Direct adaptation of cells to temperature: Similar changes of LDH isozyme patterns by in vitro and in situ adaptations in Xenopus laevis

• 1.1. Cells of Xenopus laevis were cultured in vitro chronically at different temperatures and their isozyme patterns of lactate dehydrogenase (LDH) were compared with those of liver of temperature-acclimated toads.
• 2.2. Relative increase of cathodic isozyme by cold adaptation was observed both in two independent cell lines and in toad tissue.
• 3.3. This supports the concept that adaptation of cells to local body temperature would be responsible to the change in isozyme pattern by temperature adaptation of organisms.
• 4.4. Such changes would not be a direct result of elevated pO₂, judging from tissue distribution of isozymes.

     

LDH isozymes in amazon fish—III. Distribution patterns and functional properties in Serrasalmidae (Teleostei: Ostariophysi)

• 1.1. The patterns of distribution of LDH isozymes in five tissues from 12 Serrasalmidae species have been studied through two-fold serial dilution (Klebe's test) as well as the pyruvate inhibition of LDH enzyme in skeletal and heart muscles (low/high ratios).
• 2.2. The species' electrophoretic patterns differ by orthologous A₄ isozyme mobilities since the orthologous B₄ isozymes present similar electrophoretic migration. These differences between Ldh-A and Ldh-B products reflect three-, four-, and five-banded patterns. Thus, different LDH isozyme numbers formed from A and B subunits should not be used as an evolutionary or phylogenetic characteristic from different taxonomic groups.
• 3.3. Out of 66 pairs of species only five pairs showed significant differences in the distribution patterns in all five analyzed tissues, while no pair of species showed the same distribution in these tissues. This variation was explained as differential regulation of structural genes among tissues and/or species.
• 4.4. Functional properties showed significant between the LDHs from heart and skeletal muscles, and are consistent with a preference for aerobic metabolism. We suppose that by selecting B-like subunits these fishes are able to maintain good control of aerobic/anaerobic transitions, maintaining predominantly oxidative metabolism even in hypoxic waters, with which they have to cope.

     

The influence of ehrlich ascites tumour growth on the turnover of lactate dehydrogenase in tissues of the mouse

• 1.1. The influence of Ehrlich ascites tumour growth on the turnover of total soluble protein and lactate dehydrogenase (LDH) in mouse tissues has been studied.
• 2.2. Turnover parameters were determined by means of double-labelling technique, with the enzyme (LDH) being isolated by affinity chromatography.
• 3.3. Tumour growth was accompanied by a decreased rate of synthesis of total protein in all tissues.
• 4.4. Lactate dehydrogenase by contrast snowed an increased rate of synthesis in all tissues but kidney.
• 5.5. These directions of change, in combination with the lesser response of degradation constants, resulted in a consequent conservation of enzyme activity in all tissues except kidney.
• 6.6. A generalized shift in the LDH isozyme pattern of these tissues was also observed during tumour growth with an increased contribution of A-type subunit.
• 7.7. These results have been discussed in relation to the redirection of protein synthesis and degradation, the occurrence of foetal isozymes, and possible mechanisms involved in the redistribution of protein resources in the animal during tumour development.

     

Evolution of LDH isozymes during programmed cell death

• 1.1. Lactic dehydrogenase dehydrogenase isozymes and other respiratory enzymes were studied in degenerating intersegmental muscles of Manduca sexta and Antheraea polyphemus.
• 2.2. Total activities of the different enzymes (isocitric dehydrogenase, malic dehydrogenase, catalase, lactic dehydrogenase) decline at varying rates, starting before the rapid phase of involution.
• 3.3. One isozyme of LDH, an M-type isozyme, increases several-fold during the final three days prior to the emergence of the insect.
• 4.4. The same isozyme appears very transiently or not at all in muscles which do not break down, and is present in degenerating silk glands at the time of their most rapid involution.
• 5.5. The data suggest that limitation of oxidative metabolism plays a role in the involution of the muscles.

     

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.

     

Lactate dehydrogenase isozymes of the leopard danio,Brachydanio nigrofasciatus: their characterization and ontogeny

• 1.1. The tissue specific patterns and ontogeny of lactate dehydrogenase (LDH) are reported.
• 2.2. While all tissues (eye, brain, heart, intestine, liver, ovary and skeletal muscle) show isozymes of A and B subunit composition, only liver extracts possess isozymes resulting from C subunit synthesis.
• 3.3. The A₄ homopolymer appears simultaneously with initial muscle contractility and is correlated with the physiological function of muscular contraction.
• 4.4. The activation of the Ldh-C locus is correlated with the first functioning of liver. It is suggested that the state of differentiation of liver cells may be the stimulus required for C locus expression.

