Studies of the functional properties of the hemoglobins ofHoplias malabaricus andHoplerythrinus unitaeniatus

• 1. Hemolysates fromHoplias malabaricus andHoplerythrinus unitaeniatus show blurred hemoglobin patterns with three and four bands, respectively, by alkaline disc gel electrophoresis.
• 2. The oxygen affinity of the stripped hemoglobin fromHoplerythrinus is about a third of that fromHoplias; theP₅₀ value ofHoplias Hb is about 1.3 mm Hg (pH 6.9 and 20°C). The addition of 1 mM ATP lowers the oxygen affinity of each hemoglobin 2.6-fold.
• 3. Both hemoglobins show Root and Bohr effects;Δlog P₅₀ΔpH= −0.40 for a stripped hemoglobins for the interval pH 7–8.
• 4. The rate of dissociation of oxygen from each hemoglobin is similar and is kinetically homogeneous with rate constants decreasing from 200–250/sec at pH 6.2 to about 25–26 at pH 7.7 with or without 1 mM ATP.
• 5. The CO combination reaction forHoplias hemoglobin is kinetically heterogeneous at all pH values and forHoplerythrinus hemoglobin below pH 7.5. The fast and slow phases each account for about half the observed reaction. The kinetic heterogeneity is maximal at low pH for both hemoglobins. The fast phase forHoplias hemoglobin is more than twice as fast as that forHoplerythrinus hemoglobins.

     

The isolation and characterization of the hemoglobin ofBrachyplatystoma sp.: A tropical catfish

• 1. The single hemoglobin component ofBrachyplatystoma sp. has been isolated. The CO-hemoglobin has an apparent molecular weight of 69,000 as determined by gel filtration.
• 2. The hemoglobin displays both acid and alkaline Bohr effects, as organic phosphate effect and no Root effect. The whole bloodp_1/2 for oxygen shifts from 10.7 mm Hg in air equilibrated solutions to 25.1 mm Hg after the addition of 5.6% CO₂ to the equilibration gas. Thep_1/2 of purified hemoglobin varies from 0.3 mm Hg at pH 8.4 to 4.5 mm Hg at pH 5.9. The Bohr effect measured for stripped hemoglobin between pH 8.0 and 7.0 isΔlog p_1/2/ΔpH= −0.23. Additions of 1 mM ATP induce a shift in the Bohr effect toΔlog p_1/2/ΔpH= −0.58 over the same pH range.
• 3. Then value of stripped hemoglobin solutions varies from 1 at pH 5.9 to 1.7 at pH 7.0. Additions of 1 mM ATP shift the variation inn to higher pH values, and cause an increase in then value (n = 2 at pH 7.4).
• 4. The kinetics of carbon monoxide binding and oxygen dissociation are pH dependent. The CO_on rate becomes autocatalytic as the pH is lowered, indicating positive subunit interactions. The O_2off rate was homogeneous at all pH values.
• 5. The Bohr effects ofBrachyplatystoma hemoglobin and other pimelodid hemoglobins are greater than those determined for the unfractionated hemoglobins of more sedentary species from other catfish families such as the Loricariidae and Callichthyidae.

     

The hemoglobin ofpseudodoras,a South American catfish: Isolation,characterization and ligand binding studies

