Properties Of Lactate Dehydrogenase Research Articles

Physicochemical and Regulatory Properties of Lactate Dehydrogenase from Pea (Pisum sativum L.) Leaves under Oxygen Deficiency

Lactate dehydrogenase activity (LDH, EC 1.1.1.27) was induced in pea (Pisum sativum L.) leaves by culturing plants under flood conditions. The enzyme was purified to an electrophoretic homogeneous state with a multistage purification process including ammonium sulfate fractionation, ion exchange chromatography on DEAE-sephacel, and gel chromatography on Sephadex G-200. The LDH preparation had a specific activity of 41.9 E/mg protein, with a purification rate of 101 and a yield of 26.8%. Its physical and chemical features were studied. The molecular weight of the native LDH molecule (148 kD) was determined, and the enzyme was shown to consist of four subunits, the molecular mass of which was determined by PAGE-Na electrophoresis in the presence of SDS-Na equal to 37 kD. The kinetic and regulatory properties, such as the Michaelis constants, the substrate inhibition constants, the effects of hydrogen-ion and temperature on the direct and reverse reactions, were studied.

Каталитические свойства лактатдегидрогеназы в органах крыс с термической травмой при воздействии глутатион-содержащих динитрозильных комплексов железа

Каталитические свойства лактатдегидрогеназы в органах крыс с термической травмой при воздействии глутатион-содержащих динитрозильных комплексов железа

젖산탈수소효소 eye-specific C4동위효소의 생화학적 특성: 파랑볼우럭(Lepomis macrochirus)과 큰입우럭(Micropterus salmoides)

The properties of lactate dehydrogenase (LDH, EC 1.1.1.27) eye-specific isozyme were studied by polyacrylamide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme kinetics. Furthermore, we proposed the optimal conditions for measuring the activity of LDH eye-specific isozyme. The isozymes were detected in the cytosol of eye tissues from Lepomis macrochirus and Micropterus salmoides and were more similar to the than the isozyme. LDH/CS in the eye tissue of L. macrochirus was increased in September, so the ratio of anaerobic metabolism was high. The electrophoretic patterns of mitochondrial LDH were similar to those of cytosolic LDH in the eye tissues of L. macrochirus and Micropterus salmoides. LDH eye-specific isozyme from eye tissue was purified by preparative native-PAGE. The activities of LDH eye-specific isozymes in L. macrochirus and M. salmoides were reduced at concentrations greater than 0.2 mM and 0.1 mM of pyruvate, respectively. These concentrations remained at 5.2% and 15.8% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The LDH activities of eye tissues were reduced at concentrations greater than 22 mM and 24 mM of lactate, respectively, in L. macrochirus and M. salmoides. The of eye-specific was 0.088 mM in L. macrochirus and it was 0.033 mM in M. salmoides. The activities of cytosolic and mitochondrial eye-specific isozymes were high in -ketobutyric acid. Furthermore, the activities of eye tissue and eye-specific isozyme had to be measured with 0.5 mM of pyruvate and a buffer solution of pH 7.5. As a conclusion, the eye-specific isozyme in M. salmoides has a high affinity for pyruvate and exhibits maximum activity at a lower concentration of pyruvate and at higher concentration of lactate than that in L. macrochirus. Therefore, it seems that the energy produced by the LDH eye-specific isozyme in M. salmoides was used at the first stage of predatory behavior.

