Functional studies on the single component hemoglobin from an Amazon knife fish,Sternopygus macrurus

• 1. The whole blood of the non-air-breathing gymnotid teleost,Sternopygus macrurus, is half-saturated with oxygen at 5.2 mm Hg (apparent value) at 30°C in the absence of CO₂. Addition of 5.6% CO₂ causes the apparentP₅₀ value to increase over 3-fold.
• 2. The oxygen affinity of the stripped single-component hemoglobin at 20°C increases about 20-fold between pH 5.8 and 8.6 in the absence of ATP. This difference increases to 100-fold in the presence of 1 mM ATP.
• 3. A substantial Root effect is present: the stripped hemoglobin is only 70% saturated with O₂ at pH less than 6 when equilibrated with air.
• 4. The value of the Hill coefficient,n, is maximal near pH 7.0–7.5, and approaches 1.0 at high pH. The value is about 1.5 at low pH in the absence of ATP and 1.0 in the presence of 1 mM ATP.
• 5. The O₂ dissociation kinetics are heterogeneous at all pH values but most heterogeneous at low pH. The rate increases substantially as the pH decreases.
• 6. The CO combination kinetics as measured by the stopped flow technique are largely homogeneous except at high pH, but the CO combination kinetics after flash photolysis are markedly heterogeneous.

     

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

     

The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Kinetics of oxygen and carbon monoxide binding to the hemoglobins of the water snakes Liophis miliaris and Helicops modestus

• 1.1. Liophis miliaris and Helicops modestus are water snakes having different respiratory adaptiations to their specific habitats. L. miliaris is more active and spends more time on land than H. modestus. Knowledge of the equilibrium and kinetics of ligand binding to their hemoglobins leads to better understanding of molecular aspects of this adaptation.
• 2.2. Both snakes contain several hemoglobin types in their blood. Studies on the kinetics of oxygen dissociation and carbon monoxide combination with these hemoglobins were performed by stopped-flow and flash-photolysis experiments at various pH values, both in the presence and absence of adenosine triphosphate.
• 3.3. The oxygen dissociation kinetics of L. miliaris hemoglobins show a strong pH dependence and cooperative interactions between chains are indicated by autocatalytic time-courses at pH 7.0. In contrast, H. moledstus hemoglobins show nearly pH independent rate constants for oxygen dissociation and cooperative interactions between chains were not apparent. The hemoglobins of H. modestus show increased pH dependence in the presence of adenosine triphosphate.
• 4.4. The carbon monoxide combination kinetics differ for the hemoglobins of L. miliaris and H. modestus in general agreement with the differences found in the kinetics and equilibria of oxygen binding. Both the kinetic and steady-state difference between these hemoglobins may be advantageous in light of the behavioral differences of these two water snakes.

     

Equilibria and kinetic of oxygen and carbon monoxide binding to the haemoglobin of the South American lungfish,Lepidosiren paradoxa

• 1. The haemoglobin of the South American lungfishLepidosiren paradoxa has a single component.
• 2. The equilibria of this respiratory protein with oxygen have been investigated both in the blood and with the purified haemoglobin. There is a substantial, normal, alkaline Bohr effect and marked sensitivity to organic phosphates in the haemoglobin solutions.
• 3. Studies on the pH dependence of the kinetics of oxygen dissociation can be interpreted in terms of a normal Bohr effect.
• 4. The kinetics of combination of carbon monoxide have an unusual pH dependence.
• 5. These findings are discussed in terms of the two-state model of Monodet al. (1965)

     

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.

     

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 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.

     

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.

     

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.

     

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.

     

Purification,catalytic and regulatory properties of malate dehydrogenase from the foot of Patella caerulea (L.)

• 1.1. Malate dehydrogenase has been purified from the foot muscle of Patella caerulea by ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Blue Agarose and gel filtration on Sephadex G-150.
• 2.2. The yield was 23.5% of the initial activity with a final specific activity of 257 U/mg of protein.
• 3.3. The apparent mol. wt of the native enzyme is approx. 75,000 and it consists of two subunits of mol. wts in the range of 36,000–39,000.
• 4.4. The enzyme exhibits hyperbolic kinetics with respect to oxaloacetate, NADH and l-malate. The K_m values were determined to be 0.055 mM for oxaloacetate, 0.010 mM for NADH and 0.37 mM for l-malate. The pH optima are around 8.4 for the reduction of oxaloacetate and 9.2–9.6 for the reduction of oxaloacetate and 9.2–9.6 for the l-malate oxidation. V_max and K_m values for oxaloacetate change in an opposite manner with respect to pH values.
• 5.5. Of the various compounds tested, only α-ketoglutarate, citrate and adenylate phosphates were found to inhibit the enzyme activity.
• 6.6. From the above properties it appears that the reaction of cytoplasmic malate dehydrogenase of P. caerulea foot muscle is a key reaction in the anaerobic pathway and it occurs with the production of malate.

