A one-step pmr determination of hydrogen transfer stereospecificity of NADP+-linked oxidoreductases

• 1.1. A simple, facile one-step method has been devised to measure the stereospecificity of NADP⁺-linked oxidoreductases. The procedure involves coupling the test enzymes to enzymes of known stereospecificity in the presence of deuterated substrates. The regenerated NADP⁺ in the coupled reactions is analyzed by PMR for its deuterium content at the carbon-4 position of the nicotinamide ring.
• 2.2. It is found that malate dehydrogenase (EC 1.1.1.37). lactate dehydrogenase (EC 1.1.1.27) and glycerate dehydrogenase (EC 1.1.1.29) are A-side stereospecific whereas glutamate dehydrogenase (EC 1.4.1.3) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) are B-side stereospecific.
• 3.3. Enzymes which can utilize both NAD⁺ and NADP⁺ have the same stereospecificity with respect to the coenzyme.

     

Distribution of opine dehydrogenases and lactate dehydrogenase activities in marine animals

• 1.1. Opine dehydrogenases (OpDHs) and lactate dehydrogenase (LDH) activities were determined in various marine animals. OpDHs were detected in six marine invertebrate phyla; Porifera, Coelenterata, Annelida, Mollusca, Arthropoda and Echinodermata in phylogenic sequence.
• 2.2. Among several OpDHs, tauropine dehydrogenase (TaDH) occurred widely in marine invertebrates, from Porifera to Echinodermata.
• 3.3. With a few exceptions, total OpDHs activities exceeded that of LDH activity in the marine invertebrates investigated.
• 4.4. With respect to anaerobic glycolysis, OpDHs are indicated to play an important role in phylogenically lower invertebrates, whereas LDH is more important in higher animals.

     

Control of pyruvate kinase flux during gluconeogenesis in isolated liver cells

• 1.1. With pyruvate as the gluconeogenic substrate, pyruvate kinase flux, estimated isotopically, and lactate formation were inhibited by glucagon, but only slightly affected by epinephrine.
• 2.2. The glucagon effect was unchanged in the absence of calcium.
• 3.3. Ethanol increased lactate formation from pyruvate, but depressed pyruvate kinase flux.
• 4.4. These results support the role of pyruvate kinase m the cyclic mechanism which transfers mitochondrial reducing hydrogen to the cytosol.
• 5.5. Glucagon and, to a lesser degree, epinephrine inhibit lactate formation from fructose or dihydroxyacetone.
• 6.6. Ethanol also inhibits lactate formation from these substrates, suggesting the possibility that NADH may in some manner regulate pyruvate kinase flux.

     

On the comparative turnover rates of the lactate dehydrogenase isozymes in rat tissues

• 1.1. An affinity chromatography technique for the determination of turnover parameters has been utilized for the derivation of new data for lactate dehydrogenase and its isozymes in a wide variety of tissues from female rats.
• 2.2. This methodology allowed the establishment of relative turnover characteristics in all tissues examined, and the evaluation of rate constants for synthesis and degradation and half-life values in the majority of these enzyme sources.
• 3.3. Considerable variation was evident in the turnover characteristics of total protein and lactate dehydrogenase in the different tissues, with the synthesis of the enzyme being highest in reproductive tissues, heart and liver, and degradation most prominent in reproductive tissues.
• 4.4. With regard to the isozymes, marked differences in half-lives were evident not only between the same isozyme in different tissues, but also between the various isozymes in any one tissue.
• 5.5. Bi-directional trends in the half-life values for the isozyme sequences in several tissues, indicated a distinctive contribution to turnover from the different cell types present.
• 6.6. These data have been discussed in relation to the available comparisons in the literature, and the known physiological correlations of lactate dehydrogenase in these tissues.

     

Anaerobic amino acid metabolism in the locust nervous system

• 1.1. Changes in the concentration of amino acids and other metabolites have been followed in the isolated locust nervous system under anaerobic conditions.
• 2.2. The results (accumulation of alanine, pyruvate, lactate, succinate and α-glycerophosphate, decline in asparate, glutamine and glycogen) are qualitatively similar to those found in many other invertebrate and vertebrate tissues, although the pathways in the insect tissue show some differences.
• 3.3. The locust nervous tissue does not show that very large accumulation of α-glycerophosphate shown by whole insects, and which is due to the influence of the flight muscle.
• 4.4. It can be concluded that the locust nervous system has only a limited capacity for anaerobic metabolism, and cannot support normal nervous activity for more than a few minutes in the absence of O₂.