큰입우럭(Micropterus salmoides) 조직의 젖산탈수소효소 및 Monocarboxylate 수송체(MCT) 1, 2, 4

The properties of lactate dehydrogenase (EC 1.1.1.27, LDH) and expression of monocarboxylate transporters (MCTs) 1, 2, and 4 were studied in tissues from Micropterus salmoides. Native-PAGE revealed that the LDH isozyme was predominantly located in skeletal muscle. The LDH , , and isozymes were detected in heart, liver, eye, and brain tissues, while eye-specific isozyme was detected in eye tissue. In September, strong LDH isozyme activity was detected in heart tissue. High isozyme activity was noted in all other tissues except heart tissue. However, in November, strong isozyme activity was detected in heart tissue. The LDH/CS (Citrate synthase, EC 4.1.3.7) ratio in skeletal muscle and heart tissues indicated that anaerobic metabolism was high in those tissues. Native-PAGE after immunoprecipitation showed that eye-specific isozyme was more similar to the than the isozyme. The LDH isozyme was purified by affinity chromatography. The molecular weight of subunit A was 37,200. The LDH activity in tissues was consistently 11.05~28.32% due to inhibition by 10 mM pyruvate. The of LDH in eye tissue was very low. The optimum pH for LDH in tissues was pH 7.5~8.0. The LDH isozyme was detected in mitochondria of skeletal muscle, whereas the and isozymes were detected in heart tissue mitochondria. Western blot analysis indicated that MCTs 1, 2, and 4 were located in the plasma membrane and mitochondria of skeletal muscle and heart tissues. The sizes of MCTs 1, 2, and 4 in skeletal muscle were 60, 54~38, and 63 kDa, while those in heart tissue were 57, 54~38, and 55.5 kDa, respectively. In conclusion, M. salmoides appears to use anaerobic metabolism predominantly when adapted to a hypoxic environment. In highly activated skeletal muscle and heart tissue, energy production is controlled by inward and outward flows of pyruvate and lactate through MCTs 1, 2, and 4 in the plasma membrane and mitochondria, with effective adjustment by LDH isozymes.

Properties of lactate dehydrogenase from the isopod, Saduria entomon

Saduria entomon lactate dehydrogenase (LDH-A* 4) from thorax muscle was purified about 89 fold to specific activity 510 μmol NADH/min/mg using Cibacron Blue 3GA Agarose and Oxamate-Agarose chromatographies. The enzyme is a tetramer, with molecular weight of 140 kDa for the native enzyme and 36 kDa for the subunit. The isoelectric point was at pH 5.7. The enzyme possesses high heat stability ( T 50=71.5°C). The optimum pH for pyruvate reduction reaction was 6.5, while for lactate oxidation one, the maximum activity was at pH 9.1. The K m for pyruvate was minimal at 5°C, the average environmental temperature of the isopod. The K m values determined at 30°C and optimal pH for pyruvate reduction and lactate oxidation were 0.18 and 90.04 mM, respectively. Amino acid compositional analyses showed the strongest resemblance of the isopod isoenzyme to cod ( Gadus morhua) LDH-C 4.

Lactate dehydrogenase of Mugil sp. (Mugilidae, Perciformes). Lack of electrokinetic, thermostability and kinetic differences among individuals with different number of scales

The scale number in lateral sets (SNS) of Mugil sp. (Mugilidae, Perciformes) collected in the lagoon-estuarine region of Cananéia, State of São Paulo ranges from 33 to 39. Electrokinetic, kinetic and thermostability properties of lactate dehydrogenase (LDH) were tested to determine if individuals with different SNS correspond to different species or populations of mullet. As in many other teleosts, LDH-A*, LDH-B*, and LDH-C* loci were detected. Through a two-fold serial dilution method applied to 10 different tissues of Mugil sp., a bidirectionally divergent expression of these loci was suggested. No association among LDH electrophoretic pattern, thermal inactivation, kinetic responses and different SNS was observed. The apparent Km (pyr) values obtained here were similar to Km values obtained by other authors for muscle and heart LDH or their purified isoforms. The effect of NaCl on Km and Vmax values of Mugil sp. (35 and 39 SNS individuals) indicates that this salt behaves as a competitive inhibitor, since it decreases enzyme-substrate affinity. Thus, electrokinetic and thermostability behavior, Km and Vmax values and the effect of NaCl do not permit us to consider these mullets, with SNS ranging from 33 to 39, as belonging to different populations or species.

Functional Properties of Lactate Dehydrogenase from Dunaliella salina and Its Role in Glycerol Synthesis

The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) Vmax of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle.