• 1. The hemoglobin of the Amazonian catfishPseudodoras sp. was isolated and characterized; it comprises a single component.
• 2. The hemoglobin's subunit composition is similar to that of other teleost hemoglobins. The apparent native molecular weight as determined by gel filtration is 66,000. The apparent subunit molecular weight is 14,300 by sodium dodecyl sulfate electrophoresis. The hemoglobin does not polymerize after oxidation by potassium ferricyanide.
• 3. The hemoglobin lacks a Root effect. A small Bohr effect is evident in the phosphate-free hemoglobin:Δlog p_1/2/ΔpH is no more than about −0.1 to −0.2 and increases toΔlog p_1/2/ΔpH = −0.4 in the presence of 1 mM ATP. The cooperativity, as determined byn of the Hill equation, is low, varying from 0.8 to 1.7 between pH 6.1 and 8.6.
• 4. Thep_1/2 values of stripped hemoglobin solutions are extremely low, less than 0.5 mm Hg at all pH values examined between pH 6.1 and 9.0. The high oxygen affinity is reflected primarily in the CO combination rate which resembles that found in myoglobins and isolated subunits of human hemoglobin.
• 5. Both the CO combination rate and the O₂ dissociation rate determined by stopped-flow spectrophotometry are pH and phosphate sensitive. Between pH 6.2 and 8.1 the CO_on rate increases about 5-fold in the phosphate-free hemoglobin. Addition of 1 mM ATP causes a depression in the rate at all pH values examined. The O_2off rate decreases 7-fold going from pH 6.0 to 8.2 in stripped hemoglobin solutions. Addition of 1 mM ATP induces a 10-fold decrease over the same range. At pH values below 6.0 a depression in the O_2off rate occurs in the stripped hemoglobin, indicative of an acid Bohr effect.

     

Effect of pH on the kinetics of oxygen and carbon monoxide reactions with hemoglobin from the air-breathing fish,Loricariichthys

• 1.Loricariichthys sp., an air-breathing fish from the Amazon River has one major hemoglobin component.
• 2. Quantitative studies on the kinetics of O₂ dissociation and CO combination to the protein were performed by stopped-flow experiments at different pH values and a constant ATP concentration of 1.25 mM.
• 3. The oxygen dissociation shows a simple first order behavior and a strong pH dependence.
• 4. The CO combination kinetics, on the other hand, were homogeneous and fast at higher pH values and slow and clearly autocatalytic at pH values below 7.0.

     

Functional studies on the separated hemoglobin components of an air-breathing catfish,Hoplosternum littorale (Hancock)

• 1. The two hemoglobins, Hb I and II, of the obligate air-breathing catfish,Hoplosternum littorale have been isolated.
• 2. The unfractionated stripped hemoglobin has a high oxygen affinity, a normal alkaline Bohr effect, and a Root effect.
• 3. Both the Bohr and Root effects are enhanced by 1 mM ATP.
• 4. Stripped Hb I has a relatively high oxygen affinity, a reversed Bohr effect between pH 6.0 and 8.0 (Δlog P₅₀2DpH> 0), but no Root effect. Addition of 1 mM ATP to Hb I causes a marked reduction in the oxygen affinity, a change to a normal alkaline Bohr effect (Δlog P₅₀ΔpH< 0), but no Root effect.
• 5. Stripped Hb II has a lower oxygen affinity at low pH and a higher oxygen affinity at high pH than does Hb I. Hb II shows a large alkaline Bohr effect which is only slightly increased by 1 mM ATP and a Root effect at low pH which is enhanced by 1 mM ATP.
• 6. The observed rates of O₂ dissociation and of CO combination with Hbs I and II show differences which parallel those observed in the oxygen equilibrium measurements.

     

Some kinetic properties of pyruvate kinase from Phycomyces blakesleeanus

• 1.1. Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555(−) has been partially purified and some kinetic properties has been investigated at pH 7.5.
• 2.2. Positive homotropic interactions were observed with phosphoenolpyruvate and Mg²⁺, showing Hill coefficient values of 2.8 and 2.5, respectively, whereas hyperbolic kinetics are found when ADP was the variable substrate.
• 3.3. Fructose 1,6-bisphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form.
• 4.4. ATP inhibits pyruvate kinase from mycelium of Phycomyces blakesleeanus. ATP appears to be a non-competitive inhibitor with respect PEP and competitive inhibitor with respect ADP.

