Use of differences in substrate inhibition for determination of the interrelationship between molar fractions of lactate dehydrogenase subunits]

A kinetic method of estimating the ratio of mole quota of H and M human lactate dehydrogenase (LDG) subunits is proposed, based on changes in substrate inhibition of LDG isoenzymes with lactate. Stability of kinetic constants for a long period of time is demonstrated. The dependency of activities ratio under low and high substrate concentration on the contribution of mole quota of LDG M subunits is studied. The correlation of experimental and theoretical values is shown to be: r=0.998 p less than 0.001. A comparison is carried out of the content of LDG subunits molar quotas in artificial mixtures with electrophoretic experimental data, a good coinsidence of these values being registered. The informative importance of the method described with standard methods of the estimation of LDG isoenzyme systems is discussed. No effect of components of human diploid cells homogenate and an insignificant effect of blood serum components on kinetic constants of LDG isoenzymes is registered. A dependency of variation coefficients on the enzyme activity is studied, minimal omegan value being 0.6%. The applicability of the method described for the calculation of quantitative content of both LDG subunits in natural objects (blood serum, diploid cell homogenate etc.) is demonstrated.     

Lactate dehydrogenase isoenzymes of sperm cells and testes

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDH_x from human sperm cells and rat testes were studied. 2. LDH_x shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H₄ and LDH-M₄ isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18·8% in the head fraction. LDH_x was found in all particulate fractions of sperm cells. The middle piece contained 41·0% of total LDH_x activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7·5 and with NADPH as coenzyme at pH6·0. At pH6·0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD⁺. 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD⁺/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDH_x. 9. The findings described are consistent with the idea that LDH_x is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.     

Additional lactate dehydrogenase (LDH) isoenzymes in normal testis and spermatozoa of adult man

Summary Adult human testicular tissue contains up to six previously undescribed lactate dehydrogenase (LDH) isoenzymes in addition to the five LDH isoenzymes normally found and the sixth found in spermatogenic cells and spermatozoa, LDH-X. Additional LDH isoenzymes were also found in spermatozoa but not in seminal fluid or in serum. After electrophoresis one additional LDH isoenzyme of testicular tissue was localized between LDH-1 and LDH-2, two between LDH-2 and LDH-3, two between LDH-3 and LDH-4, and two between LDH-4 and LDH-5. These localizations indicate that the additional LDH isoenzymes are tetramers combining the A and B subunits of the five normal LDH isoenzymes and the C subunit of LDH-X. The additional LDH isoenzymes may be important in the metabolism of spermatogenic germ cells and spermatozoa.     

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.     

Kinetic studies on human lactate dehydrogenase isoenzyme-catalyzed lactate-to-pyruvate reaction

In order to evaluate the functional differences that may exist in human lactate dehydrogenase (LDH) isoenzymes widely used for clinical examination the kinetic and thermodynamic properties of the lactate to pyruvate reaction that they catalize were examined. Small but significant differences in the kinetic properties of the three isoenzymes were observed. The difference in the rate constants might affect the activity measurement of the individual isoenzyme as the initial velocity for the L-P reaction catalyzed will not be the same for an equal amount of enzyme. Equilibrium constants for the overall reaction in the presence and absence of pyruvate have been determined. On the basis of transition-state theory, the standard enthalpy and free-energy changes for formation of ternary activated complex were positive, while the standard entropy change was negative. Although the standard free-energy change was the same for activation by the three isoenzymes, the enthalpy and entropy changes for the LDH-3-catalyzed reaction were different from the respective values for others. A large positive value for the free-energy change and a negative value for the entropy change indicated unfavorable production of the activated complex (K _infeq. ^sup╪ =1.89×10^-16). The enzyme appears to stabilize and retain the activated complex until it dissociates into the products.     

Changes in the mole fraction ratio of lactate dehydrogenase subunits in the lymphocytes of Down's syndrome patients]

A comparative study of the total activity and mole quota ratio of lactate dehydrogenase subunits in lymphocytes of 14 patients with Down's syndrome (trisomy-21) and in 10 healthy persons is carried out. Differences in the total activity in both groups were insignificant. In patients with Down's syndrome the mole quota ratio of H and M subunits of LDH was found to be significantly altered (p greater than 0.999): H = 33.2%, M - 66,8%, as compared to 51.5% and 48.4% in the control (healthy) group respectively. These differences are evaluated as a result of changed gene expression of both loci controlling H and M polypeptide chains of heteromeric enzyme molecule.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

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.     

