Phosphofructokinase from the anterior byssus retractor muscle of Mytilvs edulis: Modification of the enzyme in anoxia and by endogenous protein kinases

• 1.1. Anoxia exposure resulted in a stable modification of the kinetic properties of 6-phosphofructo-1-kinase (PFK) from the anterior byssus retractor muscle (ABRM) of the sea mussel Mytilus edulis L.
• 2.2. Compared to the aerobic enzyme, the anoxic form of PFK. showed a reduced affinity for both substrates, fructose-6-phosphate (F6P) and ATP, and an increased sensitivity to inhibition by phosphoenolpyruvate.
• 3.3. To analyze the involvement of protein kinases in the modification of PFK, extracts from aerobic or anoxic muscle were incubated with ATP and Mg²⁺ plus protein kinase second messengers cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP) or Ca²⁺ plus phorbol 12-myristate 13-acetate (PMA).
• 4.4. Both forms of the enzyme responded to the presence of cAMP with a strong increase in affinity for F6P.
• 5.5. In response to cGMP affinity of the aerobic enzyme for F6P decreased whereas that of the anoxic enzyme form was not affected (at 0.5 mM ATP) or increased (at 3 mM ATP).
• 6.6. Incubation with Ca²⁺ + PMA had only a limited effect on PFK kinetics but appeared to enhance the response to cGMP when the three compounds were given together.
• 7.7. Treatment of PFK-aerobic with alkaline phosphatase resulted in a strong decrease in enzyme activity and affinity for F6P; subsequent treatment with cAMP reversed the effect on S_0.5 F6P.
• 8.8. The data indicate that PFK activity is altered during the aerobic-anaerobic transition by a change in the phosphorylation state of the enzyme and that cAMP and cGMP act oppositely to regulate PFK activity, and thereby alter glycolytic rate, during this transition.

     

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.

     

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—II. High energy phosphate compounds and the role of glycolytic and gluconeogenetic enzymes

• 1.1. ATP, ADP, AMP, energy charge potential and total adenylates in heart, kidney and muscle are relatively unaffected by environmental hypoxia. In the liver, hypoxia causes a 90% drop in ATP, a rise in ADP and AMP, and a drop in energy charge potential and total adenylates. In the muscle tissue ATP concentration is stabilized by a large creatine phosphate pool.
• 2.2. Hexokinase activity in the heart is 20 times higher than in the swimming muscle, and thus the heart has a high potential for utilizing exogenous glucose as an anaerobic substrate.
• 3.3. The role of creatine phosphate in regulating muscle glycolysis is discussed on background of the strong inhibition of muscle phosphofructokinase by physiological concentrations of creatine phosphate.
• 4.4. Flounder heart has a dominating M-type lactate dehydrogenase which is identical to the muscle enzyme by electrophoretic and kinetic criteria. This improves the anaerobic capabilities of the flounder heart compared to other fish hearts.
• 5.5. Both liver and kidney have high activities of the gluconeogenetic enzymes glucose-6-phosphatase, fructose-1,6-diphosphatase, and phosphoenolpyruvate carboxykinase and both are capable of synthesizing glucose from [¹⁴C]lactate. Because of more favorable energy conditions in the kidney this organ may substitute the liver as a gluconeogenetic organ during hypoxia.

     

Seasonal adaptations in oxygen transport in brown trout Salmo trutta fario

• 1.1. Blood parameters determining oxygen capacity and oxygen affinity were measured in brown trout at different times of the year.
• 2.2. Haematological data indicate a slight decrease in blood oxygen capacity during the warm seasons. 3. Oxygen affinity increases significantly during summer and decreases in winter.
• 3.4. The changes in P₅₀ exhibited a positive correlation with the amount of anodic haemoglobin components, and a negative correlation with the amount of cathodic haemoglobin components.
• 4.5. The changes observed in the [ATP]/[Hb] molar ratio were not correlated with oxygen affinity and gave values near one.
• 5.6. We conclude that the oxygen affinity increase could be a physiological adaptation to oxygen transport during the wanner period. A possible mechanism is discussed.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—I. Utilization of glycogen and accumulation of glycolytic end products in various tissues

• 1.1. Resting oxygen consumption at 10°C did not change from normoxia (150 mm Hg) down to an oxygen tension of 55 mm Hg for the flounder, Platichtys flesus.
• 2.2. Flounders exposed to hypoxia showed increased levels of blood glucose and lactate, dependent on the degree of hypoxia.
• 3.3. Due to hypoxia glycogen was depleted in the liver and swimming muscle but in the heart there was no significant change.
• 4.4. Liver glucose increased after 7 hr of hypoxia. Heart and muscle glucose did not change but the absolute glucose concentration in the heart was five times higher than in the muscle.
• 5.5. There is a transient accumulation of lactate in heart, liver and kidney after 7 hr of hypoxia while lactate accumulation in the swimming muscle is significant only after 21 hr of hypoxia.
• 6.6. Succinate only accumulated in the liver while alanine accumulated in muscle, heart and liver.

