Seasonal changes in antioxidant defence system of liver and gills of Salmo trutta caspius, Salmo trutta labrax and Salmo trutta macrostigma

Seasonal changes in antioxidant enzyme activities (superoxide dismutase, SOD, EC 1.15.1.1; catalase, CAT, EC 1.11.1.16; glutathione peroxidase, GPx, EC 1.11.1.9; glutathione reductase, GR, EC 1.6.4.2; glucose-6-phosphate dehydrogenase, G6PD, EC 1.1.1.49 and glutathione S -transferase, GST, EC 1.5.1.18) and lipid peroxidation (LPO) levels of livers and gills of female Caspian trout Salmo trutta caspius , Black Sea trout Salmo trutta labrax and mountain trout Salmo trutta macrostigma were investigated. SOD, CAT, GPx, G6PD and GST activities were higher in liver compared to gills of all sub-species; concomitantly, the GR activity was also higher in the livers of S. t. caspius and S. t. labrax , but the reverse was seen in S. t. macrostigma . LPO levels were higher in the gills compared to the liver of all sub-species. There was no general trend in the seasonal changes in the gill antioxidant enzyme (AE) activities or LPO levels. Higher AE activities, however, were found in the liver of each sub-species during autumn, and this coincided with an increase in the gonado-somatic index.     

The effect of temperature acclimation on NADP-dependent dehydrogenase activity in the carp liver and various mechanisms of its regulation

Acclimation of carp both to the temperature fall (from 20 to 5 degrees C) and rise (from 20 to 30 degrees C) induces an increase in activity of cytoplasmic liver NADPH-generating enzymes--glucose-6-phosphate dehydrogenase (G6PDG) and malic-enzyme (ME) 6-phosphogluconate dehydrogenase (6PGDG) and NADP-isocitrate dehydrogenase (NADP-IDG) activities are unchanged. Actinomycin D does not prevent cold activation of G6PDG but blocks activation of ME. "Warm" G6PDG has minimal Km value for glucose-6-phosphate and "warm" ME has minimal Km value for glucose-6-phosphate and "warm" ME has minimal Km value for malate at 25 degrees C "Cold" G6PDG and ME have the warmest Km values at 5 degrees C. Isozyme composition of cytoplasmic G6PDG (2 bands with Rf 0.16 and 0.20) does not change within the limits of 5-30 degrees C. The prolactin action on G6PDG and ME is similar to the effect of cold acclimation (activity increases Km value decreases, isozyme pattern (for G6PDG) remains unchanged). It is supposed that activation of G6PDG and ME during cold adaptation may be a result of the prolactin action on substrate-binding ability without changes in the enzyme biosynthesis and isozyme pattern.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

G6PD Sendagi: A new glucose-6-phosphate dehydrogenase variant associated with congenital hemolytic anemia

A new glucose-6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was discovered. It was found in a 2-year-old male who had a hemolytic crisis after an upper respiratory tract infection. The enzyme activity of the variant was 8.4% of that of the normal enzyme. The enzymatic characteristics were slower than normal anodal electrophoretic mobility, low Km G6P, normal Km NADP, increased utilization of substrate analogues, high Ki NADPH, decreased heat stability, and an alkaline pH optimum. From these results, this was considered to be a new variant and was designated G6PD Sendagi.     

Comparative reactivity of carbamyl phosphate and glucose 6-phosphate with the glucose-6-phosphatase of intact microsomes

The ability of glucose 6-phosphate and carbamyl phosphate to serve as substrates for glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) of intact and disrupted microsomes from rat liver was compared at pH 7.0. Results support carbamyl phosphate and glucose 6-phosphate as effective substrates with both. Km values for carbamyl phosphate and glucose 6-phosphate were greater with intact than with disrupted microsomes, but Vmax values were higher with the latter. The substrate translocase-catalytic unit concept of glucose-6-phosphatase function is thus confirmed. The Km values for 3-O-methyl-D-glucose and D-glucose were larger when determined with intact than with disrupted microsomes. This observation is consistent with the involvement of a translocase specific for hexose substrate as a rate-influencing determinant in phosphotransferase activity of glucose-6-phosphatase.     

