Lactate Dehydrogenase Variants Research Articles

Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak

Huang, L., Jin, S-Y., Xu, Y-O., Li, Y-P., Lin, Y-Q. and Zheng, Y-C. 2012. Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak. Can. J. Anim. Sci. 92: 291–296. The main objective of the present study was to analyze quantitatively the alternative splicing of the lactate dehydrogenase C (ldhc) gene in the testes of yak (Bos grunniens) and male sterile yak hybrid. RT-PCR amplification of ldhc cDNA in the testes revealed eight splice variants formed by the deletion of one or more exons in the mRNA transcripts. The deleted exons occur mostly in exons 7 and 4. The deletion of exons caused reading frame shift and formation of stop codon in some variants. Quantitative real-time RT-PCR analysis using ldhc variant-specific primers showed that the mRNA level of full length ldhc decreased dramatically in the testes of sexually immature yak calf (n=6) and male sterile hybrid cattle–yak (n=4) compared with that of adult yak (n=14). The proportions of the ldhc variants assayed differed significantly among adult yak, yak calf and cattle–yak; more ldhc transcripts were spliced in immature or sterile testes. Our results suggest that the alternative splicing could play a role in the regulation of ldhc expression in testes, and could be one factor that plays a role in infertility of yak hybrids.

Identification of yak lactate dehydrogenase B gene variants by gene cloning

Native polyacrylamide gel electrophoresis showed that two types of lactate dehydrogenase (LDH) existed in yaks. Based on the electrophoresis characteristics of LDH isoenzymes, yak LDH variants were speculated to be the gene mutation on H subunit encoded by B gene. According to the mobility in electrophoresis, the fast-band LDH type was named LDH-Hf and the slow-band LDH type LDH-Hs. In order to reveal the gene alteration in yak LDH variants, total RNA was extracted from heart tissues of yaks with different LDH variants, and cDNAs of the two variants were reverse transcripted. Two variants of B genes were cloned by RT-PCR. Sequence analysis revealed that four nucleotides differed between LDH-Bf and LDH-Bs, which resulted in two amino acids alteration. By Deepview software analysis of the conformation of yak LDH1 variants and H subunit, these four nucleotides altered two amino acids that generated new hydrogen bonds to change the hydrogen bonds network, and further caused subtle conformational changes between the two LDH variants.

Cloning and Sequence Analysis of Multiple Splice Variants of Lactate Dehydrogenase C in Yak Testes

Lactate dehydrogenase C (LDH-C) has been reported to play a role in the energy metabolism of mammal spermatozoa. However, the functions and expression patterns of LDH-C still remain unclear. In order to elucidate the functions and expression patterns of LDH-C, we cloned the cDNA of yak LDH-C. Total RNA was extracted from yak testes and reverse transcribed and amplified by PCR. The full length open reading frame (ORF) of LDH-C and its five splice variants were obtained. The full length ORF contained 999 bp encoding a 332-amino-acid protein that showed 100% identity with bovine LDH-C. Compared with the full length ORF of LDH-C, the five variants used the same start codon as the full length ORF and encoded 5 putative proteins. In detail, variants 1 (missing the coding sequence of exon 6 and 7) and 2 (missing the coding sequence of exon 7) bear the entire nicotinamide-adenine dinucleotide (NAD) binding domain and an active site. Variants 3 (missing the first 42 nuleotides of exon 4) and 4 (missing the coding sequences of exons 5, 6 and 7) lack part of the NAD binding domain but contained the entire active site. Variant 5 (missing the coding sequence of exons 4 and 7) lacks a large part of the NAD binding domain and the entire active site. Native polyacrylamide gel electrophoresis was performed to determine if the splice variants can be translated into proteins. However, native PAGE detected no specific bands from yak testes and bovine spermatozoa. This study suggests the alternative splicing of LDH-C is ubiquitous in bovine testes and might be involved in regulation of LDH-C expression. The findings also help to elucidate the functions of LDH-C.

N-Terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well ( R 2=0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.

Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase

The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide–propylene oxide random copolymer (EO30PO70). Five different LDH variants were constructed with N-terminal tags containing tyrosines (Y3 and Y6), tyrosines and prolines (Y3P2 and Y6P2), and only prolines (P2). LDH fused with tags containing tyrosines increased the partitioning coefficient, and the more tyrosines added to the protein, the larger improvement in partitioning. When prolines were added between the tyrosine-rich tag and the protein, a further increased partitioning was obtained. The enhanced partitioning was attributed to the rigid structure of the proline, which in turn led to an increase in the exposure of the tag to the surroundings. The best tyrosine tag, Y6P2, increased the partition coefficient four times and additionally, a higher thermostability was observed.

Immobilisation of lactate dehydrogenase on poly(aniline)–poly(acrylate) and poly(aniline)–poly(vinyl sulphonate) films for use in a lactate biosensor

The immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)–poly(acrylate) (PANi–PAA) or poly(aniline)–poly(vinyl sulphonate) (PANi–PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD + to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface. In solution, the activity of the C-tagged enzyme (LDH–CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi–PAA and PANi–PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH–PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations.

Arginine to tryptophan substitution in the active site of a human lactate dehydrogenase variant — LDHB ∗GUA1: postulated effects on subunit structure and catalysis

A variant of lactate dehydrogenase (LDHB ∗GUA1) was previously identified among the Guaymi Indians of Panama and Costa Rica. The LDHB ∗GUA1 variant is enzymatically inactive; however, the variant subunits alter the electrophoresic mobility of the tetramers that include active LDHA and LDHB subunits. The kinetic properties of the tetramerc enzyme, comprised of inactive B plus active A subunits, are similar to properties of the heterotetramers with active B subunits, except for the reduced specific activity. We have determined that a single C · G to T · A transition changes an Arg to a Trp at amino acid residue 106. This substitution explains the increase in net negative charge observed by protein electrophoresis. This Arg 106 residue is absolutely conserved throughout evolution. Published high-resolution crystal structures of LDH reveal that this residue is within the hinge of a loop that closes over the active site of the subunit upon binding of substrate and cofactor and also has a direct role in catalysis. Computer modeling of the variant enzyme suggests that replacement of this Arg residue with a Trp does not induce significant change in the structure of the active site. However, this substitution would result in disruption of enzyme activity through the inability of the uncharged tryptophan side-chain to polarize the substrate carbonyl bond. This would explain the loss of catalytic function with maintenance of normal kinetic properties in the heterotetramers containing the variant subunits. The ability to maintain normal, tissue-specific kinetic properties could explain the absence of clinical manifestations in the homozygous LDHB ∗GUA1 individuals.

Fast-type electrophoretic variant of lactate dehydrogenase M(A) and comparison with other missense mutations in lactate dehydrogenase M(A) and H(B) genes

An electrophoretic variant of lactate dehydrogenase (LD) M(A) subunit was discovered in a female patient with chest pain. Her LD activity in serum was within the normal reference interval, and analysis of her LD isoenzyme pattern showed an abnormal migration indicating a fast-type LD-M(A) subunit variant. DNA analysis of the mutant LD-M gene detected a single base substitution, an A to G transition at codon 220 (AAA-->GAA). This mutation resulted in the replacement of a lysine by a glutamic acid (mutation K220E) and produced a subunit variant (electrophoretic fast type). This missense mutation was also observed in the patient's son, and genotypes of mother and son were consistent with their biochemical phenotypes, as evaluated by LD isoenzyme analysis.

Lactate Dehydrogenase lsozymes in Barley: Polymorphism and Genetic Basis

Hypoxia induces a set of five lactate dehydrogenase (LDH) isozymes in roots of barley (Hordeum vulgare) and most lines of H. spontaneum, the wild progenitor. Barley LDH isozymes are tetramers. A survey of 30 diverse H. spontaneum lines identified three electrophoretic LDH variants; in two variants the five isozymes were displaced as a group (fast or slow) relative to the normal set, whereas in the third there was only one major lsozyme. Immunoblot analysis established that the latter variant lacked LDH protein as well as enzyme activity from the normal band positions. All variants bred true for LDH phenotype. In crosses among variants, the F1 banding patterns were consistent with those predicted by a model in which the products of two Ldh loci associate freely in all possible combinations. However, no recombinants were found in the F2 progenies, and F2 ratios of parental to F1-hybrid phenotypes were 1:2:1. These data indicate that barley has two closely linked Ldh genes, each with several alleles that have been fixed in particular combinations in natural H. spontaneum populations.

