Malate dehydrogenase in subtropical fish belonging to the orders characiformes,siluriformes and perciformes I. Duplicate gene expression and polymorphism

• 1.1. Electrophoretic analysis of the soluble malate dehydrogenase (sMDH) from 22 subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes, collected in 10 reservoirs of São Paulo State and in two lakes of Minas Gerais State, Brazil, indicates that at least two sMDH loci, MDH-A¹ and MDH-B¹, are active. In addition to this latter locus, in Hoplias malabaricus (Erythrinidae, Characiformes), a MDH-A 1,3¹ isoloci is proposed in order to explain the six-banded pattern detected in all the individuals screened.
• 2.2. In attempting to explain the multiplicity of compounds detected in 87% of the Geophagus brasiliensis (Cichlidae, Perciformes) specimens analyzed, three hypotheses are proposed: the event of duplication in processing the presence of three loci with a null allele within the MDH-B¹, and overdominance.
• 3.3. In 87% of the species here studied, a bidirectionally divergent pattern of expression of the sMDH loci was observed, in which the least anodal isozyme A₂ predominated in liver, and the most anodal isozyme B₂ predominated in skeletal muscle. In two siluriform species, Pimelodela gracilis and Hypostomus regani, and in one perciform, Tilapia rendalli, a unidirectionally divergent pattern, in which the isozyme A₂ predominated in every tissue analyzed, was observed.
• 4.4. Polymorphism in at least one of the sMDH loci was detected in 9% of the species studied here: Leporinus friderici (Characiformes) at the MDH-A¹ and P. gracilis at both sMDH loci. In L. friderici and Pimelodus maculatus (Siluriformes), rare alleles at the MDH-B¹ locus were detected. Polymorphism at the mitochondrial locus was detected in Tilapia rendalli.

     

Subunit homologies of lactate dehydrogenase (LDH) isoenzymes between amphibians (Xenopus laevis) and mammals (Wistar rat)

• 1.1. The subunit distribution and subunit homologies of LDH isoenzymes were studied in the amphibian Xenopus laevis and in Wistar rats.
• 2.2. Several of the 11–15 isoenzymes of the pattern in Xenopus, separable by vertical starch gel electrophoresis, were purified, hybridized, and the cross-reaction of antibodies against the most positively charged isoenzyme with the isoenzymes present in tissue extracts of both species was tested.
• 3.3. The isoenzyme with the highest positive charge in Xenopus is a M-homotetramer homologous to mammalian LDH₅.
• 4.4. The multibanded pattern of Xenopus LDH isoenzymes is very probably due to heterozygoty of the gene locus controlling the synthesis of M-subunits rather than to an epigenetic subbanding phenomenon.

     

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.

     

Electrophoretic identification of bird species involved in collisions with aircraft

• 1.1. The utility of biochemical genetic methods of bird identification was investigated for some common species which create a hazard for commercial aviation in Ireland.
• 2.2. Sixteen enzyme loci were assayed in eight species, using starch gel electrophoresis; three larids, three corvids and two columbids.
• 3.3. Genera were distinguishable using all but two loci.
• 4.4. Differences within genera were small, but all species except for the gulls Larus argentatus and L. marinus, could be identified using one or more loci.
• 5.5. Arising from the success of the method using fresh specimens, a protocol for the electrophoretic identification of traumatized remains of strikes is suggested.

     

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.

     

Calcutta-1 LDH: Kinetic and thermodynamic properties of an electrophoretic variant of human LDH

• 1.1. Kinetic constants determined for the purified heterozygous variant LD₁ were closely similar to those of normal human LD₁.
• 2.2. Calcutta-1 homozygote LDH differed from normal LDH in K_m NADH and in Arrhenius activation energy.
• 3.3. The normal B subunits confer stability on the mutant subunits in the heterotetramers of Calcutta-1 LD₁.

     

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.

     

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.