Purification and Characterization of the Main Isozyme of Polyphenol Oxidase in Mung Bean (Vigna mungo) Seedlings

The induction of NADPH-generating enzymes by polychlorinated biphenyls (PCB) in rats was investigated. The administration of PCB to rats for 3 and 14 days increased the activities of malic enzyme (ME, EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44) about 2-fold above the control level in the liver. Hepatic mRNA levels of ME, G6PD, and 6PGD, except for G6PD mRNA of the 14-day group, were also elevated to the same degree as the enzyme activities in PCB-treated rats. In rats fed a PCB-containing diet for 1 day, the hepatic mRNA levels of ME and G6PD were elevated prior to the induction of enzyme activity. In the kidney, lung, spleen, heart, and testis, the mRNA levels of ME, G6PD, and 6PGD were not affected by PCB. The induction of hepatic NADPH-generating enzymes would imply an increased demand of NADPH in the liver of rats fed with a PCB-containing diet.     

Microsomal hexose-6-phosphate dehydrogenase in injured liver.

Hexose-6-phosphate dehydrogenase (H6PD) in rat liver microsomes was clearly differentiated kinetically, immunologically and electrophoretically from glucose-6-phosphate dehydrogenase (G6PD) localized in liver supernatants. Although the soluble G6PD activity increased upon liver injuries induced by CCl4 and thioacetamide, the H6PD activity decreased markedly 1-2 days following administrations of these hepatotoxins. The specific activity of H6PD remained fairly constant under other experimental conditions where the levels of the soluble G6PD activity increased.     

Daily changes in parameters of energy metabolism in liver, white muscle, and gills of rainbow trout: dependence on feeding

We assessed the daily patterns of parameters involved in energy metabolism in liver, white muscle, and gills of rainbow trout. Where daily rhythms were found, we analyzed the potential influence of feeding. Immature rainbow trout were randomly distributed in 3 groups: fish fed for 7 days, fish fasted for 7 days, and fish fasted for 7 days and refed for 4 days. On sampling day, fish of fed and refed groups were fed at 11.00 h, and all fish were sampled from each treatment group using the following time schedule: 14.00, 18.00, 21.00, 00.00, 04.00, 07.00, 10.00 and 14.00 h. The results obtained from metabolic parameters can be grouped into four different categories, such as i) those displaying no daily changes in any group assessed in liver (acetoacetate and lactate levels), white muscle (protein levels, and low Km (glucose) hexokinase (HK) and HK-IV activities) and gills (protein levels), ii) those displaying no 24 h changes in fed fish but in refed or fasted fish in liver (glucose, glycogen, amino acid and protein levels, and HK-IV activity), white muscle (glycogen and amino acid levels) and gills (glucose levels), iii) those displaying 24 h changes that were apparently dependent on feeding since they disappear in fasted fish in liver (Low Km (glucose) HK, lactate dehydrogenase (LDH-O), glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (FBPase) , alpha-glycerophosphate dehydrogenase (G3PDH), glutamate dehydrogenase (GDH) and aspartate aminotransferase (Asp-AT) activities), white muscle (glucose levels, and pyruvate kinase (PK), LDH-O, G3PDH and Asp-AT activities) and gills (glycogen and lactate levels, and Low Km (glucose) HK, HK-IV, LDH-O and Asp-AT activities), and iv) those parameters displaying 24 h changes apparently not dependent on feeding in liver (lactate levels and PK activity) and gills (amino acid levels, and PK and GDH activities). In general, most 24 h changes observed were dependent on feeding and can be also related to daily changes in activity.     