     

Multiple leucine aminopeptidases in the oocysts of Eimeria tenella and their changes during sporulation

• 1.1. There was little neutral protease activity but high levels of leucine aminopeptidases (LAP) in the oocysts of Eimeria tenella.
• 2.2. By electrophoretic analysis, there were three apparent LAP isozymes I, II and III in unsporulated oocysts.
• 3.3. They all diminished with the simultaneous emergence of a new, fast-moving isozyme V during late phase of sporulation.
• 4.4. The enzyme V was unlikely to have resulted from de novo protein synthesis and was predominantly in the cytoplasm surrounding the sporocysts.
• 5.5. It differed from the other isozymes by a slightly higher pH optimum, more dependence on Mn²⁺ or Mg²⁺ in the assay and higher susceptibility to chelating agents.
• 6.6. The possible biological function of these isozymes remain unknown. Since they were not found in sporozoites or merozoites of E. tenella, they may be needed only for sporulation and, possibly, excystation.

     

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.

     

Immunochemical homologies among l-α-hydroxyacid oxidase isozymes

• 1.1. Antisera were prepared in rabbits against purified (>95%) rat L-α-hydroxyacid oxidase (HAOX) A₄ and B₄ isozymes and used in immunochemical titration and immunodiffusion experiments.
• 2.2. Immunochemical homologies were demonstrated between mammalian HAOX isozymes.
• 3.3. This provides further evidence that the loci (Hao-1; Hao-2) encoding these isozymes are products of a recent gene duplication event.

     

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.

     

Lactate dehydrogenase in amoeba,Acanthamoeba sp. in different phases and conditions of culture

• 1.1. The activity and kinetic changes of amoeba LDH in different phases and conditions of culture were investigated.
• 2.2. LDH of the amoeba is specific against d(−)LDH irrespective of the hypoxic conditions created.
• 3.3. In hypoxic conditions it was not possible to visualize the presence of another LDH isozyme of muscle type by kinetic or electrophoretic analysis.
• 4.4. However, the changes in the K_m value and the L:H ratio as well as the decrease of electrophoretic mobility of LDH band indicate the change in kinetic properties of the enzyme from an obviously heart type in oxygenated culture in the direction of a muscle type LDH in strongly hypoxic culture conditions.
• 5.5. The influence of factors producing either environmental or metabolic hypoxia on possible repression or induction of LDH in amoeba is discussed.

     

Phylogeny and ontogeny of the phosphoglycerate mutases—I. Electrophoretic phenotypes of the glycerate-2,3-P2 dependent phosphoglycerate mutase in vertebrates

• 1.1. The electrophoretic phenotype of phosphoglycerate mutase in tissues from different classes of vertebrates at several stages of development have been analyzed on cellulose acetate.
• 2.2. Mammals, reptiles, amphibians and fish show a common three-banded isozyme pattern.
• 3.3. The three isozymes vary in their relative distribution from tissue to tissue and during growth.
• 4.4. In birds electrophoretically distinguishable phosphoglycerate mutase isozymes have not been detected.
• 5.5. The results support a genetic basis for the phosphoglycerate mutase isozymes and suggest that gene duplication may have occurred early in vertebrate evolution.

     

Preparation of two neurotoxic esterases from the chick central nervous system

• 1.1. A standard procedure for lipid-extraction of lyophilized hen brain material is decribed.
• 2.2. Nine carboxylesterase isoenzymes (EC 3.1.1.1) are identified in lipid-extracted lyophilized material (LELM) using kinetic analysis of organophosphate inhibition. Total phenyl valerate (PV) hydrolysing carboxylesterase activity in LELM is 43.3U.g⁻¹
• 3.3. Two carboxylesterase isoenzymes of LELM are classified as neurotoxic esterases (NTE_A and NTEg_B).
• 4.4. Using n-octylglucoside 51% of the water-insoluble neurotoxic esterase activity from LELM are solubilized.
• 5.5. Six carboxylesterase isoenzymes including NTE_A (6.5 U-l⁻¹) and NTE_B (4.2 U-l⁻¹) are present in the solubilized preparation.
• 6.6. Throughout purification and separation steps carboxylesterase isoenzymes are identified by their rate constants for the reaction with organophosphorus inhibitors.

     

Phosphonomonoesterase activity in Metridium senile L.

• 1.1. An esterase which hydrolyzes 4-nitrophenyl(phenyl)phosphonic acid (4-NPPP) was purified from M. senile (sea anemone).
• 2.2. The enzyme showed no 5′-nucleotide phosphodiesterase activity with 5′-(4-nitrophenyl) TMP or phosphomonoesterase activity with 4-nitrophenylphosphate.
• 3.3. Addition of excess Zn²⁺ restored activity after inactivation by EDTA.
• 4.4. Thiol reagents and phenylmenthanesulfonylfluoride did not inactivate, whereas, dithiothreitol inactivated.
• 5.5. Aminoethylphosphonic acid (AEP) was a competitive inhibitor of 4-NPPP indicating possible activity with phosphonomonoesters of AEP.

