Detection of lactate dehydrogenase B4 in human hemolysate of patients deficient in lactate dehydrogenase B subunit activity using enzyme immunoassay

We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)- B₄ with antiacetylated LDH-B₄ Fab-horse-radish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B₄ concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B₄ concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.This work was supported in part by grants in aid for Scientific Research from the Ministry of Education, Japan (59570998), and from the Clinical Pathology Research Foundation of Japan.     

LDH calcutta-1: A mutation of the B subunit of human lactate dehydrogenase

The electrophoretic variant of human LDH, Calcutta-1, occurs at phenotypic frequencies of 0–4% throughout India. The variant was examined by various electrophoretic techniques and by heat stability studies. The LD₁ (B₄) isoenzyme was purified from normal and variant bloods by affinity chromatography and ion-exchange chromatography. A minimum of five Calcutta-1 LD₁ bands was demonstrated by isoelectric focusing. Electrophoresis of variant LD₁ in high-molar urea-acrylamide denaturing gels resulted in two Calcutta-1 B subunit bands, while normal gels yielded only a single band. Homozygote Calcutta-1 LDH from red cells demonstrated a decreased heat stability, while heterozygote variant LDH showed a normal heat stability. This effect was confirmed when purified LD₁'s were compared. Evidence is presented suggesting a B-subunit variant showing thermolability in the homozygous form.The author was supported by an Australian National University Scholarship.     

Glucose-6-phosphate dehydrogenase in the population of northern Thailand: Evidence for two common electrophoretic variants with deficient enzyme activity

Summary Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, identified by a dye decolorization test, was found in 101 (12.5 percent) of 811 male subjects from northern Tailand. Blood samples from 169 subjects with normal G-6-PD activity and from all 101 subjects with G-6-PD deficiency were examined by electrophoresis on cellulose acetate gel with the following results: In all samples with normal G-6-PD activity the enzyme had the electrophoretic mobility of type B G-6-PD. 73 of the 101 G-6-PD deficient samples had the same mobility and are therefore probably identical with the common Mediterranean variant B-. 16 of the 101 deficient samples contained an electrophoretically fast G-6-PD, and 1 sample a slow variant. In 11 deficient samples the enzyme could not be made visible. Kinetic studies on crude hemolysates suggest that the fast variant has a higher mean activity and heat stability in comparison to the B- variant.Established and supported by Stiftung Volkswagenwerk, Hannover.     

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.     

X linkage of phosphoglycerate kinase in the mouse

The levels of phosphoglycerate kinase (PGK), glucose 6-phosphate dehydrogenase (G6PD), and lactate dehydrogenase (LDH) were measured in one-cell embryos from X/0 and X/X females. Since the level of both PGK and G6PD was dependent on the number of X chromosomes present in the mother, these two enzymes are most likely coded for by X-linked genes. The level of LDH was the same in both types of embryos, indicating autosomal linkage. A search for an electrophoretic variant of PGK was not successful.     

Enzyme specificity: “pseudopolymorphism” of lactate dehydrogenase in Drosophila melanogaster

An electrophoretic variant in the LDH (l-lactate:NAD oxidoreductase, E.C.1.1.1.27) of Drosophila melanogaster was observed on starch (or polyacrylamide) gels. This variant was found to exhibit an identical isozymic pattern (three isozymes with a decreasing staining density) on starch gel and map position as the Adh locus. On the other hand, anodal polyacrylamide gel electrophoresis in crude extracts has shown LDH to consist of nine bands and ADH of four bands. We have shown that ADH (Alcohol:NAD oxidoreductase, E.C.1.1.1.1) also oxidizes l(+)-lactate or d(–)-lactate with the NAD, while LDH oxidizes ethanol. By using various genetic and biochemical techniques, we have shown that the observed Ldh electrophoretic variant was not a real one and could be attributed to the presence of ADH. We have called this phenomenon pseudopolymorphism, and the problem of enzyme specificity has been examined. The appearance of a band in an assay using lactic acid as a substrate is not sufficient evidence for the presence of LDH. Hence, caution is called for before characterizing an electrophoretic band on a gel as being equivalent to the presence of a genetic locus. Out of the nine electrophoretic zones of activity observed on polyacrylamide gel (or out of the six previously observed) using crude extract, only two (one major and one minor) belong to LDH, as revealed by purified enzyme preparations. Furthermore, purified LDH exhibits activity in two bands on starch gel (out of three observed in crude extracts), which appear in different positions as compared with those of ADH. Finally, one band which responds to the presence of d(–)-lactate but not to l(+)-lactate has been revealed.     

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.     

