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.

     

Malate dehydrogenase (sMDH) in Amazon cichlid fishes: evolutionary features

• 1.1. The sMDH isozyme system was studied in five species of cichlid fishes found in the Amazon hydrographic basin (Astronotus ocellatus, Cichla monoculus, Geophagus cff harreri, Cichlassoma severum and Mesonauta insignis). All studied specimens presented a six-banded electrophoretic pattern, suggesting the existence of three gene loci (sMDH-A1, sMDH-B11 and sMDH-B21).
• 2.2. Klebe's serial dilutions, thermostability tests and tissue specificity performed on the sMDH of studied species indicated no divergence between B11 and B21 loci products, suggesting that these genes probably undergo the same regulatory gene action and that the duplication event occurred recently, after A1 and B1 divergence.
• 3.3. The appearance of the same characteristics in all specimens, and the chromosomic picture of the family, suggest the occurrence of an event of duplication “in tandem” in the ancestors of Amazon cichlids.

     

Functional properties of the two major hemoglobin components from Leporinus friderici (Pisces)

• 1.1. The hemoglobins of Leporinus friderici were separated by liquid chromatography on DEAE-Sepharose in order to isolate the two major electrophoretic components.
• 2.2. The chromatographic fraction I (electrophoretically slow anodic) showed no Bohr effect and no nucleoside triphosphate modulation.
• 3.3. The chromatographic fraction III (electrophoretically fast anodic) showed a normal Bohr effect and addition of nucleoside triphosphate decreased oxygen affinity but did not alter the Bohr effect.
• 4.4. The whole hemolysate showed a normal Bohr effect and phosphate modulation altered both Bohr effect and oxygen affinity.
• 5.5. No or little difference between the effect of adenosine or guanosine triphosphates on hemoglobin function was observed.

     

Kinetic properties of primitive vertebrate carbonic anhydrases

• 1.1. Carbonic anhydrases from the red cells, ocular secretory tissues, and rectal gland of species of Myxine, elasmobranchii, and Teleostii, were examined using rates of CO₂ hydration. The enzyme is absent from corneal endothelium and lens of elasmobranchs and salt water teleosts, but present in fresh water fish.
• 2.2. The red cell carbonic anhydrase of the representative elasmobranch, Squalus acanthias, is a single enzyme of relatively low activity, K_cat = 2 × 10⁴ sec⁻¹ at 1°C. The ciliary folds and rectal gland of S. acanthias contain carbonic anhydrases with turnover numbers five times higher than the red cell enzyme.
• 3.3. Both red cell and secretory enzymes of S. acanthias are susceptible to inhibition by sulfonamides within a 10-fold range of mammalian secretory carbonic anhydrases. In general, they are moderately sensitive to anion inhibition; a notable exception is enzyme from rectal gland which is insensitive to halion inhibition.
• 4.4. In teleosts, both red cell and secretory carbonic anhydrases have a high turnover number, and are susceptible to sulfonamide inhibition. In red cells there appear isozyme(s) of lower activity, in considerable concentration.
• 5.5. Taken with earlier ion transport work in S. acanthias, the basic vertebrate pattern of aqueous humor formation, both chemically and physiologically, appears to be established in this “primitive” species.
• 6.6. The finding of at least three different types of carbonic anhydrases in S. acanhias suggests that separate loci for the enzyme have existed throughout most of vertebrate evolution.

     

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.

     

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.

     

Mixed function oxidase activity in the harbour seal (Phoca vitulina) from sable is., N.S.

• 1.1. One adult male, eight pups (including two full term foetuses) and nine adult female harbour seals (Phoca vitulina) were analysed for indices of mixed function oxidase (MFO) activity.
• 2.2. MFO activity was present in liver samples, but was at or below detection limits in samples of kidney, lung and pancreas.
• 3.3. Hepatic ethoxyresorufin O-de-ethylase and benzo[a]pyrene hydroxylase activities were similar to those reported in other seals and in other mammals.
• 4.4. Cytochromes P-450 and b₅ concentrations were slightly lower than those observed in other mammals.
• 5.5. MFO activities in newborn pups and foetuses were significantly lower than those in adult females.
• 6.6. No qualitative differences in cytochrome P-450 isozyme distribution between foetal and adult samples could be discerned by electrophoresis.