     

Measurement of lactic acid in nanomolar amounts reliability of such a method as an index of glycolysis in pancreatic islets

• 1.1. The output of lactate is measured in isolated pancreatic islets.
• 2.2. The method for lactate assay is reproducible, with a coefficient of variation between duplicate measurements not exceeding 10%. The biological factors of variability are more important.
• 3.3. In islets exposed to glucose (16.7 mm), the output of lactate accounts for 45–50% of the uptake of glucose and its further conversion to triosephosphates.
• 4.4. Rapid, marked changes in lactate output occur when the glucose concentration of the incubation medium is modified.
• 5.5. The reliability of lactate output as an index of glycolysis in the islets is discussed.

     

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—I. Utilization of glycogen and accumulation of glycolytic end products in various tissues

• 1.1. Resting oxygen consumption at 10°C did not change from normoxia (150 mm Hg) down to an oxygen tension of 55 mm Hg for the flounder, Platichtys flesus.
• 2.2. Flounders exposed to hypoxia showed increased levels of blood glucose and lactate, dependent on the degree of hypoxia.
• 3.3. Due to hypoxia glycogen was depleted in the liver and swimming muscle but in the heart there was no significant change.
• 4.4. Liver glucose increased after 7 hr of hypoxia. Heart and muscle glucose did not change but the absolute glucose concentration in the heart was five times higher than in the muscle.
• 5.5. There is a transient accumulation of lactate in heart, liver and kidney after 7 hr of hypoxia while lactate accumulation in the swimming muscle is significant only after 21 hr of hypoxia.
• 6.6. Succinate only accumulated in the liver while alanine accumulated in muscle, heart and liver.

     

Interaction of lipogenic substrates in porcine adipose tissue in vitro

• 1.1. Porcine adipose tissue was incubated with radiolabeled glucose, acetate or lactate. Saturation curves indicated that lactate > glucose > acetate in providing two-carbon units for fatty-acid synthesis.
• 2.2. Competition between individual substrates indicated that lactate was the best lipogenic substrate.
• 3.3. Incubation of all three substrates at concentrations observable in serum indicated that at 5.56mM, glucose was the preferred lipogenic substrate in the presence of 0.1 mM acetate and 1.0 mM lactate.
• 4.4. At elevated concentrations (18.52mM glucose, 1.0 mM acetate and 10.0 mM lactate), acetate and lactate were preferred to glucose as lipogenic substrates.

     

Reduction of ferrihemoglobin in erythrocytes of birds and mammals

• 1.1. Species differences exist in ferrihemoglobin reduction rates in bird and mammalian red cells, bird erythrocytes being very active reducers.
• 2.2. Glucose and lactate enhance ferrihemoglobin reduction. In horse and quail red cells β-hydroxybutyrate has this effect as well.
• 3.3. Malate and pyruvate do not enhance ferrihemoglobin reduction.
• 4.4. Plasma addition to red cell suspensions enhances ferrihemoglobin reduction; addition of lactate mimics this effect in all species except the dog.
• 5.5. Incubation conditions are very important for measuring ferrihemoglobin reduction. Especially the presence of bicarbonate ions is essential. In our experiments no inhibition of reduction rates by chloride ions is found.
• 6.6. Mitochondrial NADH production does not play a role in ferrihemoglobin reduction in bird red cells.

     

1H NMR study of metabolic alterations in the small intestine of rats infected with Hymenolepis diminuta

• 1.1. 1H NMR spectra of the duodenum, jejunum and ileum tissues of the small intestine of a rat showed metabolic gradients.
• 2.2. The concentrations of metabolites in these gut regions were altered by the presence of the tapeworm Hymenolepis diminuta.
• 3.3. In the infected duodenum there was significantly less glycogen, glucose and phosphocreatine/creatine, but significantly more lactate than in the corresponding controls.
• 4.4. Infected jejunum contained significantly less betaine but significantly more succinate, alanine and lactate.
• 5.5. Infected ileum had significantly less glycogen and taurine but significantly more alanine and lactate.