     

The effect of temperature on oxygen equilibrium curves of trout blood (Salmo gairdnerii)

• 1.1. Oxygen equilibrium curves were measured on trout red blood cell suspensions at pH 7.8 and 8.4 at 15, 20 and 25 C. Normal red cells and red cells that had been depleted of their ATP content were used.
• 2.2. The equilibrium data were fitted to the Adair's model and the enthalpy (ΔH) and entropy (ΔS) changes for the first and fourth steps of oxygenation and for overall oxygenation were calculated from the temperature dependencies of the Adair constants.
• 3.3. For normal red blood cells, the apparent heat for the first oxygenation step, δh₁, is close to zero.
• 4.4. Temperature insensitivity of this step at physiological pH, combined with a large pH dependence, probably denotes a property of Hb4, the Root effect Hb of trout blood.
• 5.5. At pH 7.8, ΔH₄ is about —4kcal/mol, a small value which may be attributed to the large release of Bohr protons that occurs at the last oxygenation step and corresponds to an endothermic process which opposes to the exothermic oxygenation of the haem.
• 6.6. The ΔH₄ value appears to have a large influence on the enthalpy for overall oxygenation.
• 7.7. Results for ATP-free red cells are consistent with a mere increase in the intracellular pH and suggest that ATP has no specific effect at and above pH_i ~ 7.7.
• 8.8. Effects of temperature and pH on trout red blood cell isotherms emphasize the primary importance of the major component of trout blood, namely Hb4, in trout blood functional properties.

     

Separation and characterization of the hemoglobin components ofPterygoplichthys pardalis,the acaribodo

• 1. The four main hemoglobin components of the hemolysate ofPterygoplichthys pardalis have been isolated and characterized.
• 2. The functional properties investigated for the isolated components comprise the effect of pH and ATP on (i) the O₂ equilibrium, (ii) the O₂ dissociation kinetics, (iii) the CO combination kinetics.
• 3. Component I, corresponding to approx 50% of the total hemoglobin, is characterized by functional properties which are distinctly different from those of Components II, III and IV, which are alike
• 4. Thus it is shown, once more, that multiple components in an hemolysate fall into categories of hemoglobins characterized by distinct and complementary functional properties

     

Oxygen binding of intact coelomic cells and extracted hemoglobin of the echiuran Urechis caupo

• 1.1. The oxygen affinity of Urechis caupo coelomic cells is the same in normoxic and in hypoxic animals. There is no Bohr effect between pH 6.8 and 8.0.
• 2.2. The oxygen affinity of intact coelomic cells is the same as that of extracted, stripped hemoglobin. The oxygen binding properties of stripped hemoglobin are not affected by 1 mM ATP, IMP, or hydrogen ions between pH 6.8 and 8.0, nor do they clearly show cooperativity. The heat of oxygenation. ΔH, = −13.1 kcal/mol between 10 and 25 C.
• 3.3. Although U. caupo coelomic cell hemoglobin is tetrameric and intracellular, it apparently exhibits neither heterotropic nor homotropic interactions.

     

Equilibria and kinetics of oxygen and carbon monoxide binding to the haemoglobin of the teleostSynbranchus marmoratus

• 1. The haemoglobins of the air-breathing fishSynbranchus marmoratus have multiple components and demonstrate polymorphism.
• 2. The equilibrium of oxygen with whole cell and isolated haemoglobin has been compared. There is a considerable alkaline Bohr effect and this is more marked in stripped haemoglobin to which 1 mM ATP has been added.
• 3. The kinetics of oxygen dissociation from oxyhaemoglobin and of carbon monoxide combination to deoxyhaemoglobin have been studied, and show surprisingly uniform kinetic time courses for a system composed of multiple components.
• 4. These findings are discussed within the framework of the requirements of the animal in its mixed aerial-aquatic environment.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

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.

     

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.