     

Possibilities of biochemical taxonomy of bats using hemoglobin,lactate dehydrogenase,esterases and other proteins

• 1.1. Bat hemoglobin resembles other mammalian Hb's in its physiological properties, and Hb differences among bat species are minor.
• 2.2. One polymorphism of the H chain of lactate dehydrogenase occurs in Myotis lucifugus and M. keenii, and another occurs in Eptesicus fuscus. Bat heart and muscle have identical LDH isozyme profiles.
• 3.3. Esterases and major low ionic strength extractabe proteins show a number of differences at the generic level, as well as some polymorphisms which cross species lines.
• 4.4. The protein studies indicate that bats have great individual variation, often of a type comparable to known genetically based protein polymorphisms in other species, but apparently have not had time to accumulate extensive divergent species specificity.

     

Comparative distribution of nitrogenous compounds in Acipenser transmontanus and Hydrolagus colliei: Enzyme kinetics and isozyme ratios alterations

• 1.1. Time patterns of intravenously administered [¹⁴C]urea in primitive fishes showed generalized but not quantitatively equivalent tissue distribution within defined concentration limits which were species specific (Rasmussen & Rasmussen, 1978). Comparative patterns are presented here for other ¹⁴C-labelled organics, such as thiourea, demonstrating temporal and quantitative differences in tissue distribution.
• 2.2. Demonstrable species differences between [¹⁴C]TMA and [¹⁴C]urea distribution are seen between H. colliei and A. transmontanus.
• 3.3. The tissue distribution of [¹⁴C]urea of H. colliei maintained in sea water with 0.1 M urea plus minor amounts of [¹⁴C]urea is presented; especially to be noted is the rapid distribution to the ocular fluid.
• 4.4. Finally, the effects of elevated concentrations of selected organics including urea, TMAO, guanidine-HCl on serum and CSF levels of peroxidase, lactic dehydrogenase (LDH), on some kinetic parameters of LDH, and on LDH isozyme ratios are reported. Especially enhanced by extra urea is the fastest electrophoretically migrating LDH band in ratfish CSF and serum.

     

Temperature acclimation in amphibians: Changes in lactate dehydrogenase activities and isoenzyme patterns in several tissues from adult Discoglossus pictus pictus (Otth.)

• 1.1. The effect of cold (8 ± 2°C) acclimation on the lactate dehydrogenase activities and isoenzyme patterns from sartorius muscle, liver, heart and brain of adult Discoglossus pictus pictus (Otth.) was studied.
• 2.2. Two groups of animals were studied: one set of animals was trapped in October and another set in December. In both cases some of the animals were sacrificed upon collection and some others subjected to 5 months of acclimation at 8 ± 2°C before being sacrificed for analysis.
• 3.3. A general trend towards a decrease in LDH specific activity was observed during cold acclimation. The magnitude of change, but not the direction, depends on both the tissue examined and the season at which the experiment was initiated.
• 4.4. A complex LDH isoenzyme reorganization was also found in liver, heart and brain. In liver from Experiment 1 and in heart from both experiments, a relative maintenance in M-type LDH activity during cold acclimation was observed. However, in brain there was a relative maintenance of LDH₃ activity in both experiments.
• 5.5. The low behavioral activity (and its metabolic consequences) and the existence of an intrinsic annual rhythm in D. pictus metabolism are suggested as responsible for the observed enzymatic changes.

     

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.

     

Soluble cyclic amp-dependent protein kinases from chick liver: Effects of NaCl and heat-stable modulator

• 1.1. Two cyclic AMP-dependent protein kinases—Fraction I and II—have been isolated from chick liver soluble preparation on DEAE-cellulose.
• 2.2. Both fractions have an apparent K_m for ATP of 2 × 10⁻⁶M, are stimulated maximally by 5 × 10⁻⁸ M cyclic AMP and phosphorylate mainly basic proteins—histone and protamine.
• 3.3. They exhibit various pH values for optimal activity and show differences with respect to both sensitivity to NaCl and substrate specificity.
• 4.4. The heat-stable protein modulator inhibits the cyclic AMP-dependent protein kinase activity of both fractions, but with cyclic GMP one kinase is stimulated and the other inhibited.
• 5.5. Slight differences in histone triggered holoenzyme dissociation as well as the lack of difference between their ability for subunit reassociation do not allow to classify these isozymes as protein kinases of Type I and II, according to Corbin et al. (1975).

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.