Purification and properties of lactate dehydrogenase from liver of Uromastix hardwickii

Lactate dehydrogenase isoenzyme-1 was purified from liver of Uromastix hardwickii using colchicine-Sepharose and heat-inactivation methods. The crude enzyme showed four isoenzymes by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after native AGE and SDS-PAGE corresponding to a molecular weight of 34 kDa. The enzyme did not bind with DEAE-Sepharose at pH 7.2. The optimum pH for forward reaction was 7.5, while for reverse reaction, the maximum activity was at pH 9.5. The K m values for pyruvate, NADH, lactate and NAD + were 0.105, 0.045, 9.0 and 0.011 mM, respectively. The pyruvate showed maximum activity at about 150 μM and then starts showing inhibition at higher concentration. Pre-heating of enzyme showed that it was stable at 8°C for 30 min and at 100°C it became inactive immediately. Oxalate, glutamate, Cu 2+, Co 2+, Mn 2+, and Mg 2+ have shown inhibitory effects both for forward- and reverse-reactions. From these properties, we suggest that LDH-1 from Uromastix liver may be quite different from that of other vertebrates.

Исследование рН-зависимых структурных и функциональных свойств лактатдегидрогеназы M4 при действии ионов цинка

Исследование рН-зависимых структурных и функциональных свойств лактатдегидрогеназы M4 при действии ионов цинка

Properties of lactate dehydrogenase in a psychrophilic marine bacterium.

Lactate dehydrogenase (EC 1.1.1.27) from Vibrio marinus MP-1 was purified 15-fold and ammonium activated. The optimum pH for pyruvate reduction was 7.4. Maximum lactate dehydrogenase activity occurred at 10 to 15 degrees C, and none occurred at 40 degrees C. The crude-extract enzyme was stable between 15 and 20 degrees C and lost 50% of its activity after 60 min at 45 degrees C. The partially purified enzyme was stable between 8 and 15 degrees C and lost 50% of its activity after 60 min at 30 degrees C. The thermal stability of lactate dehydrogenase was increased by mercaptoethanol, with 50% remaining activity at 42 degrees C.

Properties of lactate dehydrogenase in a psychrophilic marine bacterium

Lactate dehydrogenase (EC 1.1.1.27) from Vibrio marinus MP-1 was purified 15-fold and ammonium activated. The optimum pH for pyruvate reduction was 7.4. Maximum lactate dehydrogenase activity occurred at 10 to 15 degrees C, and none occurred at 40 degrees C. The crude-extract enzyme was stable between 15 and 20 degrees C and lost 50% of its activity after 60 min at 45 degrees C. The partially purified enzyme was stable between 8 and 15 degrees C and lost 50% of its activity after 60 min at 30 degrees C. The thermal stability of lactate dehydrogenase was increased by mercaptoethanol, with 50% remaining activity at 42 degrees C.

Some properties of lactate dehydrogenase from the foot muscle of the giant African snail ( Achatina achatina)

1. 1. Lactate dehydrogenase in extracts of the foot muscle of the giant African snail Achatina achatina was found to be a d(−)-lactate-specific tetramer with a molecular weight of 139,000 ± 2000, and a subunit molecular weight of 35,000 ± 2000 was calculated for the predominant isoenzyme from its electrophoretic behaviour. 2. 2. The purified major isoenzyme showed optimal activity at pH 6.4–6.6 in the direction of lactate production but at pH 9.0 in the direction of pyruvate production. 3. 3. The Michaelis constants for pyruvate, d(−)-lactate, NADH and NAD + were calculated to be 0.412, 2.130, 0.001 and 0.025 mM respectively. 4. 4. High concentrations of pyruvate ( > 0.5 mM ) inhibited the enzyme whereas 200 mM dl-lactate did not.