     

Variations in the lactate dehydrogenase activity during the development of the shrimp Palaemon serratus

• 1.1. The lactate dehydrogenase (LDH) from Palaemon serratus muscle has been studied throughout the development of the animal.
• 2.2. Enzymatic activities have been traced by polyacrylamide gel electrophoresis and kinetic studies.
• 3.3. The existence of two enzymes (L₁ and L₂) has been demonstrated.
• 4.4. During the larval development, both L₁ and L₂ remain at a low level.
• 5.5. After the larvae hatch L₁ and L₂ gradually rise although L₁ is predominant.
• 6.6. Measurement of kinetic parameters shows that the general behaviour of the enzymes of the embryo resembles that of the adult enzymes.
• 7.7. However, one can observe during the development a constant increase in the affinity of the enzyme towards its substrate, lactate.

     

Isolation of allantoin and adenosine from the marine sponge Tethya aurantia

• 1.1. Propanol extracts of the sponge Tethya aurantia (Demospongiae) were fractionated, guided by bioassay, for a component with negative chronotropic and inotropic activity on isolated guinea pig atria.
• 2.2. The bioactive component was found to be adenosine. These extracts also contained allantoin.
• 3.3. This intermediate in the sequence of degradation of purines was unexpected, since it has been reported only once before to occur in marine invertebrate animals.

     

The effects of oxalate and glucose on lipogenesis by isolated hepatocytes from normal and biotin-deficient chicks (Gallus domesticus)

• 1.1. Isolated hepatocytes synthesize fatty acids and cholesterol from lactate and acetate with lactate being the more effective substrate.
• 2.2. Biotin deficiency decreased fatty add synthesis from both substrates but stimulated cholesterogenesis.
• 3.3. Exposure of intact hepatocytes to oxalate inhibited fatty acid and cholesterol synthesis from lactate, this effect was enhanced in biotin-deficient chicks. A similar effect was not observed when acetate was the substrate.
• 4.4. Synthesis of fatty acids from lactate and acetate was stimulated by glucose, biotin deficiency increased this response. Cholesterogenesis was reduced in control but not biotin-deficient chicks.

     

Glycolytic enzymes of fowl and turkey spermatozoa

• 1.1. It was confirmed that, under anaerobic conditions, fowl spermatozoa formed lactate from glucose thirteen times faster than turkey spermatozoa.
• 2.2. The profiles of glycolytic enzyme activities were similar for spermatozoa from both species; however fowl spermatozoal activities were generally 2- to 4-fold higher.
• 3.3. Exceptions were glycerophosphate mutase and lactate dehydrogenase activities which were respectively 9.5 and 41 times greater in fowl spermatozoa.
• 4.4. In both species, spermatozoal glyceraldehyde-3-phosphate dehydrogenase had the lowest activity of the glycolytic enzymes.

     

Fetal lung metabolism: Response to maternal fasting

• 1.1. Fetal lung metabolic response to maternal fasting late in gestation was investigated.
• 2.2. Maternal fasting 4 days before term was associated with low fetal plasma glucose and insulin levels but increased levels of fetal plasma glucagon, glycerol, lactate and fatty acids.
• 3.3. Fetuses from fasted mothers showed a significant decrease in body weight (30%), lung weight (30%) and lung glycogen (46%), but no change in lung protein, phospholipid or total lung DNA, suggesting that lung size is affected more than maturation.
• 4.4. Fetal lung slices incubated in vitro showed that lactate oxidation to CO₂ equalled that of glucose in control fetal lungs and was unaffected by maternal fasting, while glucose oxidation was depressed (23%).
• 5.5. Maternal fasting significantly decreased in vitro incorporation of [U-¹⁴C]-glucose, [U-¹⁴C]lactate and [1-¹⁴C]palmitate into lung phospholipids.
• 6.6. Fetal lungs from fasted mothers showed increased conversion of lactate to glucose, indicating gluconeogenic potential by fetal lung.
• 7.7. These studies show that plasma lactate serves as an important energy fuel and substrate for lipid synthesis for the fetal lung, and maternal fasting markedly alters fetal lung metabolism.

