lactate dehydrogenase-A isozyme

Eight cDNA clones for lactate dehydrogenase-A isozyme (LDH-A) were isolated from a human fibroblast cDNA library, characterized, and no sequence heterogeneity was found. Four cDNA clones appear to contain nearly full-length cDNA inserts and the complete nucleotide sequence of 1710 base pairs consists of the protein-coding sequence (999 base pairs), the 5' (97 base pairs) and 3' (565 base pairs) untranslated regions and poly(dA) tail (49 base pairs). The predicted amino acid sequence of the human LDH-A polypeptide shows 92% homology (27 differences out of 331 amino acids compared) with that of the pig LDH-A subunit determined by direct protein sequencing [Kiltz et al. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 123-127]. Human genomic clones containing an LDH-A pseudogene were isolated and the nucleotide sequence of 1635 base pairs from an intronless pseudogene was determined. The presence of two termination codons, two deletions of three nucleotides each and the replacement of three arginine residues at the active site (nos 98, 105 and 168) by other amino acids renders its coding region incapable of producing a functional LDH-A protein. A comparison between human LDH-A cDNA and the pseudogene sequences reveals 12.9% differences (114 transitions, 65 transversions and 36 deletions/insertions). Further, only four out of the 25 dCpdG dinucleotides present in the cDNA sequence remain unchanged, although the sequences possess 87.1% homology.     

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.     

Supernumerary chromosomes and spermatogenesis in a human male carrier

Screening for lactate dehydrogenase (LDH) subunit deficiencies was performed on 2880 blood samples from healthy individuals in the Fukuoka Prefecture in Japan by means of electrophoresis. The frequencies of heterozygotes with either LDH-A or LDH-B deficiency were found to be 0.104% at each locus. These estimated frequencies of either LDH-A or LDH-B deficiencies were slightly lower than, but not significantly different from, those found previously in Shizuoka Prefecture. The genetic mutations in individuals heterozygous for LDH-B deficiency were analyzed by the polymerase chain reaction and DNA conformation polymorphism. Abnormal migration patterns were observed in individuals heterozygous for LDH-B deficiency. Subsequent sequence determination of the mutant alleles revealed three novel mutations: an eight-base duplication in exon 3, a four-base duplication in exon 4, and a one-base deletion in exon 7 of the LDH-B gene. These three mutations result in frameshift translation and premature termination. In addition, the mutations resulting in the duplication of eight or four nucleotides appear to cause a decrease in the levels of LDH-B mRNA.     

Lactate dehydrogenase expression at the onset of altered loading in rat soleus muscle.

Both functional overload and hindlimb disuse induce significant energy-dependent remodeling of skeletal muscle. Lactate dehydrogenase (LDH), an important enzyme involved in anaerobic glycolysis, catalyzes the interconversion of lactate and pyruvate critical for meeting rapid high-energy demands. The purpose of this study was to determine rat soleus LDH-A and -B isoform expression, mRNA abundance, and enzymatic activity at the onset of increased or decreased loading in the rat soleus muscle. The soleus muscles from male Sprague-Dawley rats were functionally overloaded for up to 3 days by a modified synergist ablation or subjected to disuse by hindlimb suspension for 3 days. LDH mRNA concentration was determined by Northern blotting, LDH protein isoenzyme composition was determined by zymogram analysis, and LDH enzymatic activity was determined spectrophotometrically. LDH-A mRNA abundance increased by 372%, and LDH-B mRNA abundance decreased by 43 and 31% after 24 h and 3 days of functional overload, respectively, compared with that in control rats. LDH protein expression demonstrated a shift by decreasing LDH-B isoforms and increasing LDH-A isoforms. LDH-B activity decreased 80% after 3 days of functional overload. Additionally, LDH-A activity increased by 234% following 3 days of hindlimb suspension. However, neither LDH-A or LDH-B mRNA abundance was affected following 3 days of hindlimb suspension. In summary, the onset of altered loading induced a differential expression of LDH-A and -B in the rat soleus muscle, favoring rapid energy production. Long-term altered loading is associated with myofiber conversion; however, the rapid changes in LDH at the onset of altered loading may be involved in other physiological processes.     

Localization of lactate dehydrogenase isozymes in human muscle tissues by the mixed aggregation immunocytochemical technique.

Lactate dehydrogenase (LDH) isozyme composition and localization was determined in sections of skeletal, heart and smooth muscle by the mixed aggregation immunocytochemical method using first antibody directed against purified human LDH-A4 (M4) or LDH-B4 (H4) followed by the enzymes LDH-A4 and LDH-B4, respectively. An even distribution of the two monomers in all fibres was seen with heart muscle and smooth muscle. Heart muscle had a low concentration of A-monomers and a high concentration of B-monomers, whereas the smooth muscle had equal concentrations of the two monomers. In contrast, skeletal muscle from m. quadriceps femoris was found to be composed of two muscle fibre types, one containing mainly A-, the other mainly B-monomers. On the basis of succinate dehydrogenase activity it was shown that the red (type 1) fibres contain mainly B-monomers and the white (type 2) fibres mainly A-monomers of LDH.     

Genotypic analysis of families with lactate dehydrogenase A(M) deficiency by selective DNA amplification

Summary Genomic DNA prepared from LDH-A-deficient whole blood was amplified by the polymerase chain reaction technique using two primers specific for the active human LDH-A gene. The amplified fragment was examined by direct agarose gel electrophoresis, and a deletion of 20 base pairs (bp) in exon 6 of the LDH-A gene was found. The results permitted a clear distinction between the homozygous mutant, the heterozygous mutant, and wild-type genotypes. Moreover, HinfI digestion and direct sequencing of the amplified product confirmed the results from direct agarose gel electrophoresis. Four families, including 18 individuals, were shown to contain the same mutation, that is a 20-bp deletion in exon 6. All genotypes were consistent with their biochemical phenotypes as evaluated by the ratio of LDH-B to LDH-A subunits in erythrocytes. Thus, all four known affected families in Japan have been shown to carry the same mutant gene, which may have been derived from a single mutational event.