     

Purification and properties of tetralysine endopeptidase from Escherichia coli AJ005

• 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
• 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
• 3.3. The optimal pH was about 7.5
• 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
• 5.5. The apparent K_m value was 2.5 × 1O⁻³ M for tetralysine.

     

Reabsorption and accumulation of α-amino-iso-butyric acid in the nephridia of Sabella pavonina Sa vigny (Annelida polychaeta)

• 1.l. High amino acid concentrations were found in the anterior coelomic fluid of a Polychaeta (Sabella pavonina Savigny).
• 2.2. The concentrations being much higher in the fluid which penetrates the nephrostomia into the nephridia lumen than in the final urine indicates that the nephridia reabsorbs large amounts of amino acids.
• 3.3. Nephridial perfusion experiments showed that an amino acid analogue (α-amino-iso-butyric acid, AIB) is transported by the nephidia.
• 4.4. The transport took place across the nephridial wall owing to the presence of a carrier-mediated transport system and a diffusion system.
• 5.5. For the carrier-mediated transport, the V_max was 0.234 ± 0.025 nmol·min⁻ and the K_m 3.715 ± 0.315mmol·l⁻.
• 6.6. AIB accumulated in the nephridial cells up to a maximum rate of 01.17 nmol·min⁻.
• 7.7. Intracellular accumulation stopped increasing when the V_max for reabsorption was reached.
• 8.8. These results indicate that the carrier-mediated transport of AIB is located at the apical membrane of the nephridial cell, and that AIB transport by simple diffusion takes place through the paracellular pathway.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Isolation and characterization of lipoamide dehydrogenase from mackerel dark muscle

• 1.1. Lipoamide dehydrogenase was purified 1500-fold from mackerel dark muscle.
• 2.2. The enzyme was homogeneous as judged by acrylamide gel electrophoresis in the presence and absence of SDS.
• 3.3. Molecular weights of 102,000 and 55,000 were estimated for the native and denatured enzyme, respectively.
• 4.4. Optimal activity for the enzyme was obtained at around pH 5.7 and enhanced with citri acid.
• 5.5. Loss of activity was less than 5% by incubating the enzyme at 70°C for 20 min.
• 6.6. An apparent K_m of 3.1 × 10⁻³ M was obtained for dl-lipoic acid and 1.5 × 10⁻⁵ M for NADH.
• 7.7. The properties of lipoamide dehydrogenase from mackerel dark muscle observed in this investigation were very similar to those reported for the enzyme from other sources.

     

An electrophoretic study of genetic variation in populations of Yarrow's spiny lizard Sceloporus jarrovi (Sauria,Iguanidae)—I. Esterae characterization and polymorphism

• 1.1. Muscle esterase variation in Sceloporus jarrovi, sampled from 25 locations in southeastern Arizona, was investigated employing acrylamide gel electrophoresis.
• 2.2. Three distinct esterase phenotypes were observed, presumably resulting from the expression of two gene loci, Est-1 and Est-2.
• 3.3. Lizards sampled from all 25 locations were found to be monomorphic with respect to esterase encoded at Est-1. Further, lizards sampled from the Santa Rita and Pinaleno Mountains were also found to be monomorphic for esterases encoded at Est-2, whereas those sampled from the Chiricahua and Huachuca Mountains proved to be polymorphic.
• 4.4. Characterization of the esterases utilizing eserine sulfate, diisopropylfluorophosphate, and sulfhydryl-group inhibitors revealed the EST-1 isozyme to be an arylesterase and the EST-2 isozymes to be carboxylesterases.

     

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.