The importance of arginine 171 in substrate binding by Bacillus stearothermophilus lactate dehydrogenase

A variant of lactate dehydrogenase from Bacillus stearothermophilus has been engineered by site-directed mutagenesis in which an active-site arginine residue at position 171 in the protein sequence is replaced by lysine. Replacement of this arginine by lysine has no effect on co-enzyme binding, a relatively small effect on the rate of turnover of the enzyme, but causes a 2000-fold increase in the Michaelis constant for pyruvate, a 6000-fold increase in the dissociation constant for oxamate and results in a Michaelis constant for lactate which is too high to measure. The decrease in binding energy for these carboxylate-containing substrates caused by this mutation is very large, around 5.5 kcal.mol-1 and in part, is explained by the small increase in the distance of a lysine-substrate carboxylate interaction at this site and the absence of the additional hydrogen bond from a two-point arginine-carboxylate interaction. Consistent with this last observation, the ability of this mutant enzyme to stabilize an NAD+-sulphite compound in its active site (an alternative enzyme-substrate complex which does not involve bifurcated bonding to arginine) is only reduced 14-fold.

Defining the locus of origin of a genetically determined electrophoretic variant of a multilocus enzyme system; the Calcutta-1 of human LDH system is a B-locus variant.

Six (four Hindus, one Sikh, and one Muslim) outr of 213 individuals originating from different parts of the Indian subcontinent (namely, Andhra Pradesh, Maharashtra, Uttar Pradesh, East Punjab, and West Punjab) were found to be Calcutta-1 (CAL1) variants of lactate dehydrogenase (LDH). The CAL1 variant was originally described (and thus, generally believed at present) as an allelic variant at the LDHA locus in chromosome 11. By using an improved Cellogel electrophoretic procedure the isozyme patterns observed in the erythrocytes and leukocytes of the variant have indicated that the CAL1 is not variant of LDHA but that of LDHB, a chromosome 12 marker. The suggestion was supported by the isozyme patterns of LDH in a set of segregating clones of man-mouse somatic cell hybrids with the variant as human partner. Moreover, the variant cosegregated consistently with the human chromosome 12 and with the markers firmly assigned to the latter but not with human chromosome 11 or its markers in these hybrids. These results confirmed that the CAL1 is an LDHB variant.

Screening for Haemoglobin and Lactate Dehydrogenase Variants in the Icelandic, Swedish, Finnish, Lappish, Mari and Greenland Eskimo Populations

Haemolysates from 9,589 North Europeans (Swedes, Finns, Icelanders and Lapps), Maris (Cheremissses) in the USSR, and an Eskimo population in NW Greenland were screened for haemoglobin (Hb) and lactate dehydrogenase (LDH) variants. No Hb variants were observed. In 2 of the 1,769 native Finns examined, LDH variants of the LDH-A subunit were detected. The incidence of Hb and LDH variants detectable by electrophoresis seems to be rather low in circumpolar populations. This fits with the fact that the two Finns with LDH variants had common ancestors around the year 1700.

A B sub-unit variant of lactate dehydrogenase in Bulgaria.

One example of a B sub-unit variant of lactate dehydrogenase was found among 138 samples from Sofia, Bulgaria. Thismutation probably represents a random sporadic mutation amongBulgarians.

Caste and age variations of the incidence of LDH variants in the Bengali Hindus.

In continuing our studies on the incidence of the genetically determined variants of Lactate dehydrogenase (LDH) among the Indian populations, it has been possible to collect some suitable material presented here to examine the question of caste and age variations in the incidence rates of the commonly occurring variant, LDH Cal-1, among the Bengali Hindu. It is a “Faster A-subunit variant”.

Four types of genetic variants of LDH found in India.

