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.     

Genetic polymorphism of blood proteins in the troops of Japanese macaques,Macaca fuscata: II. Erythrocyte lactate dehydrogenase polymorphism inMacaca fuscata

In investigating the genetic marker for population genetics of Japanese macaques by electrophoresis, the author found the erythrocyte lacate dehydrogenase (LDH) polymorphism existing in some troops. There were four kinds of variations which seemed to be controlled by two loci, controlling A and B subunits of this enzyme. The variant phenotypes were named LDH-A_mac2-1 LDH-B_mac1-1, LDH-A_mac3-1 LDH-B_mac1-1, LDH-A_mac 1-1 LDH-B_mac2-1, and LDH-A_mac1-1 LDH-B_mac2-2. The two former types seemed to be heterozygote of a mutation controlling subunit A; the third was heterozygote, and the last homozygote, a mutation controlling subunit B. LDH-A_mac2-1 LDH-B_mac1-1 was found in Takasakiyama A, Takasakiyama B, Takasakiyama C, and Kamae troops; LDH-A_mac3-1 LDH-B_mac1-1 was in an individual of the unknown group. LDH-A_mac1-1 LDH-B_mac2-1 and LDH-A_mac1-1 LDH-B_mac2-2 were found in the Kohchi troop. All other troops were found to be monomorphic.     

Thermostability of lactate dehydrogenase LDH-A4 isoenzyme: Effect of heat shock protein DnaK on the enzyme activity

Cells exposed to temperature a few degrees higher than their growth temperature synthesize heat shock proteins (hsp) which may then compose even 20% of total protein content. This paper examined the in vitro protective effect of heat shock protein DnaK (70 kDa) from Escherichia coli against the heat inactivation of lactate dehydrogenase isoenzyme LDH-A₄. The LDH-A₄ isoenzyme was purified from fish skeletal muscle using the affinity chromatography on Oxamate-agarose. The enzyme was then heated in the absence and the presence of DnaK protein in a water bath at either 51 or 55°C. The LDH activity was determined by measuring the change in absorbency at 340 nm min⁻¹ at 30°C. The addition of DnaK protein to the LDH-A₄ isoenzyme before heat treatment can protect enzyme activity against mild thermal inactivation. Incubation of the LDH-A₄ isoenzyme at 51°C in the presence of DnaK protein stimulates its activity by about 30%. The presence of 2 mM ATP can raise LDH activity by another 10%. No significant recovery was observed when DnaK protein was added to LDH at 25°C following earlier inactivation. The maximal activities (V_max) in the presence of DnaK protein are almost twice those without DnaK protein in the case of heat-treated LDH-A₄ isoenzyme at 51°C. The observed protection of LDH-A₄ activity increased with the increasing DnaK protein concentration in the incubation medium. Results suggested that the presence of DnaK protein can protect LDH-A₄ from heat inactivation. This action may be important as a part of cellular chaperone machinery capable of repairing heat-induced protein damage. It may have a fundamental role in the acquisition of the thermotolerance to stress temperatures.     

Methods for the separation of lactate dehydrogenases and clinical significance of the enzyme

Lactate dehydrogenase (LDH), an ubiquitous enzyme among vertebrates, invertebrates, plants and microbes was discovered in the early period of enzymology. The enzyme has been dissolved in several distinguishable molecular forms. In mammals, three types of subunits encoded by the genes Ldh-A, Ldh-B and Ldh-C give rise to a selected number of tetrametric isoenzymes. LDH-A₄, LDH-B₄ and the mixed hybrid forms of the A- and B-subunits are present in many tissues but with certain distribution patterns. LDH-C₄ is confined in mammals to testes and sperm. Numerous techniques have been employed to purify, characterize and separate the different forms of the enzyme. This report deals with the main protocols and procedures of purification of LDH and its isoenzymes including chromatographic and electrophoretic methods, partitioning in aqueous two-phase systems and precipitation approaches. In particular, affinity separation techniques based on natural and pseudo-biospecific ligands are described in detail. In addition, basic physico-chemical and kinetic properties of the enzyme from different sources are summarized. In a second part, the clinical significance of the determination of LDH in diverse body fluids in respect to the total activity and the isoenzyme distribution in different organs is discussed.     