     

Crayfish retinular cells: Influence of extracellular sodium and calcium upon receptor potential

• 1.1. In crayfish, light stimulation of the retinular cells induces a depolarizing receptor potential.
• 2.2. Experiments were designed to determine the role of Na⁺ and Ca²⁺ on receptor potential during dark And light states.
• 3.3. Depolarization depends on Na⁺ and Ca²⁺ availability to the retinular cell.
• 4.4. Repolarization velocity and response duration depend on extracellular Ca²⁺ availability.
• 5.5. Light adaptation increases receptor potential dependence on calcium and sodium ions.
• 6.6. We analyse these results with respect to other invertebrate photoreceptors.

     

Metabolomic Analysis Identifies Lactate as an Important Pathogenic Factor in Diabetes-associated Cognitive Decline Rats

1. Download : Download high-res image (156KB)
2. Download : Download full-size image

Highlights

• •Quantitative metabolomics identified elevated lactate levels in diabetic brains.
• •Levels and enzymatic activity of LDH-A were also found significantly up-regulated.
• •GPR81 dependent PKA-CREB regulated cognition decline in the diabetic rats.
• •Mechanistic insights into role of lactate in diabetes-associated cognitive decline.

     

Evolution of invertebrate homeostasis: Osmotic and ionic regulation

• 1.1. Homeostasis, or the maintenance of a constant internal environment, in invertebrate organisms decreases the dependence of these organisms on the vagaries of the external environment.
• 2.2. The evolution of physiological processes influencing homeostatic conditions begins with the organism being totally dependent upon the external environment, passing through a series of intermediate stages during which fluctuation in a given internal parameter occur, proceeding finally to a condition where the internal environment is constant and independent of the external environment.
• 3.3. A hypothetical scheme for the possible evolutionary pathway of osmotic and ionic regulation in aquatic invertebrates was developed in an attempt to follow the process of homeostasis in these organisms.
• 4.4. Primitive marine cells developed mechanisms to regulate the ionic composition of the cytoplasm probably in association with the need for volume regulation.
• 5.5. The development of a body wall separated an internal body fluid from the external sea-water and the ionic composition of the body fluids was initially maintained slightly different from that of the sea-water by essentially passive means.
• 6.6. The addition of excretory organs, which arose initially through an inpushing of the body wall, brought about the ability for osmoregulation.
• 7.7. Homeostatic mechanisms must be in place before an organism can move from one environment to another.

     

The effect of d2o on glycolysis by rat hepatocytes

• 1.1. The effect of incorporating D₂O into the incubation medium on glycolysis and gluconeogenesis by hepatocytes from fasted rats was examined.
• 2.2. The substitution by heavy water, D₂O, at concentrations from 10 to 40%, stimulated glucose uptake, lactate production and CO₂ yields from glucose. At 10 mM glucose, 40% D₂O doubled glucose uptake, increased CO₂ production by 40%, and increased lactate production by 350%.
• 3.3. The stimulation of lactate production decreased at higher glucose concentrations, but was still substantial even at 80 mM glucose.
• 4.4. There was no effect on CO₂ production above glucose concentrations of 30 mM.
• 5.5. Ten percent D₂O showed little inhibition of lactate uptake, its oxidation and gluconeogenesis. At 40% D₂O the inhibition ranged from 10 to 20%.
• 6.6. No effect of D₂O on the rate of glucokinase or glucose-6-phosphatase was observed.
• 7.7. The concentration of fructose, 2,6-P was not affected by D₂O

     

“Stress” reactions in teleosts: Effects of temperature and activity on anaerobic energy production in roach (Rutilus rutilus L.)

• 1.1. In roach any kind of excitement due to swimming, handling, or to unfavourable environmental conditions, is reflected in an increase of lactate above 1.0 μ mol and a decrease of phosphocreatine (PCr) below 15.0 μ mol g⁻¹ in the muscles.
• 2.2. In fish acclimated to and tested at 4°C lactate accumulation and PCr hydrolysis after activity were only half those in fish tested at 12 and 20°C.
• 3.3. Fish swimming at submaximal speed for 60 min at 20°C had much less lactate in the anterior body musculature than fish swimming for the same length of time at 12°C.
• 4.4. It is suggested that the “sudden death syndrome” of severely exercised fish is due to rapid hydrolysis of PCr, rather than to acidosis, in sensitive tissues.