     

A kinetic study of inosine nucleosidase from Azotobacter vinelandii

• 1.1. Inhibition of inosine nucleosidase from Azotobacter vinelandii by ATP and bases can be qualitatively and quantitatively accounted for by the partial noncompetitive inhibition mechanism with ligand exclusion model.
• 2.2. The enzyme has two binding sites for the substrate with equal affinity in the absence of the inhibitor. and two species of the inhibitor sites: I₁- and I₂-sites. The I₁-site may overlap part of each substrate binding sites, and the I₂-site is separated from the substrate sites.
• 3.3. ATP binds to the I₁-site of the enzyme, and prevents the substrate from binding to either of two identical sites, producing the cooperativity with inosine, whereas binding of ATP to the I₂-site causes a noncompetitive inhibition.
• 4.4. Adenine and hypoxanthine bind to the I₂-site of the enzyme, and the EIS complex is partially active, resulting in a partial noncompetitive inhibition with Michaelis-Menten kinetics.

     

A fetal-maternal shift in the oxygen equilibrium of hemoglobin from the viviparous caecilian,Typhlonectes compressicauda

• 1. The equilibria and kinetics of oxygen binding by blood and hemoglobin from adult and fetal caecilians,Typhlonectes compressicauda, have been measured.
• 2. The oxygen affinity of fetal blood is higher than that of adult blood.
• 3. Electrophoresis of adult and fetal hemoglobins suggests that they may be identical: a major and minor component occurs in each.
• 4. Adult and fetal hemoglobins have identical oxygen equilibria. Stripped hemoglobins have a high oxygen affinity and no Bohr effect between pH 6.5 and 10.0. An “acid”, reversed Bohr effect is present below pH 6.5. The addition of 1 mM ATP reduces the oxygen affinity markedly and produces a moderate, normal Bohr effect.
• 5. The major nucleoside triphosphate in fetal and adult erythrocytes is adenosine triphosphate: about 10% of the nucleoside triphosphates is guanosine triphosphate. Adult erythrocytes contain 3 times as much ATP as do the fetal erythrocytes.
• 6. The fetal to maternal shift in the oxygen equilibrium is mediated entirely by the difference in ATP content of the maternal and fetal red blood cells.

     

Respiratory properties of blood and hemoglobin solutions from the piranha

• 1. Respiratory properties of piranha blood are distinguished from those of other fish primarily by the high CO₂ buffering capacity (ΔHCO₃/⁻ΔpH= 19.6mmol/l for oxygenated blood and 39.1 mmol/l for deoxygenated blood).
• 2. The concentration of nucleoside triphosphates (NTP) and the half-saturation tension (P₅₀) of whole blood were found to be inversely related to body size.
• 3. The higherP₅₀ in smaller fish, analogous to values obtained in previous studies involving interspecies comparisons, could be adaptive to a higher weight-specific metabolic rate.
• 4. Both ATP and guanosine triphosphate (GTP) lowered the oxygen affinity of purified hemoglobin solutions, accounting for the size-dependent correlation ofP₅₀ and NTP concentration in whole blood.
• 5. While similar in concentration in red cells, GTP is more potent than ATP as an allosteric modifier of hemoglobin function.

     

Sodium balance in adult atlantic salmon (Salmo salar L.) during migration into neutral and acid fresh water

• 1.1. In sea-water, adult salmon (S. salar) exchange an average of 12.6% of total body sodium/hr.
• 2.2. Following transfer to fresh water sodium uptake follows Michaelis-Menton kinetics. F_max = 2.40 mmol Na/1 ECF/hr, K_m = 0.26 mmol Na/1. The uptake system is fully activated immediately following transfer to fresh water.
• 3.3. Post smolts adapted to sea-water for 3 months take up sodium at only one third of the rate of adult fish following return to fresh water.
• 4.4. The concentration of prolactin in the plasma is low in sea-water adapted fish and does not rise during the first 8 hr in fresh water.
• 5.5. At pH 5 sodium uptake is reduced by almost 90%, even in the absence of aluminium, but recovers immediately on return to neutral water.
• 6.6. At pH 5 and 20 μmol Al/1 there is little further effect on sodium uptake but after 6 hr in aluminium the inhibition of sodium uptake continues after return to neutral aluminium fresh water and uptake is only 50% of normal 24 hr later.

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.