Characterization of two electrophoretic lactate dehydrogenase-A mutants inMus musculus

Two lactate dehydrogenase (LDH) mutations were recovered independently among offspring of ethylnitrosourea-treated male mice by screening for alterations of isoelectric focusing pattern in liver homogenates. Investigations of physicochemical and kinetic properties of the mutant enzymes indicated that the mutant traits resulted from point mutations at theLdh-1 structural locus. Therefore, the new alleles were designatedLdh-1 ^a-m5Neu andLdh-1 ^a-m6Neu, respectively. Both mutant alleles code for proteins which exhibit an altered stability to heat, in addition to changes in isolectric focusing pattern and a reduction in anodal electrophoretic mobility. While LDH-A^a-m5Neu proteins are markedly less heat stable, LDH-A^a-m6Neu proteins are more heat stable than the wild-type enzyme. Furthermore, a small elevation ofK _m for pyruvate, a slightly reduced inhibition by high pyruvate concentrations, and a slight acidic shift of the pH activity profile distinguish LDH-A^a-m6Neu from both wild-type and LDH-A^a-m5Neu enzymes. Significant alterations of LDH activity were detected in some tissues from LDH-A^a-m5Neu individuals but not in those from LDH-A^a-m6Neu animals. Erythrocytes and blood of LDH-A^a-m5Neu mutants revealed activity levels which were reduced by approximately 6 and 13% compared with those of wild types in heterozygous and homozygous individuals, respectively. In addition, an elevation of approximately 6% in LDH activity was found in skeletal muscle in homozygous mutants. Consistent with the unaltered or only slightly altered LDH activity in tissues, the genetic as well as the physiological characterization yielded no easily detectable effects from either mutation on metabolism or fitness of the affected individuals.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities.     

Purification and Properties of the Threespine Stickleback (Gasterosteus aculeatus) Lactate Dehydrogenase LDH-B4 and LDH-C4 Isoenzymes

In the threespine stickleback (Gasterosteus aculeatus) lactate dehydrogenase (LDH, EC 1.1.1.27) is encoded by three loci, Ldh-A, Ldh-B, and Ldh-C. LDH-B₄ isoenzyme restricted its function to eye and brain, while LDH-C₄ isoenzyme functions in the eye. In the Dead Vistula stickleback population, none of LDH loci is polymorphic. The LDH-B₄ and LDH-C₄ isoenzymes from the eye were purified to homogeneity to specific activity of 186 and 229 μmol NADH min⁻¹mg⁻¹, respectively, at 30°C. Some physico-chemical and kinetic properties revealed that eye LDH-C₄ isoenzyme was more thermostable and had a higher affinity to pyruvate than LDH-B₄ isoenzyme. Lower Km for pyruvate of eye LDH-C₄ isoenzyme distinguishes it from fish LDH-C₄ isoenzyme isolated from liver.     

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.     

Lactate dehydrogenase (LDH) in 27 species of amazon fish: Adaptive and evolutive aspects

• 1.1. Among the 27 species of Amazon fish belonging to the orders Rajiformes, Clupeiformes, Osteoglossiformes, Characiformes, Siluriformes and Perciformes here analyzed, 56% showed an electrophoretic pattern of five, 7% of four, 30% of three, and 7% of two LDH isozymes, suggesting the presence of both LDH-A¹ and LDH-B¹ loci. In addition to these loci, the third gene LDH-C¹ was detected only in the Osteoglossiform species O. bicirrhosum and in the perciform species P. squamosissimus, with a generalized expression in the first and a restricted in the second.
• 2.2. Only P. squamosissimus (Perciformes) showed a LDH reversed pattern, in which the A₄ is more anodic than the B₄.
• 3.3. Like other vertebrates, in most (93%) of the species here analyzed, a direct correlation between electrophoretic mobility and thermostability was observed. The inactivation temperatures varied from 55°C in the Rajiformes species of 70°C in the Perciformes species.
• 4.4. Polymorphism in at least one of the LDH loci was detected in 22% of the species studied here: P. castelnaena (Clupeiformes) and B.cf. cephalus (Characiformes) at the LDH-A¹ locus, R. myersi and H. unitaeniatus (both Characiformes) at the LDH-B¹ and L. agassizi (Characiformes) at both loci.
• 5.5. No modifications of the classic LDH pattern found by other authors in organisms routinely subjected to hypoxic stress were observed in these Amazon species. In 93% of the species screened here, subjected to considerable hypoxic stress, large daily oscillations in temperature, O₂ and CO₂ levels, pH, low ionic content, and seasonal drought, a bidirectional pattern of expression of the LDH loci was observed.