     

A comparative study of the respiratory properties and physiological function of haemocyanin in two burrowing and two non-burrowing crustaceans

• 1.1. Oxygen dissociation curves were constructed for the haemolymph of two non-burrowing, Galathea strigosa and Eupagurus bernhardus, and two burrowing crustaceans, C. cassivelaunus and Nephrops norvegicus. The p₅₀ at in vivo pH values and 10°C was 12.6 Torr in G. strigosa, 23 Torr in E. bernhardus, 3.1 Torr in C. cassivelaunus and 11.5 Torr in N. norvegicus.
• 2.2. The Bohr values (Δlogp₅₀/ΔpH) were high in all species ranging between −0.96 and −1.48. Cooperativity expressed as P₅₀ averaged 3.3, 3.8 and 3.8 in G. strigosa, E. bernhardus and N. norvegicus. respectively. A lower value of 2.2 was observed in C. cassivelaunus.
• 3.3. The oxygen affinity of the haemocyanin was relatively temperature independent, the values for ΔH at pH7.9 ranging between −5.1 and −18.1 kJmol⁻¹.
• 4.4. Haemolymph respiratory gas analysis showed values similar to those previously reported in crustaceans: p_aO₂ ranging between 44 and 107 Torr and p_vO₂ values between 18 and 24 Torr.
• 5.5. Pre-/post-branchial pH differences were small in G. strigosa, E. bernhardus and N. Norvegicus, but averaged 0.09 of a pH unit in C. cassivelaunus. p_aCO₂ and P_vCO₂ values ranged between 1.4 and 2.3 Torr.
• 6.6. In buried C. cassivelaunus both pre- and post-branchial oxygen tensions decreased, as did oxygen tension overall during respiratory pauses.
• 7.7. Cardiac output values were low, ranging between 59 and 71 ml kg⁻¹ min⁻¹ for all four species and calculated stroke volumes were realistic in terms of animal size.
• 8.8. In the non-burrowing species physically dissolved oxygen accounted for 5–21% of the oxygen transported to the tissues. In the burrowing species values of 40–77% were found.

     

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.

     

Functional properties of hemoglobin and whole blood in an aquatic mammal,the Amazonian manatee (Trichechus inunguis)

• 1. Hematocrit (43%) and O₂ binding capacity (18.8 ml O₂/100 ml blood) ofTrichechus inunguis blood are low compared to the values for other diving mammals but are similar to those for land mammals.
• 2. Stripped manatee Hb is similar to human Hb A in its sensitivity to pH, 2,3-diphosphoglycerate and CO₂, but less sensitive to temperature and more prone to dissociate into dimers.
• 3. The unique Hill plots exhibit no cooperativity below 30% O₂-saturation indicating a highly stabilized T or low-affinity state(s); such asymmetric Hill plots together with biphasic O₂-binding kinetics could mean chain heterogeneity.
• 4. The pH dependence of oxygen binding by the apparent T state, hemoglobin as seen in the Hill plots, is enhanced by 2,3-diphosphoglycerate but eliminated by CO₂.

     

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.

     

The effect of temperature on the acid-base status of the blood of the hatching quail (Coturnix c. japonica)

• 1.1. The ambient temperature of embryos of pipped eggs was reduced from 38 to 28°C for a period of 45 min.
• 2.2. The blood P_CO2 was lower and the blood more alkaline at 28°C than at 38°C.
• 3.3. At 28°C plasma [HCO₃⁻] ] was lower than predicted from the blood buffer line determined in vitro.
• 4.4. The plasma concentrations of strong ions and lactate were the same at both temperatures.
• 5.5. After the ambient temperature had been returned to 38°C for a period of 45 min, blood pH was more acidic than before cooling, but there was no difference in blood P_CO2.
• 6.6. The plasma [HCO₃⁻] was the same as that at 28°C and plasma [K⁺] was higher than before cooling.
• 7.7. The results arc discussed in relation to the factors affecting blood pH in embryos at this stage of development.

     

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

• 1.1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine.
• 2.2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5.
• 3.3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site.
• 4.4. The K_I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane.
• 5.5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

     

Lipoxygenase of the thermophilic bacteria thermoactinomyces vulgaris—properties and study on the active site

• 1.1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized.
• 2.2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C.
• 3.3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0.
• 4.4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5.
• 5.5. The purified lipoxygenase from Th. vulgaris showed an apparent K_m of 1 mM and V_max of 0.84 μmol diene/min/mg protein.
• 6.6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD.
• 7.7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.

     

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⁺.