6-Phosphogluconate Dehydrogenase Isoenzymes from the Developing Endosperm of Ricinus communis L

The cytosolic and proplastid isoenzymes of 6-phosphogluconate dehydrogenase were purified from the developing endosperm of the castor bean (Ricinis communis L.). No differences in physical or kinetic properties were found for the purified isoenzymes. Each was composed of two identical 55,000 subunits. They had identical pH optima of 7.8 to 8.0 and similar MgCl₂ stimulation for the oxidative decarboxylation of 6-phosphogluconate. The Km values for 6-phosphogluconate were 12 and 9.6 micromolar and for NADP⁺ were 4.1 and 5.4 micromolar for the cytosolic and proplastid isoenzymes, respectively. Therefore, the synthesis of two distinct 6-phosphogluconate dehydrogenase isoenzymes does not appear to have any kinetic significance for the developing seed. However, changes in the proplastid contribution toward carbohydrate metabolism occur in the developing seed and may necessitate independent gene expression to allow for a unique and flexible subcellular distribution of isoenzymes during development.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

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.     

Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue

Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O₂ and greatest at 0.06%, the lowest O₂ concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O₂ level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-¹⁴C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [¹⁴C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis.     

Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate.

Soluble lactate dehydrogenase (EC 1.1.1.27) extracted from brain, skeletal and cardiac muscle and liver of rats, and purified isoenzymes LDH-1 and LDH-5, were incubated with sodium deoxycholate. Deoxycholate almost totally inactivated isoenzyme LDH-5 (A4), whereas it left isoenzyme LDH-1 (B4) unaffected. Tissue lactate dehydrogenase was inactivated to different degrees depending on the origin of the enzyme. Electrophoretic isoenzyme studies of tissue lactate dehydrogenase showed the loss of activity to be quantitatively related to the overall percentage of subunit A distributed among the homotetramer LDH-5 and the heterotetramers LDH-2, LDH-3 and LDH-4. It was concluded that subunit A of lactate dehydrogenase interacts selectively with deoxycholate, irrespective of its association with subunit B. Distinct changes in electrophoretic mobilities of deoxycholate-treated isoenzymes strongly indicated an indiscriminate binding of deoxycholate by all LDH isoenzymes, probably through hydrophobic interactions. The results suggest that the inactivation of the enzyme is non-competitive, but the basis of the selectivity of deoxycholate towards subunit A is not known at present.     

Characterization of potato (Solanum tuberosum) lactate dehydrogenase isoenzymes by affinity chromatography and hybridization

The five isoenzymes of potato (Solanum tuberasum) lactate dehydrogenase have been resolved by affinity chromatography. Mixtures of isoenzymes LDH-1 and LDH-5 dissociate and reassociate during freezing and thawing to produce five isoenzymes. These results indicate that potato lactate dehydrogenase isoenzymes are primary isoenzymes of the vertebrate type, which are composed of two subunit types.     

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.     

Studies on lactate dehydrogenase activity in the shrimp Palaemon serratus. Part 1: Characterization of the enzymes from the caudal muscle of Palaemon serratus

Palaemon serratus muscle lactate dehydrogenase (LDH) has been partially purified. The electrophoretic pattern of the LDH enzymes indicates that at least two molecular structures are present. The mean molecular weight is 130,000. The substrate and coenzyme dependence of the LDH system show non-Michaelian kinetics. This can be interpreted as being due to the presence of two binding sites in the enzyme which show negative effects. The behaviour of the two molecular species of LDH appears to be nearly identical in this respect. The study of the effect of temperature on the kinetic parameters of the LDH system shows the existence of a thermal dependence of K_m values. This may be related to developmental or environmental changes in the animal.     

Properties of water-insoluble matrix-bound lactate dehydrogenase.

Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower K_m values for its substrates. K_m values for pyruvate and lactate were 8 × 10 ^?5m and 4 × 10^?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H₄) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M₄) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules.     

A novel enzyme-immobilization method for a biofuel cell

Biofuel cells utilizing biocatalysts are attractive alternatives to metal catalyst-based cells because of environmentally friendly cells and their renewability and good operations at room temperatures, even though they provide a low level of electrical power. In this study, the effect of a novel enzyme immobilization method on anodic electrical properties was evaluated under ambient conditions for increasing the power of an enzyme-based biofuel cell. The anodic system employed in the cell contained a gold electrode, pyrroloquinoline quinone (PQQ) as the electron transfer mediator, lactate dehydrogenase (LDH), β-nicotinamide adenine dinucleotide (NAD⁺) as the cofactor, and lactate as the substrate. The anodic electrical properties increased as a result of the novel enzyme-immobilization method. Furthermore, lactate, NAD⁺, or CaCl₂, which can all influence enzyme activation, were used to prevent covalent bond formation near the active site of the LDH during enzyme-immobilization. Protection of the active site of the LDH using this novel enzyme-immobilization method increased its stability, which enabled to increase power production (142 μW/cm²) in a basic enzymatic fuel cell (EFC).