     

A genetically controlled lactate dehydrogenase variant at the B locus in mink

Erythrocytes of 119 mink, and tissue extracts of three mink, were examined for electrophoretic patterns of lactate dehydrogenase (LDH). A variant was detected at the B locus. There are two alleles, LDH-B ^a and LDH-B ^b; three phenotypes, LDH-Ba, LDH-Bab, and LDH-Bb; and three genotypes, LDH-B ^a/LDH-B^a, LDH-B^a/LDH-B^b, and LDH-B ^b/LDH-B^b. The inheritance as observed in 24 families agrees with an autosomal, codominant, two-allele system at the LDH B locus.Supported by National Research Council Grant A-4442 and the Ontario Department of Agriculture and Food.     

HISTOCHEMISTRY OF LACTIC DEHYDROGENASE IN HEART AND PECTORALIS MUSCLES OF RAT

Left-ventricular heart muscle and pectoralis major muscle of the rat were studied to determine the intracellular localization of lactic dehydrogenase (LDH) isoenzymes. Fixation of tissue for 2 hr in 2% buffered formaldehyde provided the best preservation of the ultrastructure and enzyme activity. Total LDH activity was found diffusely in the ground substance of the sarcoplasm and in the mitochondria of the heart muscle. In skeletal muscle a strong reaction was noted in the sarcoplasmic reticulum, and moderate activity was seen in the ground substance of the sarcoplasm and in the mitochondria. Differentiation of the isoenzymes of LDH was accomplished by addition of 4 M urea or application of heat. Heart-type isoenzymes were mainly localized in the mitochondria and sarcoplasm, whereas muscle-type isoenzymes were localized mainly in the sarcoplasmic reticulum of the skeletal muscle. It is speculated that the sarcoplasmic reticulum of the skeletal muscle is the site of anaerobic glycolysis and that the sarcoplasm and mitochondria are involved primarily in aerobic metabolism of pyruvate.     

Yak Lactate Dehydgogenase A4: Purification,Properties, and cDNA Cloning

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.     

Different Pressure Resistance of Lactate Dehydrogenases from Hagfish is Dependent on Habitat Depth and Caused by Tetrameric Structure Dissociation

The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined. LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure of LDH-A₄ from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures.     

Lactate dehydrogenase in abdominal muscle of crayfishOrconectes limosus and shrimpcrangon crangon (Decapoda: Crustacea): Properties and evolutionary relationship

In crayfishOrconectes limosus and shrimpCrangon crangon abdominal muscle, lactate dehydrogenase (LDH, EC 1.1.1.27) is encoded by one locus. No polymorphism was detected. The enzymes were purified to homogeneity. The specific activities for purified crayfish and shrimp LDHs were 472 and 414 μmol NADH min⁻¹ mg⁻¹, respectively, at 30°C. Their physicochemical and kinetic properties did not resemble fish (Gadus morhua) LDH-A₄ isoenzyme. Their amino acid composition indicated greater similarity to fish LDH-C₄ isoenzymes.     

Lactate dehydrogenase isozymes in type I,IIA and IIB fibres of rabbit skeletal muscles

