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.

     

Salinity tolerance and sodium balance in the prawn Palaemonetes vulgaris (say) compared with P. pugio

• 1.1. Larvae (first zoeae) of Palaemonetes vulgaris are relatively stenohaline (optimum salinity = 20%.), adults euryhaline (96-hr LD₅₀ values: 0.8 and 51%.).
• 2.2. The concentration of blood sodium remains nearly constant over the salinity range 5–45%.
• 3.3. Adult P. vulgaris are less tolerant of dilute (1–20%.) media than sympatric P. pugio but equally tolerant of higher salinities (35–45%.). Palaemonetes vulgaris maintains a slightly more constant and higher (average) sodium concentration in the blood than P. pugio.
• 4.4. It is suggested that these differences contribute to habitat partitioning of these species and that they reflect the greater affinity of P. vulgaris for a euhaline milieu.

     

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⁻.

     

Amino acid composition and the protein form of procarboxypeptidase a purified from the pancreas of the sei whale Balaenoptera bolealis

• 1.1. Procarboxypeptidase (W-PCPA) was purified from the pancreas of the sei whale Balaenoptera bolealis.
• 2.2. W-PCPA was obtained as a homogeneous protein in polyacylamide gel disc electrophoresis.
• 3.3. W-PCPA has a molecular weight of 75,000.
• 4.4. Amino acid composition of W-PCPA was compared with that of bovine procarboxypeptidase as A S5 (PCPA-S5).
• 5.5. W-PCPA may be two subunits, and the aggregate form may resemble PCPA-S5.

     

Neural network regulating heart activity in Aplysia depilans and its comparison with other gastropod species

• 1.1. The efferent part of heart regulatory neural network was investigated in Aptysia depilans.
• 2.2. In the abdominal ganglion 9 identified giant neurons and 12 unidentified small cells were found which modified heart activity, 3 neurons of the RP1G. 1-1 cell of the RPG and LPG involved in heart regulation were also detected.
• 3.3. Similar to other gastropod species, the heart regulatory neural network of Aplysia depilans is scattered throughout the CNS.
• 4.4. Most of the neurons having efferent influences on the heart receive also inputs from heart. There were only six exceptions, five located in the abdominal ganglion and one in the LPG. A left side dominancy was found in the localization of heart regulatory neurons.
• 5.5. One interneurone (L10) and two motoneurons (RB_HE, LD_HI1) were identified and described earlier in other Aplysia species. Cell L10 was silent or displayed tonically active or a bursting pattern. Its tonic firing led to the inhibition of the heart. Neurone L10 received indirect information from the heart.
• 6.6. There exist mutual connections between or similar inputs in neurones located in different ganglia. The regulation of heart activity in Aplysia depilans seems to be accomplished by the aid of multifunctional neural networks, where the regulation of various organs is subordinated to the need of the whole organism.

     

Isolation and characterization of glutamine synthetase from the diazotroph Azospirillum brasilense

• 1.1. Glutamine synthetase was purified from the diazotroph Azospirillum brasilense.
• 2.2. The holoenzyme with a M_r of 630,000 is composed of 12 subunits of M_r 52,000.
• 3.3. A modified subunit of M_r 53,000 was also found by electrophoresis under denaturing conditions.
• 4.4. It is shown that the M_r 53,000 species is the adenylylated subunit.
• 5.5. The apparent K_m values for glutamate, ATP and ammonia were 2.5 ± 0.3 mM, 200 ± 20 μM and42 ± 2 μM, respectively.
• 6.6. Levels of glutamine synthetase activity in A. brasilense cells varied by a factor of 8 depending on the nitrogen source and its concentration in the growth medium.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

• 1.1. Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins.
• 2.2. The luteal and adrenal proteins had similar absorbance spectra and molecular weights.
• 3.3. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450_scc.
• 4.4. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450_scc.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

Some properties of ATPase activity in the intact cells of yeast Saccharomycopsis fibuligera

• 1.1. The properties of ATPase activity were studied with the cells at the early stationary phase of Saccharomycopsis fibuligera.
• 2.2. Optimal pH for the activity was approximately 7.
• 3.3. The activity was stimulated by Mg²⁺.
• 4.4. The activity was inhibited by NaF, DCCD, oligomycin, NaN₃, NaVO₃, or PCMB but not inhibited by ouabain.

     

Partial characterization and subunit analysis of major phosphoproteins of egg yolk in the fish,Oryzias latipes

• 1.1. Molecular weight estimation and subunit analysis of four yolk phosphoproteins (PP1-PP4) in medaka (Oryzias latipes) eggs were performed.
• 2.2. PP1 (M_r ≈ 210,000) and PP2 (M_r ≈ 180,000) were found to be heterodimers composed of subunits of 113,000 and 94,000 and subunits of 84,000 and 72,000, respectively.
• 3.3. PP3 and PP4 [phosvitins of medaka (Murakami et al., 1990, Devl. Growth Differ.32, 619–627)], were monomeric phosphoproteins having mol. wts of about 40,000 and about 20,000, respectively.
• 4.4. Lipid composition of the mixture of PP1 and PP2, vitellogenin and yolk were found to be almost the same. PP1 and PP2 are probably lipovitellins of medaka.

     

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.

     

Conodipine-P1-3, the First Phospholipases A2 Characterized from Injected Cone Snail Venom*

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Highlights

• •Three novel Conodipines P1-3 in the injected venom of Conus purpurascens.
• •Conodipines P1-3 have consensus catalytic characteristics of sPLA₂.
• •We determined multiple modification sites in Conodipines P1-3.
• •Evaluated the activity of Conohyal-P1 by a MS-based method.

     

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.

     

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.

     

Purification and characterization of the d-lactate dehydrogenase from Leuconostoc lactis

• 1.1. The d-lactate dehydrogenase from Leuconostoc lactis has been purified in high yield.
• 2.2.The enzyme is a dimer of subunits of M_r = 39,000 and each subunit contains a single thiol group. The N-terminal residue is methionine.
• 3.3. The amino acid composition has been determined and is typical of that of a soluble globular protein.

     

Purification,isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes

• 1.1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP)3 isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL.-6B chromatography and isoelectric focusing using carrier ampholytes. pH 4–6.
• 2.2. The isoenzyme has an isoelectric point of 5.00 ± 0.05 and could be purified 33.000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme.
• 3.3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges.
• 4.4. At 4°C the isoenzyme is more stable in the pH range of 7–9 than at acid pH values.
• 5.5. Incubation at 30 and 40°C for 4 hr does not affect the activity of the isoenzyme.
• 6.6. It has a K_m-value of 0.28 mM for phosphoglycolate (PG) as substrate.

     

Further studies on the phylogenetic distribution of pyruvate oxidoreductase activities

• 1.1. The phylogenetic distribution of lactate, octopine, alanopine and strombine dehydrogenase activities (respectively, LDH, ODH, ADH and SDH) was examined in over 60 species from seven phyla and from three continents.
• 2.2. The results confirm and extend previously published data. Consistencies of distribution are observed at the levels of phyla, class, order and family.
• 3.3. Major observations include prominent SDH in the Porifera; LDH only in the Polyplacophera, Nudibranchia and Myidae (Mollusca) and nereid worms (Polychaeta); ODH and SDH in the marine pulmonate Melampus bidentatus (Basommatophora); high ADH to SDH ratios in marine gastropods; high ODH in active molluscs; and apparent SDH in the barnacle Lepas anatifera.
• 4.4. The results are discussed in relation to theories of opine pathway evolution and the newly discovered tauropine and β-alanopine opine dehydrogenases.

     

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.