     

Respiration in the eastern water dragon,Physignathus lesueurii (agamidae)

• 1.1. Some aspects of the gas exchange system of a diving lizard, Physignathus lesuewii were studied.
• 2.2. Breathing patterns were analysed.
• 3.3. Breathing rate increases logarithmically with temperature and Q₁₀ = 1.8. LogBR = −0.237 + 0.0256 T.
• 4.4. Gas tensions in lung air and arterial and venous blood were measured. Arterial pH declines with increasing temperature.
• 5.5. Temperature has a marked effect on oxygen affinity of the blood (ΔH = −10.1 kcal mol⁻). A Bohr effect was also noted.
• 6.6. CO₂ equilibrium curves were drawn.
• 7.7. The results are considered with a view to anticipating the efficiency of the gas exchange system of this species under conditions of variable temperature and during diving.

     

Isotopic labeling of taurine; implications for its synthesis in selected tissues of Homarus americanus

• 1.1. The abdominal muscle and ventral nerve chord of Homarus americanus were incubated with radioactive precursors.
• 2.2. ³⁵SO₄ and ¹⁴C-CO₂ were not incorporated into amino acids in the abdominal muscle.
• 3.3. ¹⁴C-glucose was incorporated into serine, cysteine, cysteine-sulfinic acid and taurine in the abdominal muscle.
• 4.4. ¹⁴C-CO₂ appeared to be slightly incorporated into taurine in the ventral nerve chord.
• 5.5. ¹⁴C-glucose incubation with the ventral nerve chord resulted in labeling of the same amino acids as in the abdominal muscle.
• 6.6. The results are discussed in view of the catabolism of carbohydrates and the synthesis of taurine.

     

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.

     

Comparative studies of sodium transport and its relation to hydrostatic pressure in deep and shallow water gammarid crustaceans from Lake Baikal

• 1.1. Freshwater gammarids from 900–1400 m depths lose Na at 1 atm, 4°C, while related shallow water gammarids are near neutral Na balance.
• 2.2. Na⁺ influx rates are similar at 1 atm, 4°C, for abyssal and shallow water gammarids of similar weight.
• 3.3. Na⁺ efflux is faster for abyssal gammarids than for comparable shallow water gammarids.
• 4.4. Compressing abyssal gammarids to 90–140 atm increases Na⁺ influx rates enough to restore neutral Na balance, while in shallow water crustaceans, compression decreases Na⁺ influx.
• 5.5. Na⁺ influx rates in Baikalian gammarids vary with the 0.55 power of weight.
• 6.6. The equation F_{ma × t} = 1.3 × W^0.55 μEq/hr/animal applies to freshwater crustaceans over the weight range from 0.03 to 35 g.

     

Human placental atp-diphosphohydrolase: Biochemical characterization,regulation and function

• 1.1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M_r and pI values of both ATPase-ADPase activities.
• 2.2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher.
• 3.3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DBS and slightly by DCCD.
• 4.4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A.
• 5.5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of −SH, COO⁻, −OH, and probably also Tyr and Trp.
• 6.6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes.
• 7.7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

     

Modulation of active potassium transport by aldosterone

• 1.1. The role of aldosterone on active potassium transport across lizard colon under voltage-clamped conditions has been investigated.
• 2.2. Control colons exhibited no net potassium flux (J^k_net) despite of the existence of active opposite unidi ectional fluxes.
• 3.3. An important net secretory potassium flux was found in short-circuited aldosterone-stimulated colons.
• 4.4. Mucosal amiloride did not change (J^k_net) either in control or aldosterone-stimulated colons.
• 5.5. Luminal barium alters K ⁺ transport in a manner consistent with the presence of barium-sensitive conductances at the apical membrane of both control and aldosterone-treated colons.
• 6.6. The effects of ouabain and barium on control and aldosterone-induced potassium flows were consistent with a model involving basolateral uptake by an Na ⁺-K ⁺-ATPase and conductive exit across the apical membrane.
• 7.7. The stimulatory effect of aldosterone on potassium secretion is associated with parallel increases of both basolateral K ⁺ entry and the apical conductive pathway.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.