The four types of genetic variants of lactate dehydrogenase (LDH) found so far in the Indian populations surveyed by starch-gel electrophoresis of haemolysates are described, pointing out their characteristic features. Two of these variant types, namely, ‘Calcutta-1’ and ‘Madras-1, were reported earlier. The other two discovered subsequently and named trivially as ‘Calcutta-2’ and ‘Delhi-1 are now reported for the first time. A comparison between the four LDH variants found in India and those by others outside India has also been drawn.

Two erythrocytic lactate dehydrogenase variants in the house mouse, Mus musculus

SummaryTwo erythrocytic LDH variants have been found in the house mouse, Mus musculus. One, involving the presence and absence of the next to the slowest electrophoretically separable isozyme, is controlled by a regulatory locus which may be similar to Ldr‐1 (Shows and Ruddle, 1968). The second appears to be indirectly controlled by the Hbb locus and may be attributed to the accumulation of a storage product in the lysates. Ldr‐1 is polymorphic in natural populations of the house mouse.

The distribution of serum protein and enzyme group systems among North Indians.

The distribution of electrophoretic variants in 4 serum protein and 12 red cell enzyme systems has been determined among nearly 500 persons belonging to various castes in North India. No electrophoretic variants were found for transferrin or for the enzyme systems malate dehydrogenase, peptidase B, ‘oxidase’, phosphoglycerate kinase and phosphoglucomutase-locus 2. Two persons with abnormal albumins were detected, and the slower moving band has been identified provisionally as albumin Gainsville. One person with an unusual caeruloplasmin was found also, and this has been identified provisionally as Cp ANigera. In the haptoglobin system the overall frequency of Hp2 = 0.20 is similar to that found previously in North India. The lowest Hp2frequency (0.12) was found among the Scheduled Castes. The PHsc gene in the red cell acid phosphatase system was of low frequency (0.005) as also was the PGDC allele (0.02) in the 6-phosphogluconate dehydrogenase system. Three phenotypes were present in the adenylate kinase system, and the AK2 gene frequency of 0.09 is among the highest values reported so far. For phosphoglucomutase-locus 1, in addition to the common alleles PGM11 and PGM21 there were several examples of the rarer PGM31, PGM61 and PGM71 alleles. PGM11 frequencies varied from 0.63 to 0.79 in different caste groups. Seven examples of heterozygotes for the Calcutta-1 variant of lactate dehydrogenase were detected giving an overall frequency of LDHCal. = 0.007. A single case of Pep A 2–1 was observed in the peptidase A system. Three other enzyme systems showing electrophoretic variation had been studied previously only on Indians living in England. In the present study 3 phenotypes were present for adenosine deaminase, and the ADA2 allele had a range of frequency of 0.06 to 0.20. The latter figure, for the Arora, is the highest frequency so far reported for this allele. Only rare variants were present in the phosphohexose isomerase and NADH diaphorase systems. There were 6 PHI 3–1, 1 PHI 5–1 and 1 PHI 9–1 phenotypes in a total of 490 samples tested. Half of these variants were found among the 83 persons belonging to the Scheduled Castes. In the diaphorase system there were 6 2–1 and 1 4–1 phenotypes distributed between the caste groups.

Further studies on the distribution of genetic variants of lactate dehydrogenase in India.

Further studies on the distribution of genetic variants of lactate dehydrogenase in India.

Erythrocyte lactate dehydrogenase variants in the Icelandic and Swedish populations.

Erythrocyte lactate dehydrogenase variants in the Icelandic and Swedish populations.

A variant of lactate dehydrogenase in somatic tissues of pigeons: physicochemical properties and genetic control.

AbstractThree genetic loci (a, b, and c) control lactate dehydrogenase synthesis in many mammalian and avian species. A survey of the lactate dehydrogenase isozyme composition of somatic tissues from approximately 1,000 wild pigeons revealed three phenotypic classes. The frequency distribution of the three classes in wild pigeons and the results of appropriate breeding experiments demonstrated that the phenotypic differences were due to alleles at the b‐locus. One of the alleles produces a B' polypeptide with an electrophoretic mobility identical to that of the A polypeptide, but with substrate affinity and heat inactivation properties similar to those of the B polypeptide.