‘Gd(-) Hôtel Dieu’: A new G-6PD variant with chronic hemolysis in a Negro patient from Senegal

Summary A G-6PD deficiency was detected in a Negro patient from Senegal suffering from congenital nonspherocytic hemolytic anemia.The main characteristics of this variant were: profound defect of G-6PD activity in the red cells, decreased immunologic specific activity, fast electrophoretic mobility, decreased K_m-G-6P and normal K_m-NADP⁺, normal inhibition by ATP and NADPH, slightly increased utilization of the substrate analogues, slightly biphasic pH curve, high heat lability, subnormal activation energy.The characteristics of this variant being unique, it was called G-6PD Hôtel Dieu.     

Differential activity and synthesis of lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) in mouse spermatogenic cells

Spermatogenic cells isolated from adult and prepubertal mice by unit gravity sedimentation were used to examine enzyme activities and synthesis of the lactate dehydrogenase (LDH) isozymes during spermatogenesis. The synthesis and activity of LDH-C4, the germ cell-specific isozyme, was detected earliest in isolated preleptotene and leptotene/zygotene spermatocytes prior to the mid-pachytene stage of meiosis reported previously. The LDH-C4 isozyme was prominent in pachytene spermatocytes, round spermatids, and condensing spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. In addition, somatic-type LDH isozymes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. This is in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli cells were further shown to exhibit comparable amounts of five tetrameric LDH isozymes formed by combination of muscle-type LDH-A and heart-type LDH-B subunits.     

Isolation of a lactic dehydrogenase-A-deficient CHO-K1 mutant by nylon cloth replica plating.

A mutant Chinese hamster ovary cell deficient in lactate dehydrogenase A activity has been isolated using a nonselective technique. The method uses histochemical staining to examine colonies directly for enzyme activity and nylon cloth replica plating to recover particular clones. The mutant cell has an apparent Km (pyruvate to lactate) that is nearly tenfold higher than the parental cell, while its Vmax has been reduced more than 80-fold. In mutant cell extracts with added porcine LDH-B enzyme, molecular dissociation and recombination of subunits produces two new active LDH tetramers (A1B3, A2B2). The electrophoretic mobility of at least one of the tetramers (A1B3) was different from those formed in the parental extracts. The evidence suggests the variant cell contains a mutation in the structural gene for LDH-A.     

Variation of glucose 6-phosphate dehydrogenase in the HeLa and Detroit 98 cell lines and their clonal derivatives

Contrary to other reports, we have found that the A type G6PD found in two permanent cell lines—HeLa (Gey), with its single cell clonal derivative HeLa S3, and Detroit 98, with its four clonal derivative lines—is not a single variant but rather at least three different isozymes. One is heat stable with normal specific activity and normal A type electrophoretic migration, another is heat labile with normal specific activity and normal A type electrophoretic migration, and the third is heat labile with reduced specific activity and slightly slow A type electrophoretic migration. We also found that in a mosaic cell population with respect to G6PD phenotype, the predominant G6PD phenotype varied randomly over a 5-month period, that the G6PD phenotype might be mutable in permanent cell lines, and that spontaneous human cell lines might not be HeLa cell contaminants as has been suggested.Aided by the National Institutes of Health General Research Support Grant # 5 S01 FR05507.     

Molecular characterization of genetic mutations in human lactate dehydrogenase (LDH) B (H) variant

Summary We have previously detected a single base substitution of G by A at the Arg codon CGC in exon 4 of the mutant lactate dehydrogenase (LDH) gene, an unstable LDH-B variant (case 1). Here, we use the polymerase chain reaction (PCR) to amplify genomic DNA of two cases (the original case 1 and a new patient, case 2). We were able to confirm that case 1 is homozygous for the mutation, causing a replacement of the conserved Arg by His at residue 173. The resulting LDH-B variant subunit is unstable in vivo. Whereas the mutation in exon 4 was not observed in case 2, a different single base substitution of A by C was detected at the Ser codon AGT in exon 3. This mutation causes a replacement of the conserved Ser by Arg at residue 131. Genomic analysis of the family of case 2 by mismatched PCR showed that the missense mutation was consistent with their biochemical phenotypes. The replacement results in a conformational change of the residues near the Ser, probably because the side chain of Arg is much more bulky than that of Ser. The change may affect the arrangement of the cofactor binding site and result in the loss of enzyme activity. The experimental observations are consistent with computer graphics analyses.     

A missense mutation found in human lactate dehydrogenase-B (H) variant gene

A human lactate dehydrogenase-B mutant gene was isolated from a genomic DNA library constructed from a patient with unstable LDH-B (heart) subunit. The nucleotide sequences of seven protein-coding exons were determined and a single nucleotide substitution of G by A at Arg codon CGC in exon 4 was found. This mutation results in an amino-acid replacement of a conserved arginine by histidine at the residue "173," which is involved in an anion-binding site at the P-axis of LDH subunits.     