Summary Lactate dehydrogenase (LDH) isozyme patterns were analysed by polyacrylamide (PAA) slab gel electrophoresis in extracts prepared from various rabbit skeletal muscles of defined fibre composition and by PAA microelectrophoresis of microdissected, histochemically typed single muscle fibres. The results obtained by electrophoresis of whole muscle extracts generally agreed with the data obtained from single fibre electrophoresis, i.e. the LDH isozyme pattern corresponded to that of the predominant fibre type. Type I Fibres from soleus and semitendinosus muscles were characterized by a unique pattern of all 5 LDH isozymes with a predominance of LDH-1, 2 and 3. The major fraction (80%) of the type II fibres from extensor digitorum longus and tibialis anterior muscles contained only LDH-5 (M₄). About 20% of the type II fibres contained in addition to LDH-5 small amounts of LDH-4 and LDH-3. The fraction of fibres containing LDH-5, LDH-4, and LDH-3 was similar (ca. 20%) in the histochemically defined IIA and IIB subpopulations In view of the fact that the major fractions of rabbit IIB fibres display low and of IIA fibres high aerobic oxidative capacities (Reichmann and Pette 1982), these data indicate that the expression of the H-subunit of LDH is not correlated with the aerobic-oxidative capacity of the fibre. It also appears not to be correlated with the presence of different myosin isoforms in IIA and IIB fibres.     

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.     

Isoenzyme spectra of lactate dehydrogenase in tissues of mammals of the Mustelidae family

A comparative analysis of distribution of lactate dehydrogenase (LDH) isoenzymes in the heart, kidneys, lungs, spleen, liver and skeletal muscle was performed in 4 species of carnivorous mammals (Mustelidae family): American mink (Mustela vison Schr.), polecat (Mustela putorius L.), sable (Martes zibellina L.) and pine marten (Martes martes L.). It was found that in the mink, unlike pure terrestrial mustelids (polecat, sable, pine marten), the anaerobic LDH-5 fraction was predominant in the LDH isoenzyme spectrum in the liver, skeletal muscle, lungs and spleen. In all species studied, aerobic B(H) subunits of LDH were found to prevail in the heart and kidney, whereas anaerobic A(M) subunits prevailed in the liver and spleen. The tissue- and species-specific features of LDH isoenzyme spectra revealed in different mustelids reflect the biochemical adaptation of the animals to environmental conditions.     

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO₂) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO₂. However, subsequent addition of NAD⁺ significantly increased JO₂, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD⁺-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.     

High-Altitude Adaptation of Yak Based on Genetic Variants and Activity of Lactate Dehydrogenase-1

This study investigates the molecular mechanism by which yaks (Bos grunniens) adapt to hypoxia based on lactate dehydrogenase (LDH). Three LDH1 variants of the yak were revealed in tissue extracts by electrophoresis, including LDH1-F, LDH1-M, and LDH1-S. Kinetic analysis using purified LDH1 variants showed that the yak LDH1-M variant exhibited a similar K _m (NADH) and the same mobility on a gel as bovine LDH1, and the LDH1-F variant showed significant differences in K _m values for NADH or pyruvate from the other two variants of yak LDH1 and bovine LDH1. Among the three muscles assayed, yak longissimus dorsi showed the highest LDH activity and the lowest malate dehydrogenase (MDH) activity; heart muscle was exactly the opposite. Our results suggest that the three LDH1 variants might play an important role in the adaptation to hypoxia.     

乌鳢组织内三种同工酶的研究

本研究对乌鳢９种组织内的ＬＤＨ、ＭＤＨ和ＡＴＰａｓｅ同工酶作了分析，结果表明，乌鳢各组织内的ＬＤＨ、ＭＤＨ有明显的组织特异性，ＬＤＨ由两个基因编码。骨骼肌中的ｓ－ＭＤＨ也有两个基因编码。ＡＴＰａｓｅ同工酶存在于乌鳢的多种组织中，其基因编码数有待进一步探讨。     

Differences in some properties of lactate dehydrogenase from muscles of the carp Cyprinus carpio and trout Salmo gairdneri

Some catalytic and kinetic properties of lactate dehydrogenase (LDH) isolated from trout and carp skeletal muscles were compared. The specific activity of LDH in the carp muscle was lower by about one third than the activity in the trout muscle. Temperature and pH optima for LDH isolated from the carp muscle were higher than those for the trout muscle LDH. Moreover, in direct reaction, the carp muscle LDH had a higher affinity both for pyruvate and for NADH, i.e., it had lower K _M values. Instead, the trout muscle LDH showed the positive kinetic cooperativity (the Hill coefficient > 1) of the substrate and coenzyme binding sites. Thus, the carp LDH seems to function more effectively under anaerobic conditions and at higher temperatures.