Propionyl-CoA synthetase in mammary gland and liver of cows.

Propionyl-CoA synthetase of liver and mammary gland from calf and midlactation cow was investigated. No activity of this enzyme was detected in calf mammary gland, but it was detected in calf liver. Propionyl-CoA synthetase was found in both, liver and mammary gland of the cow, although mammary gland activity was about 25% of that found in liver. The effects of pH and temperature on enzyme activity and stability were also investigated in crude extracts of liver and mammary gland tissues. The results suggest a different behaviour of the enzyme from both origins. Kinetic studies of the enzyme were also carried out, showing differences, depending on the organ, in the apparent substrate KM values.

Comparative analysis of propionyl-CoA carboxylase from liver and mammary gland of mid-lactation cow.

1. Propionyl-CoA carboxylase from mid-lactation cow liver and mammary gland has been purified. 2. In both organs, the molecular mass was estimated to be approximately 450 kD, and two molecular subunits of 77 and 64 kD could be observed. 3. Physico-chemical and kinetical properties for the enzyme from both organs were similar, showing an allosteric behaviour in relation to ATP and Mg2+. 4. The presence of propionyl-CoA carboxylase in mid-lactation cow mammary gland with similar properties to the liver enzyme, could indicate the existence of a gluconeogenic metabolism in this organ exactly when a high demand of glucose for milk lactose is required.

Phosphofructokinase during haemopoiesis of the rainbow trout ("Salmo gairdneri R."): its isoenzymatic forms.

Phosphofructokinase (EC 2.7.1.11) from trout haemopoietic cells and erythrocytes exhibits biphasic behaviour, with respect to fructose 6-phosphate and MgATP in extracts filtered through Sephadex G-25. Two different values of Hill coefficient and S0.5 have been found for each cellular population. Two forms of the enzyme with high and low affinity for the substrates, were obtained after affinity chromatography in each cellular population. The kinetic behaviour of these forms is different and may be due to a distinct composition and/or proportion and contribution of phosphofructokinase isoenzymes in both types of cells.

Role of phosphofructokinase during trout haemopoiesis: physiological regulation of glycolysis.

1. The regulatory properties of phosphofructokinase (PFK) has been investigated in two cellular population representatives of trout haemopoiesis; haemopoietic cells (capable of replication and differentiation) and erythrocytes (highly specialized cells). 2. The intracellular levels of substrates and effectors have been quantified and their effect on PFK activity determined. 3. Fructose 1,6-bisphosphate anc cyclic AMP show a higher activation of the PFK from haemopoietic cells than the enzyme from erythrocytes. 4. AMP and phosphoenolpyruvate act as activators of the haemopoietic cell PFK while for erythrocytes PFK, AMP is an inhibitor and phosphoenolpyruvate does not display any effect. 5. Citrate inhibits PFK activity from haemopoietic cells but was not assayed in erythrocytes since it was not detected in these cells. 6. The differences in PFK regulation in both cellular populations may be attributed to the intracellular levels of the effectors and/or different isoenzymatic patterns. 7. The different regulation of PFK together with the higher enzymatic activity of PFK and pyruvate kinase from haemopoietic cells are related to the higher glycolytic flux that exhibits the haemopoietic cells. 8. The results shown in this investigation allow us to conclude that PFK has a specific role depending on the energetic requirements of the cellular population in which the enzyme is present. 9. The requirements are related to the physiological function of each type of cell.

Thermic and pH modulation of phosphofructokinase during trout (Salmo gairdneri R.) haemopoiesis: influence of fructose 6-phosphate.

Thermic and pH modulation of phosphofructokinase (EC 2.7.1.11) activity with respect to fructose 6-phosphate has been studied comparatively in trout (Salmo gairdneri R.) haemopoietic cells and erythrocytes. Phosphofructokinase of both cellular populations displays a biphasic kinetic behaviour with respect to fructose 6-phosphate at two values of pH and temperature. In haemopoietic cells, when pH decreases the enzyme-substrate affinity increase while an opposite effect is found in erythrocytes. Decreases in temperature act as a positive modulator in haemopoietic cells while in erythrocytes this effect is observed only at low fructose 6-phosphate concentrations. Therefore a different pH and temperature modulation of phosphofructokinase during trout haemopoiesis has been established.

Regulation of glycolysis in sea bass liver: phosphofructokinase isozymes.

Sea bass (Dicentrarchus labrax L.) liver phosphofructokinase (PFK) presents biphasic kinetics with respect to fructose-6-phosphate (F-6-P) in experiments carried out with crude extract. After the enzyme had been purified, two isozymes have been detected after chromatographic treatment. The two isozymes present different kinetic behaviour PFK-L1, the first eluted phosphofructokinase activity shows positive cooperativity with respect to fructose-6-phosphate and PFK-L2, the second activity fraction, has a Hill coefficient of 0.38 (negative cooperativity). The first isozyme shows less affinity for fructose-6-phosphate than that shown by PFK-L2. The joint kinetics of both isozymes produces a biphasic kinetics with respect to fructose-6-phosphate, similar to that observed in crude extracts.