‘GPI Roma’, a new glucose phosphate isomerase deficient variant

Summary In a 5-year-old Italian girl with severe congenital hemolytic anemia, red cell GPI deficiency was proven, and found to be due to a new variant, GPI Roma. The parents are first cousins and have been proven to be heterozygous for this variant.GPI Roma was slightly unstable to heat and exhibited a slightly increased Michaelis constant for fructose-6-phosphate. A single predominant fastmigrating GPI form existed in the patient's white blood cells, while the electrophoretic pattern in the red cells was composed, in addition to this fast band, of a major band migrating as normal GPI and of an additional slow band. It is shown that this phenomenon may be ascribed to postsynthetic events modifying the charge of the mutant enzyme.With the technical assistance of R. Kernemp     

Patterns of gene expression during teleost embryogenesis: Lactate dehydrogenase isozyme ontogeny in the medaka (Oryzias iatipes)

The ontogeny of the lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes during medaka (Oryzias latipes) embryogenesis was determined after the genetic and molecular bases of this multilocus isozyme system were established. Three LDH loci are differentially expressed among the tissues of the adult medaka. The LDH-A locus was expressed almost exclusively in the white skeletal muscle, the LDH-B locus in all tissues examined, and the LDH-C locus in the eye and brain. The contribution of each of these LDH loci was quantitatively determined throughout early medaka embryogenesis by using a combination of electrophoretic, immunochemical, and spectrophotometric procedures. LDH-B₄ is present throughout embryogenesis and is the predominant LDH isozyme during this period. LDH-C subunit activity was first detected 146 hr after fertilization (26°C), 142 hr prior to hatching. LDH-A subunit activity, however, was not detected until after hatching and, then, only as heterotetramers containing LDH-B subunits. The pattern of LDH gene expression during medaka embryogenesis was compared with the patterns of LDH gene expression during early development in five other teleost species. Some common patterns of differential LDH gene expression appear to exist among the teleosts. In all species examined, isozymes encoded in at least one LDH locus, A and/or B, were present throughout development. Those isozymes present continually during embryogenesis also tend to be active in a wide variety of differentiated tissues in the adult fish. Conversely, LDH isozymes which are active in a restricted number of adult tissues are detected only later in embryogenesis. The initiation of LDH-C gene expression, however, is closely coupled with morphological and functional differentiation of those cells in which this locus is predominantly expressed in the adult.     

A new locus regulating the expression of the Ldh-2 gene in mouse liver

Patterns of lactate dehydrogenase (LDH) isozymes of tissues from various mouse strains were examined. An interstrain polymorphism for LDH isozymes of liver was established. One phenotype (CBA/Lac and AKR/J mice) yielded a five-banded LDH pattern, another phenotype (DBA/1J, DBA/2J, C57BL/6J and C3H/He) showed a three-banded one. Immunochemical evidence was obtained indicating that differences in the LDH pattern are mainly due to different contents of the B subunit of LDH. Linkage tests indicated that the locus Ldr-2 determining the amounts of the LDH B subunit in mouse liver tissue is located in chromosome 6, 19 ± 4.1 cm away from the earlier described Ldr-1 locus. The effect of locus Ldr-2 is strictly tissue-specific; it is manifest only on days 6–8 after birth.     

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.     

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。     

6-phosphogluconate dehydrogenase activity variants inMusca domestica L.: A further allele at thePgd locus as proved by densitometric assay

A new electrophoretic variant of 6-phosphogluconate dehydrogenase (6PGD) has been detected in flies of a laboratoryMusca domestica strain. This variant is to be added to the two already described, PGD-A and PGD-B, identified by a fast-weak and a slow-thick electrophoretic band, respectively. The new variant, PGD-C, has the same mobility as PGD-A but provides a more intensely stained band; therefore it can be described as a fast-thick phenotype. The staining intensity of PGD-C is slightly lower than that of PGD-B. Genetic and densitometric tests have shown that the different levels of enzymatic activity of the two fast variants A and C are inherited as alternative genetic units, and they have been interpreted as one aspect of the phenotypic expression of twoPgd alleles, namely,Pgd ^A andPgd ^C. These alleles determine both the rates of electrophoretic mobility (fast in both cases) and the levels of activity (low for A, strong for C; shown by weak or thick stained electrophoretic bands). Similarly, the two distinctive features of PGD-B, namely, slow mobility and high activity level, are always jointly inherited and appear as two pleiotropic aspects of the phenotype coded for by thePgd ^B allele. ThePgd ^B/Pgd^C heterozygous flies provide a slightly asymmetrical three-banded zymogram, while thePgd ^A/Pgd^C combination leads to a single-banded pattern, showing the same mobility as the parents and an intermediate staining intensity. The quantitative analysis of enzyme activity of 6PGD zymograms, performed through densitometric methods, has led to the recognition of three different activity levels coded for byPgd alleles, one of which, namely,Pgd ^C, would not have been detected using electrophoretic methods alone.