     

Adaptative features of amazon fishes: Hemoglobins,hematology, intraerythrocytic phosphates and whole blood Bohr effect of Pterygoplichthys multiradiatus (Siluriformes)

• 1.1. Hemoglobin, hematological parameters, intraerythrocytic phosphates and whole blood Bohr effect of Pterygoplichthys multiradiatus, from the Amazon river, were studied in three different conditions: in their natural environment, acclimated to normoxia and acclimated hypoxia conditions.
• 2.2. Nine anodal hemoglobin fractions were detected on starch gel electrophoresis. No qualitative differences in the Hb electrophoretic patterns were detected in the three studied groups.
• 3.3. Hematocrit, hemoglobin concentration, MCV, MCHC and MCH were different among studied conditions.
• 4.4. GTP was almost absent in the blood of animals in natural conditions and acclimated to hypoxia, but was present at a concentration similar to ATP in normoxic acclimated animals.
• 5.5. There is a tendency for higher Hb-O₂ affinity for hypoxic acclimated/acclimatized animals.

     

Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.

     

Structures of functional interest in the myocardium of lower vertebrates

• 1.1. The functional relevance of the diversity in myocardial organization among lower vertebrates has been elucidated by comparing special features of the ventricular wall and the sarcoplasmic reticular network in higher and lower vertebrates.
• 2.2. Temperature effects on the contraction-relaxation cycle in isolated atrial preparations of some poikilotherms (frog, trout, flounder, shark and hagfish) are reported, providing circumstantial evidence for functional adaptations in the mechanisms involved in calcium sequestration and energy production.
• 3.3. It is suggested that the poikilotherm hearts may serve as excellent, experimental models for elucidations of the plasticity of the vertebrate myocardium, notably of its ability to handle necrotic stimuli such as excessive adrenergic stimulation at high pCO₂ and low oxygen.

     

Activities of hexokinase,phosphofructokinase and pyruvate kinase in the body wall,pyloric caeca and tube feet of Asterias vulgaris: Evidence of body wall as a major source of glycolytic activity

• 1.1. The activities of hexokinase (HK) and pyruvate kinase (PK) were significantly higher than the activity of phosphofructokinase (PFK) in the body wall, pyloric caeca and tube feet.
• 2.2. When expressed as a function of wet weight, the specific activity of HK was highest in the pyloric caeca. When expressed as a function of cytosolic protein, the specific activity of HK was highest in the body wall.
• 3.3. Specific activities PFK and PK, expressed as functions of cytosolic protein, were highest in the tube feet. These enzyme activities should reflect the high energetic requirements of the tube feet.
• 4.4. Highest total activity of PFK was found in the body wall. These results support the conclusion that the body wall is a metabolically active organ and that the energetic requirement of the body wall is a significant component of the energetic requirements of the organism.

     

Sublethal effects of hypoxia in the abalone (Haliotis rufescens) as measured by in vivo31P NMR spectroscopy

• 1.1. Effects of hypoxia were investigated in red abalones (Haliotis rufescens) using a flow-through exposure system and in vivo³¹P NMR spectroscopy.
• 2.2. Following seawater acclimation, abalones were exposed to air for 1 hr, then seawater for 2.5 hr to check recovery; parallel controls were performed without air exposure.
• 3.3. In foot muscle, hypoxia produced a decrease in phosphoarginine concentration and intracellular pH, an increase in inorganic monophosphate concentration, and no change in that of ATP; upon resubmergence, all effects generally recovered.
• 4.4. The changes induced by hypoxia during normal tidal changes are consistent with the blockage of mitochondrial oxidative phosphorylation.
• 5.5. Use of in vivo NMR allows measurement of the biochemical effects of natural stress factors in live, intact aquatic organisms in the laboratory.

     

Renin activity in rainbow trout (salmo gairdneri rich.) and effects of environmental ammonia

• 1.1. A working model for renin assay in trout is described.
• 2.2. Renal renin activity increases in trout exposed to various un-ionized ammonia (UIA) environmental concentrations, with the only exception of 20 μgN/l UIA treated specimens.
• 3.3. Such effect of the toxic substance seems to be proportional to the logarithm of the environmental ammonia concentration and reaches a maximum in overturned specimens.
• 4.4. Results contribute new findings concerning correlation between renin activity and diuresis in fresh water fish.

     

Cooperative atp inhibition and fructose-1,6-biphosphate activation of rat liver pyruvate kinase

• 1.1. For the determination of relationship between FDP and ATP in the rat liver pyruvate kinase regulation, kinelic studies have been carried out at several ATP and FDP concentrations.
• 2.2. The results obtained on FDP activation show a great cooperativity for FDP saturation with a Hill coefficient of h = 2.79.
• 3.3. Kinetic studies on ATP inhibition also show a great cooperativity for ATP saturation (h = 2.84) at high FDP concentrations.
• 4.4. These results may contribute to explain the regulation of rat liver pyruvate kinase accounting for the activity of this enzyme at high FDP concentrations modulated by small changes in ATP concentrations.