     

The oxygen consumption rates of three gastrotrichs

• 1.1. The oxygen consumption rates for three sympatric species of marine gastrotrichs (anatomically similar, except that one contains hemoglobin) were measured with a Cartesian diver microrespirometer.
• 2.2. The rates for the two species without hemoglobin, Turbanella ocellata and Dolichodasys carolinensis, were 307.2 μl O₂ g⁻¹ hr⁻¹ and 108.0 μl O₂ g⁻¹ hr⁻¹, respectively, while the rate for the hemoglobin-containing species, Neodasys, was 208.9 μl O₂ g⁻¹ hr⁻¹.
• 3.3. The possession of hemoglobin by Neodasys (14% by volume) cannot be explained by an unusually high demand for oxygen.
• 4.4. Instead, the hemoglobin may be useful as an oxygen store providing continued aerobic metabolism in anoxic conditions, thus allowing Neodasys to exploit a different niche.

     

Comparative dehydrogenase activity during the ontogenesis of the yellow fever mosquito Aedes (Stegomyia) aegypti L. (diptera:culicidae)

• 1.1. α-GPDH in most active in adults, LDH in third instar larvae, and MDH in third instar larvac.
• 2.2. During pre-pupal growth, LDH is the most active enzyme, followed by MDH and α-GPDH; while during post-pupal growth, MDH is most active followed by α-GPDH and LDH.
• 3.3. Increased enzyme activity is in response to changing physiological and physical environments, and is due to temporal activation of new gene loci indicated by the production of new α-GPDH and MDH isozymes. LDH activity is probably controlled by the temporal action of regulatory gene(s).
• 4.4. Dehydrogenase activity profiles during ontogenesis are probably species-specific.

     

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.

     

Toxicity of cadmium administration to the toad and the treatment of its poisoning with EDTA

• 1.1. The effect of cadmium administration on female Bufo regularis was studied. The median lethal doses were 22, 18, 15 and 6.2 Cd²⁺/kg after 24, 48, 72 and 96 hr respectively.
• 2.2. After a single intramuscular injection of 6.2 Cd²⁺/kg (representing 96-hr ld₅₀), the results indicated that Cd²⁺ causes severe physiological abnormalities to this experimental animal.
• 3.3. The serums alanine aminotransferase (AlAt), aspartate aminotransferase (AAt), alkaline phosphatase (A1P) and lactic dehydrogenase (LDH) were elevated while the calcium serum was not influenced by Cd²⁺ throughout the experimental period
• 4.4. On the other hand, phosphorus, total protein and total bilirubin were increased.
• 5.5. EDTA treatment (0.2 mmole/kg protected female toads from mortality up to 20 mg Cd²⁺/kg. It overcame the physiological alterations that were caused by the Cd²⁺ injection.
• 6.6. This may be due to the fact that Cd²⁺ is bound to EDTA in a strong complex which is readily excreted via the kidneys.

     

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.

     

Evidence for hyporegulation in the calanoid copepod,Acartia tonsa

• 1.1. The euryaline calanoid copepod, Acartia tonsa, maintains haemolymph Na below that of the external medium in salinities above 34^o_oo (475 mM Na).
• 2.2. The measured transepithelial electrical potential. −9.97 ± 1.0 mV, indicates that Na is regulated out of electrochemical equilibrium.
• 3.3. Water osmotically lost in hyporegulation is replaced by Na-dependent absorption by the gut.
• 4.4. High osmotic water permeability is evidenced by the fact that with an increase in external salinity from 475 mM Na to 580 mM Na the copepod's drinking rate nearly doubles.
• 5.5. Sodium efflux measurements indicate that ionic permeability is much lower than other hyporegulating crustaceans.
• 6.6. The energetic advantage of hyporegulation in this species is considered.

     

Comparative examination of the soluble muscle proteins of two species of angler-fish (Lophiidae): Lophius piscatorius (L.) and Lophius budegassa (Spinola)

• 1.1. A comparative examination of sarcoplasmic proteins of the two nominal European species of angler-fish, Lophius piscatorius and L. budegassa was carried out using isoelectric focusing techniques.
• 2.2. Two protein bands differing in isoelectric point proved diagnostic for L. budegassa (pI 4.40 and pI 5.75) while a third characterized L. piscatorius (pI 4.65).
• 3.3. These species-specific protein profiles provide a method of species discrimination independent of morphological criteria.
• 4.4. Within-species heterogeneity of banding pattern suggested the presence of polymorphic gene loci of potential use in studies of population structure.

     

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.

     

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.

     

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.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.