Metabolic regulation of glycolysis in sea bass (Dicentrarchus labrax L.) muscle. I. Kinetic study and characteristic modulators of pyruvate kinase.

White muscle pyruvate kinase from sea bass presents positive cooperativity with respect to PEP substrate. The enzyme is regulated by F-1.6-P2 and L-Phenylalanine. The activator effect of F-1.6-P2 in experiments carried out for the substrate PEP with crude extract seems to indicate that the enzyme is activated in vivo by this compound. The enzyme was not inhibited by either alanine or ATP but was inhibited by L-phenylalanine. Therefore this enzyme presents kinetic and regulatory properties similar to those of the mammalian isozyme M2.

Trout (Salmo gairdneri R.) liver 5-aminolevulinate synthetase: subcellular distribution and properties.

The subcellular distribution of trout liver 5-aminolevulinate synthetase has been studied. A cytosolic form of the enzyme has been found. Its activity was a 30% of the mitochondrial enzyme. The cytosolic form has a molecular weight of 110,000, larger than the mitochondrial enzyme (70,000). The two enzyme forms showed a pH optimum of 7.5. The kinetic characteristic of both forms suggest that the cytosolic form is a precursor of the mitochondrial ALA-synthetase.

Erythroid 5-aminolevulinate synthetase from trout (Salmo gairdneri R.).

A mitochondrial and cytosolic erythroid ALA-synthetase have been found in trout. The polypeptide existent in the cytosol is probably a precursor of the 90,000 mol. wt mitochondrial ALA-synthetase. The erythroid ALA-synthetase is about 20,000 mol. wt larger than the hepatic enzyme. The differences in mol. wt and catalytic properties between erythroid and hepatic enzyme support the existence of two forms of the ALA-synthetase in teleostei.

Pyruvate kinase of sea bass liver: interrelationship in regulating properties.

Pyruvate kinase of sea bass liver show a joint modulation of both pH and temperature for PEP substrate. The effect of F-1,6-P2, alanine and ATP is not fundamentally affected by a variation in pH. The kinetic constants in the presence of ATP are not affected, but the intensity of its inhibitor effect varies with temperature. A study of different buffers: Tris-HC1, Tris-maleic and phosphate, on this enzymatic activity with or without effectors has been made.

[Pyruvate kinase in mussel foot (Mytilus edulis L.). Purification and general properties]. / Piruvatoquinasa de pie de mejillón (Mytilus edulis L.). Purificación y propiedades generales.

The specific activity of pyruvate kinase in mussel foot is markedly higher than that from mantle and digestive gland. The foot enzyme shows maximum pH activity in the range between 7.0 and 7.5 and is stable (15 min, 37 degrees C) at pH values between 7.0 and 9.0. The activation energy value is 23 kJ/mol with a Q10 coefficient of 1.4. All of these experiments were carried out using partially purified extracts with (NH4)2SO4 treatment (30-60%) and posterior dialysis with EDTA 1.2 mM. No isoenzymatic forms could be detected using the column chromatography techniques with Sephadex G-150, DEAE Sephadex A-50 and polyacrylamide gel electrophoresis.

Influence of ATP and magnesium on phosphofructokinase from sea bass (Dicentrarchus labrax L.) liver.

Glycolytic enzyme phosphofructokinase (PFK) from sea-bass liver shows inhibition for ATP4- and MG-ATP2-, and ATP4- is a competitive inhibitor with respect to MG-ATP2-. Free Mg2+ behaves as a mixed inhibitor on the kinetic with respect to the true enzyme substrate Mg-ATP2-, and eliminates the inhibition effect of this substrate. The kinetics with respect to Mg-ATP2- at non-inhibiting concentrations is not visibly affected by temperature of pH variation. The inhibiting effect of Mg-ATP2- is more marked at 22 and 10 degrees C (of three assayed temperatures 22, 15 and 10 degrees C and at physiological pH 6.8) as opposed to the maximum activity pH (8.0).

Regulation of liver sea bass pyruvate kinase by temperature, substrates and some metabolic effectors.

Pyruvate kinase of sea bass (Dicentrarchus labrax L.) shows positive cooperativity with respect to both substrates PEP and ADP. The temperature is a modulator of this activity, changing KS0.5 and Hill coefficient values for PEP. The enzyme shows alanine and ATP inhibition and F-1,6-P2 activation at 22 degrees C. F-1,6-P2 eliminates the effect of alanine but not that of ATP. These results could indicate a regulation of this enzyme by temperature and possess kinetic properties which are similar to that of L-type mammals.

Properties and function of malate enzyme from Pseudomonas putida.