     

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

• 1.1. The expected higher gill (Na⁺+K⁺)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
• 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg²⁺, 50 mM Na⁺, 2.5 mM K⁺, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg²⁺, 100 mM Na⁺, 10 mM K⁺, pH 7.5 in BW.
• 3.3. The change of the enzyme activation kinetics by Mg²⁺, ATP, Na⁺ and K⁺ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
• 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

     

The influence of environmental and incubation temperature on fatty acid synthetase from flounder (Platichthys flesus L.) liver

• 1.1. Fatty acid synthetase from liver of cold and warm adapted flounder and rabbit was purified to homogenity and compared.
• 2.2. The mol. wt of the cold and warm flounder enzyme was estimated to be about 457,000.
• 3.3. The kinetic properties were found to be similar for warm and cold adapted flounder liver enzyme and not different from the rabbit liver enzyme when measured at 5, 10, 15, 20 and 37°C.
• 4.4. Palmitic acid was the main product of both the flounder and rabbit enzyme, but significant amounts of butyric acid were also synthesized. The product composition did not change for any of the enzymes tested when the incubation temperature was changed.
• 5.5. It was concluded that fatty acid synthetase from flounder liver is similar to mammalian fatty acid synthetase with regard to molecular weight and kinetic properties.

     

Thrombocyte aggregation in rainbow trout

• 1.1. Thrombocytes from mature rainbow trout (Sahmo gairdneri) aggregated in vitro after addition of ADP, ATP, collagen, epinephrine, 5-hydroxytryptamine or thrombin to thrombocyte-rich plasma.
• 2.2. Thrombocyte aggregation was dose dependent and could be inhibited by preincubating the thrombocyte-rich plasma with adenosine, acetylsalicylic acid or prostaglandin E₁.
• 3.3. Thrombocytes released ADP and ATP when aggregated by 10 μM epinephrine. This release was blocked by 1 mM adenosine.
• 4.4. Electron microscopic observations of thrombocytes revealed them to contain numerous microtubules and electron-dense vesicles. In addition a possible shape change associated with thrombocyte aggregation was noted.

     

Amino acid substitutions within the analogous nucleotide binding loop (P-loop) of aminoglycoside 3'-phosphotransferase-II

• 1.1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme.
• 2.2. The mutations of Gly205 → Glu, Gly210 → Ala and Arg211 → Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418.
• 3.3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg²⁺ ATP.
• 4.4. Alternatively substitutions at a highly conserved basic residue (Arg211 → Lys and Arg211 → His) were not sufficient for the enzyme to sustain the activity at a level comparable to that of the wildtype.
• 5.5. Moreover, an Arg211 → His mutation drastically reduced affinity of the enzyme for Mg²⁺ ATP.
• 6.6. This argues the importance of Arg211 residue in contributing to the formation of the P-loop structure in addition to its involvement in phosphoryl transfer reaction.
• 7.7. Computer analysis of the secondary structure predicted that the APH(3')-II loop connects a β -strand to an α-helix and that the above mutations caused varying degrees of structural distortions at the corresponding regions of the protein.

     

Human seminal phosphatase: Properties and comparison with plant phosphatases

• 1.1. Phosphatase activities were determined in various seminal fluid and plant tissues.
• 2.2. Human semen showed a markedly higher phosphatase activity, and some plants and mushrooms exhibited a considerably higher phosphatase activity.
• 3.3. Phosphatase purified from human seminal fluid showed a pH optimum of 5–6 and was potently inhibited by l-(+)-tartrate.
• 4.4. Tartrate could not inhibit most plant phosphatases, but inhibited the mushroom phosphatase with a one-order lower affinity than that of the seminal enzyme.

     

Adenosine triphosphate as a determinant of magnesium levels in cytoplasm

• 1.1. Levels of free magnesium ion in various tissues are reviewed.
• 2.2. Total magnesium and ATP levels in different tissues are correlated, due to the presence of MgATP.
• 3.3. Relationships between pH and temperature in poikilotherms are such as to keep constant the amount of magnesium bound by ATP.
• 4.4. ATP hydrolysis in vivo generally tends to release magnesium ions, but conditions in some muscle may be such as to minimize this release.

     

The carotenoids of eggs of wild and farmed cod (Gadus morhua)

• 1.1. Eggs of wild cod, and of farmed cod fed (a) a diet supplemented with astaxanthin and (b) a diet supplemented with both astaxanthin and canthaxanthin, were analysed with respect to carotenoids.
• 2.2. The total carotenoid contents in eggs were 0.7 ppm for wild cod and 0.5 ppm for farmed cod.
• 3.3. Cod, having white flesh, deposit ketocarotenoids in the eggs, preferably astaxanthin.
• 4.4. Canthaxanthin can replace astaxanthin in the eggs, but astaxanthin appears to be deposited preferentially when both carotenoids are present in the diet.
• 5.5. The isomer distribution of (3S, 3′S):(3R, 3′S, meso):(3R, 3′R) astaxanthin in the eggs reflected the isomer composition of the diet.
• 6.6. Echinenone, 4′-hydroxyechinenone, adonixanthin and zeaxanthin encountered in cod eggs may represent reductive metabolites of canthaxanthin and astaxanthin.