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

     

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.

     

Oxygen consumption and haemocyanin function in the freshwater snail Marisa cornuarietis (L.)

• 1.1. The MO₂ for branchial respiration in adult snails increased from 0.24 mmol/l/O₂ kg/hr at 18°C to 0.83 mmol/l/O₂ kg/hr at 40°C. Q₁₀ values were 2.75 between 35 and 40°C and 1.8 between 18 and 30°C.
• 2.2. The haemocyanin (31.9 ± 5.8 mg/ml) has a high oxygen affinity (6.28 ± 0.8 at 25°C) with a reversed Bohr effect measured between a pH of 6.80 and 7.95 with gelchromatographed haemolymph, and measured between a pH of 7.34 and 8.10 for native haemolymph.
• 3.3. Growth rate is optimal between 27 and 30°C whilst at 24°C stunted growth was found.
• 4.4. At 25°C the same MO₂ values were found for aerial and aquatic respiration.

     

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.

     

Effects of low environmental pH and temperature on hatching and metabolic rates in embryos of Anax junius drury (Odonata: Aeshnidae) and the role of hypoxia in the hatching process

• 1.1. Studies were conducted in order to determine the combined effects of low environmental pH and temperature on embryonic survival capacity and metabolic rates in the dragonfly, Anax junius Drury. Studies were also conducted to assess the effects of hypoxia on hatching success as well as to investigate the role of hypoxia as a possible physiological triggering mechanism for hatching.
• 2.2. At water temperatures of 10–30°C, an environmental pH value of 3.0 was extremely limiting and significantly reduced hatching success.
• 3.3. Over a pH range of 3.0–5.0, a water temperature of 30°C was found to be severely limiting. Over a pH range of 6.0–7.0, hatching success was greater than 80% at test temperatures ranging from 10 to 25°C.
• 4.4. Embryos of A. junius exhibited a greater tolerance to markedly low environmental pH (3.0) than that previously reported for fish and amphibians, although survival capacity was less than 10%.
• 5.5. An environmental pH value of 3.0 has a significant detrimental effect on embryonic development. Survivorship and developmental rate increase significantly over a pH range of 4.0–5.0.
• 6.6. Oxygen consumption rates were lowest for fertilized eggs exposed to a pH of 3.0 at all test temperatures (10–30°C). Metabolic rates increased significantly at pH 4.O.
• 7.7. Embryos hatch successfully under hypoxic conditions in both aqueous and nonaqueous media. Results suggest that hypoxia acts as a triggering mechanism for hatching in this aquatic insect.

     

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.

     

Characterization of two distinct latent proteinases associated with myofibrils of crucian carp (Carassius auratus cuvieri)

:

• 1.1. Enzymatic properties of two distinct proteinases tightly associated with crucian carp myofibrils were characterized.
• 2.2. These proteinases were latent but activated at 50 and 60°C, respectively.
• 3.3. The optimum pH of 50°C-proteinase was neutral-alkaline, while that of 60°C-proteinase was weak acid-neutral pH.
• 4.4. Both proteinases required more than 1% NaCl for the activity, but 50°C-proteinase was partially inhibited at higher concentrations of NaCl.
• 5.5. Both proteinases were regarded as trypsin-like proteinases belonging to a serine proteinase family, but only 60°C-proteinase was sensitive to urea, n-butanol and iso-propanol.

     

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.

     

Effects of temperature and acid stress on hatching,survival capacity,metabolism and postural reflexes in caenid mayflies (ephemeroptera)

• 1.1. Mortality was 100% at pH 3.5 over a temperature range of 10–30°C for embryos and nymphs of Caenis diminuta and C. hilaris.
• 2.2. Hatching success for both species was highest at pH values above 4.5.
• 3.3. Survival capacities were significantly higher at 20°C over a pH range of 4.0-7.2.
• 4.4. Oxygen consumption rates increase as a function of increasing temperature and reduced acidity.
• 5.5. Loss of the nymphal righting response was observed at pH 3.5. This response can be used as a behavioral assay for acid stress.

     

Phospholipase A activity of culture supernatants from Streptococcus mutans strain 6715

• 1.1. Phospholipase A activity was found in the culture broth of growing cultures of Streptococcus mutans strain 6715.
• 2.2. The amount of enzyme activity was proportional to the cell density of the cultures.
• 3.3. The enzyme had a pH optimum of 7.0 and was inactivated at temperatures greater than 45°C.
• 4.4. The enzyme was Ca²⁺-dependent, since both EDTA and EGTA were inhibitory and Ca²⁺ was stimulatory.
• 5.5. Analysis of the fatty acid products resulting from the enzyme's action on 1-palmitoyl-2-oleoyl phosphatidylcholine indicated the enzyme to be a phospholipase A₁, (EC 3.1.1.32).

     

Electrophysiological and metabolic responses of the isolated teleost retina to changes in po2, and temperature

• 1.1. Comparisons of electrophysiological responses (ERG) were made between two different in vitro preparations of teleost retina.
• 2.2. The ERG was independent of temperature over the normal environmental range.
• 3.3. The Q₁₀ demonstrated temperature independence between 5 and 20°C.
• 4.4. The electrical response of the isolated retina was found to be independent of partial pressures of oxygen at levels above 250 mm Hg.