Some Regulatory Properties of Lactate Dehydrogenase from Germinating Pea Plants

Summary Affinity chromatography on a column of AMP-Sepharose 413 was used for the preparation of electrophoretically homogeneous lactate dehydrogenase from germinating pea plants. The kinetic equation for two-substrate reactions was applied for determining the Michaelis constants for 4 intrinsic substrates - lactate, pyruvate, NAD+ and N AD H and the dissocia.tion constants of enzymeNAD+ and enzyme-NADH binary complexes. The molecular weight of the enzyme is approximately 144,000. The enzyme is inhibited by pyruvate in l’oncentrations higher than 1 mM. AMP, ADP and ATP act as competitive inhibitors towards the coenzymes. The inhibition by A TP is stronger by two orders of ten than inhibition by AMP and depends on the concentration of hydrogen ions in the medium. The results suggest that lactate dehydrogenase is eapable of regulating the pH of the plant cell.

Ontogenic studies of intestinal lactate dehydrogenase isozymes in the hamster.

Our study of the developing small intestine presents a detailed report of the catalytic isozymic and kinetic properties of lactate dehydrogenase. These results allow for a better understanding of the properties of this intestinal enzyme during devlopment as well as relating these properties to the changing metabolic roles of this tissue during development.

Glyoxylate Oxidation in Rat Liver and Kidney

The enzyme-catalyzed oxidation of glyoxylate to oxalate by subcellular particles of rat liver and kidney, and in the cytosol fraction (100,000 × g supernatant) of rat kidney, has been investigated. Catalytic activity is associated with liver peroxisomes and rough endoplasmic reticulum, but not with liver lysosomes and mitochondria. The activity associated with the endoplasmic reticulum has the properties of lactate dehydrogenase [EC 1.1.1.27], whereas the peroxisomal activity is attributed to both glycollate oxidase [EC 1.1.3.1.] and to lactate dehydrogenase. Among the particulate fractions of kidney, catalytic activity with respect to the oxidation of glyoxylate to oxalate is due to lactate dehydrogenase associated with the endoplasmic reticulum. The possibility that some of the catalytic activity in kidney tissue is associated with the lysosomes cannot be completely excluded on the basis of the present evidence. No evidence was obtained for peroxisomal or mitochondrial enzymes with catalytic activity with respect to the oxidation of glyoxylate to oxalate in renal tissue. The catalytic activity for the oxidation of glyoxylate to oxalate in the kidney cytosol fraction was NAD+ dependent and associated with lactate dehydrogenase. These results are discussed in relation to previous work and to the problem of excessive oxalate production in man. It would be theoretically desirable to treat the hyperoxaluric diseases by reducing the excessive rate of oxalate biosynthesis in vivo. This could be most effectively done by inhibiting the last step on the oxalate biosynthetic pathway. The present evidence shows that it would be necessary to inhibit lactate dehydrogenase throughout the body as well as glycollate oxidase in order to achieve this.

Comparative Activity and Properties of Lactate Dehydrogenase, Xanthine Dehydrogenase, and Dihydrofolate Reductase in Normal and Brugia pahangi-Infected Aedes aegypti

The amount of xanthine dehydrogenase (XDH), dihydrofolate reductase (DHFR), and lactate dehydrogenase (LDH) in crude extracts of 4- to 5-day-old adult Aedes aegypti was determined, and the properties of these enzymes were partially characterized. It was then found that the amount and other selected characteristics of XDH and LDH in extracts of female Ae. aegypti processed 5 to 7 days and 12 to 14 days after they had fed upon either normal or Brugia pahangi-infected jirds were indistinguishable from those of these two enzymes in extracts of female mosquitoes that did not have a blood meal. Under the same circumstances, the selected characteristics of DHFR were also unaffected. However, there was a suggestion that the amount of DHFR was slightly increased in extracts of female Ae. aegypti processed 5 to 7 days after they had fed upon B. pahangi-infected jirds; by 12 to 14 days after the blood meal, there was a consistent 30% to 60% increase in the amount of DHFR inextracts of infected mosquitoes. DHFR activity could not be detected in a similarly prepared extract of 4,000 to 5,000 infective (L-3) B. pagangi larvae, the approximate number present in the infected mosquito extracts. It would appear, therefore, that the increased amount of turnover of DHFR in the mosquito host occurs in response to advanced infection with B. pahangi.