Malate enzyme (L-malate: NADP+ oxidoreductase (oxalacetate-decarboxylating, EC 1.1.1.40)) has been purified from Pseudomonas putida to 99 per cent homogeneity by heat, ammonium suphate fractionation, gel filtration and anion exchange chromatography. Sodium dodecylsulphate-(SDS)-polyacrylamide disc gel electrophoresis analysis showed an approximate tetrameric subunit with a molecular weight of 52,000. The purified enzyme showed a pH optimum between 8.0 and 8.5 (for Tris-HCl buffer) and required bivalent cations for catalysis; monovalent ions like K+ and NH4+ acted as very effective activators. The temperature-activity relationship for the malate enzyme from 35-80 degrees C showed broken Arrhenius plots with an inflexion at 65 degrees C. The enzyme halflife was 30s at 85 degrees C. The enzyme showed hyperbolic kinetics for both substrates with apparent Km values of 4.0 X 10(-4) M and 2.3 X 10(-5) M for L-malate and NADP+ respectively. From the study of the effects of some compounds on the enzyme, the physiological significance of those produced by fumarate, succinate and oxalacetate can be emphasized.

Nutritional regulation of glycolysis in rainbow trout (Salmo gairdneri R.).

1. Some physico-chemical constants and the nutritional regulation of pyruvate kinase (PK), phosphofructokinase (PFK) and hexokinase (HK) from rainbow trout liver was investigated. 2. The maximum activity pH for the three enzymes appears to be in a physiological range. 3. The PK-enzyme shows sigmoid kinetic with respect to PEP with a Hill-coefficient of 3.1; the other two enzymes show michaelian kinetic for their substrates. 4. The nutritional treatments show that HK-enzyme increases its level with high carbohydrate diet and decreases with high protein diet and starvation. 5. PFK-enzyme decreases with high protein diet and starvation. 6. PK-enzyme only shows a decrease in level with starvation conditions.

Some comparative properties of pyruvate kinase in haematopoietic cells and erythrocytes from rainbow trout (Salmo gairdneri R).

1. Temperature acts as a pyruvate kinase regulator in haematopoietic cells and erythrocytes. 2. Fructose-1,6-biphosphate and alanine act as allosteric modulators of pyruvate kinase in haematopoietic cells, while in erythrocytes although fructose-1,6-biphosphate exerts also allosteric effect, alanine appears to be a competitive inhibitor. ATP (1.0 mM) does not exert any clear effect on pyruvate kinase of both cellular populations. 3. The level of specific activity of pyruvate kinase in haematopoietic cells is 40-fold that of PK from erythrocytes.

[Physico-chemical properties of guinea pig liver argininosuccinate synthetase (author's transl)]. / Propiedades físico-químicas de la arginosuccinato-sintetasa de hígado de cobaya.

The behaviour of guinea pig liver argininosuccinate synthetase with respect to pH, temperature and kinetic parameters of substrates and inhibitors has been investigated. The enzyme shows the maximum activity at pH 8 and maximum stability (15 min at 30 degrees C) at pH in the range of 6.5 and 8. With respect to temperature it remains stable (15 min at pH 8) up to 40 degrees C. The results found for the Km values of the enzyme substrates L-citrulline, L-aspartate and ATP are 0.038 mM, 0.045 mM and 0.090 mM respectively. L-ornithine, diaminopimelic acid, pyrophosphate and ATP acted as inhibitors of the enzyme (competitive or not, according to the case). These kinetic studies of substrates and inhibitors were carried out with a partially purified fraction of the enzyme which had been purified 7.3 fold, which practically suppressed the ATP-ase activity, present in crude extracts.

[Physico-chemical properties of guinea pig liver arginase (author's transl)]. / Propiedades físico-químicas de la arginasa de hígado de cobaya.

Arginase (E.C. 3.5.3.1) the enzyme which catalyses the hydrolysis of arginine to ornithine and urea has been investigated in guinea pig liver in relation to the kinetic constants of its substrate and inhibitors as well as to other physico-chemical properties. The results show that the enzyme has Km value of 19.6 mM for its substrate L-arginine and is competitively inhibited by one of its reaction products, the L-ornithine, and also by L-lysine and diaminopymelic acid. Optimal activity of the enzyme occurs at 10.5 pH and maximal stability in the range of 6.5 to 7.5 pH. The mentioned arginase exhibits temperature dependent activity and stability, being 64 degrees C (15 min and pH 7.5) the half-inactivation temperature. An increase in the activity and temperature stability of the enzyme, when previously activated by heating for 5 min at 45 degrees C in the presence of 10 mM MnCl2, has been achieved.

[The influence of fasting and cortisol upon arginosuccinate synthetase, arginosuccinase and arginase levels in guinea pig liver (author's transl)]. / Influencia del ayuno y del cortisol sobre los niveles de arginosuccinato sintetasa, arginosuccinasa y arginasa de hígado de cobaya.

The influence of fasting and cortisol treatment on the levels of arginosuccinate synthetase (E.C. 6.3.4.5.), ARGINOSUCCINASe (E.C. 4.3.2.1) and arginase (C.E. 3.5.3.1) from guinea pig liver has been investigated. The results show a general increase in the levels of the mentioned enzymes, with specific enzyme synthesis for fasting, and unspecific for cortisol treatment.