     

Haemoglobin in male Daphnia magna

• 1.1. To define the respiratory function of haemoglobin in male Daphnia magna, the swimming activity, the depression of oxygen uptake by treatment with carbon monoxide and in vivo oxygenation of Hb at various oxygen pressures were investigated.
• 2.2. The P₅₀, values for the purified Hbs from male and female red animals were 2.0 and 2.7 torr in 0.1 M phosphate buffer (pH 7.2) at 20°C, respectively.
• 3.3. The isoelectric focusing patterns of the purified Hbs from male and female red animals showed only small differences in Hb components of high PI values.

     

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.

     

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)

     

Isohematoporphyrin-diesters and diethers

• 1.1. A series of diesters of isohematoporphyrin (isoHp), from dimethyl to dioctyl were prepared according to Rimington et al. (1989b). Their optical absorption, fluorescence spectra and high performance liquid chromatography (HPLC) retention times were recorded.
• 2.2. A plot of HPLC retention time against number of C atoms in the alcohol used for esterification was approximately linear at first then rising steeply from diamyl to diocyi ester, whether a gradient elution was used or only methanol: water, 95/5, at pH 7.5.
• 3.3. Preparation of the diethers of isoHp was much more difficult than that of the corresponding derivatives of hematoporphyrin (Hp). Several different methods were investigated, varying both times and temperatures.
• 4.4. These methods included reaction of isoHp or its demethyl ester with
  • 4.1.(i) a bromoalkane in presence of anhydrous K₂CO₃;
  • 4.2.(ii) reaction with bromoalkane and Ag₂O;
  • 4.3.(iii) reaction of brominated-isoHp, prepared by using thionylbromide, with the selected alcohol, or corresponding sodium alcoholate;
  • 4.4.(iv) heating of isoHp alone with an alcohol containing 20% (w/v) H₂SCO₄ (temp. range from 45° to 118°C),
  • 4.5.(v) refluxing as in (iv) at the b.p. of the alcohol; and
  • 4.6.(vi) carrying out this reaction in refluxing ethyleneglycoldimethyl ether (b.p. 85°C) or diethyleneglycoldimethyl ether (b.p. 155°C).
• 5.5. Some diether formation was observable by all these methods but yields were small, a considerable quantity of unreacted isoHp and other products remaining.
• 6.6. Examined by HPLC, the diethers consistently afforded a forked peak which on thin layer chromatography was only resolved into two very closely associated bands by a solvent mixture carefully selected for development.
• 7.7. On elution these materials had virtually identical optical absorption and fluoresence spectra.
• 8.8. The nature of the association is discussed, atropisomers (Gottwald and Ullman, 1969) and possible stacked monomer: dimers (Abraham et al., 1963) being considered as possibilities.

     

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⁺.

     

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.

     

Temperature acclimation in respiratory and cytochrome c oxidase activity in common carp (Cyprinus carpio)

• 1.1. Common carp (Cyprinus carpio) exposed to experimental temperatures of 12, 18, 24, 30 or 36°C for a 4-week period were used to investigate the effect of temperature acclimation on the frequency of opercular movement (FOM), growth and cytochrome c oxidase (CCO) activity in heart, liver and muscle.
• 2.2. An exponential relationship between FOM and temperature after the first week (10₁₀ =1.76) disappeared after the second week.
• 3.3. The initially high FOM at temperatures of 30 or 36°C and the low FOM at 18 or 12°C changed over 4 weeks to approach the FOM of fish at 24°C.
• 4.4. This change in the relationship of FOM to temperature from highly dependent to independent appeared to be thermal compensation.
• 5.5. Heart and liver CCO activities were significantly affected by temperature, with the lowest activity at the approximate optimum temperature for growth, 24°C.
• 6.6. Highest CCO activities for heart and liver occurred at both the highest and lowest temperatures.
• 7.7. Among the three tissues, heart CCO activity was generally the highest and most affected by acclimation temperature.
• 8.8. Muscle tissue had the lowest CCO activity and was unaffected by temperature.
• 9.9. The high CCO activity at a cold acclimation of temperature 12°C was probably due to thermal compensation and the high activity at 36°C may have been a result of thermal stress.

     

The digestive enzymes of the pacific brown shrimp Penaeus californiensis.: I—Properties of amylase activity in the digestive tract

• 1.1. Crude extract of the whole digestive tract from the brown shrimp (P. californiensis) was investigated for digestive amylase activity.
• 2.2. Considerable amylase activity was found at pH 6.5–8.0, with optimum pH at around 7.5.
• 3.3. Optimum temperature was found between 30–40°C, similar to amylases from other crustaceans.
• 4.4. Amylase activity was highly halotolerant, having 50% maximum activity at 3 M NaCl.
• 5.5. Maximum amylase activity was found at 0.01 M NaCl.
• 6.6. Amylase activity was partially inhibited by the divalent ions Hg²⁺, Zn²⁺, Cu²⁺ and Cr²⁺.
• 7.7. Mg²⁺ and Ca²⁺ ions seemed to enhance amylase activity.

     

Effect of starvation and experimental feeding on the proximate composition and caloric content of an antarctic teleost,Notothenia coriiceps neglecta

• 1.1. Starving Notothenia coriiceps nn/lecta at 1°C for 20 days resulted in a loss of 4.22 gcal/kcal per day.
• 2.2. During starvation energy was obtained from lipid and carbohydrate stores of the liver and red muscle.
• 3.3. Feeding N. coriiceps neglecta low lipid, high protein shrimp meat at 18.9 gcal/kcal per day at 1°C for 20 days resulted in a gain of 8.5 gcal/kcal per day.
• 4.4. The level of carbohydrate in the liver and red muscle increased five times.
• 5.5. Gross growth efficiency (K₁) equalled 0.52.
• 6.6. Net growth efficiency (K₂) equalled 0.67.