Isolation and properties of lactate dehydrogenase from germinating pea plants

Lactate dehydrogenase (LDH) was isolated from pea seedlings by means of protamine sulphate and (NH 4) 2SO 4 fractionation and chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a MW of ca 145 500. The kinetic properties studied were the lactate oxidation pH optimum (9·1) and the pyruvate reduction pH optimum (7·1). K m values were determined for four natural substrates (Lactate, pyruvate, NAD + and NADH) and for other acids (glycollate, α-ketoglutarate and glyoxylate). The K i value was determined for p-chloromercuribenzoate (PCMB) which is a noncompetitive inhibitor of LDH from pea plants, and the course of irreversible inhibition of the enzyme by iodoacetamide (IA) and n-ethylmaleimide (NEMI) was studied. Preincubation of LDH with the coenzyme protects against PCMB inhibition, indicating the important role of the sulfhydryl group in the active site.

単細胞緑藻類の他養培養における生成乳酸の旋光性と乳酸脱水素酵素について

(1) 静置条件下での他養培養における7藻株(Chlo-rella4株,Scenedesmus1株,Chlamydomonas2株)の生成する乳酸の旋光性を検討し,7藻株の生成する乳酸がすべてD(-)-乳酸であることを認めた. (2) Chdorella vulgarisA1-40の乳酸脱水素酵素はD(-)-乳酸に特異的に作用するD(-)-乳酸脱水素酵素で,至適pHが8.0,ピルビン酸,NADH2に対するKmはそれぞれ6×10-4M,1.7×10-4Mであった.

Studies on enzymes from parasitic helminths—III. Purification and properties of lactate dehydrogenase from the tapeworm, Hymenolepis diminuta

1. A procedure for the purification of lactate dehydrogenase (E.C. 1.1.1.27) from the tapeworm, Hymenolepis diminuta, is described. 2. The 128-fold purified enzyme exhibits a specific activity of 106 units/mg of protein and was adjudged to be homogeneous by rechromatography, sedimentation velocity and sedimentation equilibrium ultracentrifugation, and a variety of electrophoretic techniques. 3. Only one form of the enzyme is present in the tapeworm as demonstrated by electrophoresis on cellulose acetate, polyacrylamide-gel and isoelectric focusing. 4. The molecular weight of the native enzyme is 141,000 and exhibits a sedimentation coefficient of 6·89 × 10 -13 sec. The enzyme is a tetramer composed of subunits of molecular weight 36,000. However, under low protein concentrations (0·1 mg/ml) on analytical gel filtration, the enzyme dissociates to the dimeric state and exhibits a molecular weight of 75,000. 5. Inhibitor studies are consistent with an active center which may be catalytically and structurally similar to the enzyme from vertebrates. 6. The enzyme is kinetically similar to vertebrate heart LDH. The K m for pyruvate was 0·17 mM and for lactate was 6·3 mM. The H. diminuta lactate dehydrogenase also forms an abortive ternary complex with coenzyme and substrate. 7. The data are discussed in regard to the physiology of the parasite and the host.

Lactate dehydrogenase isozymes: kinetic properties at high enzyme concentrations.

The kinetic properties of lactate dehydrogenase (LDH) isozymes have been determined at high enzyme concentrations. Spectrophotofluorometric assays revealed that the extent of substrate inhibition of LDH-1 and LDH-5 depends on enzyme concentration. At high enzyme concentrations, in the range of those that exist in most mammalian cells, no inhibition by pyruvate occurred. Pyruvate concentrations up to and including 20.0 millimoles per liter were used for each isozyme at 25 degrees and 40 degrees C at pH 7.0 and 7.4. These results suggest that substrate inhibition of LDH may not occur in vivo but only in vitro after appreciable dilution from physiologic enzyme concentrations. These experiments provide further evidence against the theory that substrate inhibition of LDH-1 in vivo accounts for the distribution of LDH isozymes within various tissues. They raise the possibility that, for other enzymes, kinetic properties determined at highly dilute concentrations in vitro may also be quite different from kinetic properties at the much higher concentrations that exist in vivo.