Kinetics of human erythrocyte glucose-6-phosphate dehydrogenase dimers

The steady-state kinetics of human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) dimers were studied by initial rate measurement. These experiments gave intersecting double-reciprocal plots suggesting a ternary complex mechanism with a Km for NADP and glucose 6-phosphate of 11 microM and 43 microM, respectively. These studies were combined with rate measurements in the presence of one product (NADPH), dead-end inhibitors, as well as alternative substrates. The inhibition by NADPH was found to be competitive with respect to both substrates. Alternate substrates experiments gave linear double-reciprocal plots over a wide range of substrate concentrations. The results suggest that the dimeric enzyme follows either a random or a Theorell-Chance mechanism.     

Glucose 6-Phosphate dehydrogenase variants: A unique variant (G6PD Kobe) showed an extremely increased affinity for galactose 6-phosphate and a new variant (G6PD Sapporo) resembling G6PD Pea Ridge

Summary Two new glucose 6-phosphate dehydrogenase (G6PD) variants associated with chronic nonspherocytic hemolytic anemia were discovered. G6PD Kobe was found in a 16-year-old male associated with hemolytic crisis after upper respiratory infection. The enzyme activity of the variant was about 22% of that of the normal enzyme. The main enzymatic characteristics were slower than normal anodal electrophoretic mobility, high Km G6P, increased thermal-instability, an acidic pH optimum, and an extremely increased affinity for the substrate analogue, galactose 6-phosphate (Gal-6P).G6PD Sapporo was found in a 3-year-old male associated with drug-induced hemolysis. The enzyme activity was extremely low, being 3.6% of normal. In addition, this variant showed high Ki NADPH and thermal-instability.G6PD Kobe utilized the artificial substrate Gal-6P effectively as compared with the common natural substrate, glucose 6-phosphate. In G6PD Sapporo, NADPH could not exert the effect of product inhibition. The structural changes of these variants are expected to occur at the portions inducing conformational changes of the substrate binding site of the enzyme.     

A new glucose-6-phosphate dehydrogenase variant (G6PD Nagano) associated with congenital hemolytic anemia

Summary A new glucose-6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was reported. The patient, a 6-year-old Japanese male, was noticed to have hemolytic anemia soon after birth, and a diagnosis of G6PD deficiency was made at the age of 2. He had episodes of hemolytic crisis several times after upper respiratory infection. G6PD activity of the patient was 5.5% of normal. The enzymatic characteristics were examined when he was 5 years old, and his G6PD showed faster-than-normal electrophoretic mobility, low Km G6P, high Km NADP, low Ki NADPH, normal utilization of substrate analogues, heat instability, and a normal pH optimum curve. From these results, this was considered to be a new variant and was designated G6PD Nagano. Infection-induced hemolysis and chronic hemolytic anemia seem to be due to markedly impaired enzyme activity and thermal instability.     

The in vitro stability of rat liver glucose-6-phosphate dehydrogenase as a function of diet

Rat liver glucose-6-phosphate dehydrogenase (G6PD) activity was studied in starved-refed rats given diets containing three levels of fat (35%, high-fat; 5%, low-fat; 0%, fat-free). Elution characteristics from DEAE-cellulose, Km for glucose-6-phosphate, pH optimum, and molecular weight appeared to be similar. During storage or heat denaturation, stability apparently was lowest of G6PD of livers from rats refed the high-fat diet. Heat stability was enhanced by the addition of NADP, but some differences due to diet persisted. Titration of a constant amount of enzyme with heating gave inconsistent results: in two of four experiments rats refed the high-fat diet had an equivalence point twice that of rats refed the fat-free diet. This difference disappeared if the antibody titration was carried out in the cold. The diet-induced instability of the G6PD, as measured in vitro, was reversible by changing the diet of the rats.     

A new glucose 6-phosphate dehydrogenase variant (G6PD Thessaloniki) in a patient with idiopathic myelofibrosis

A new deficient glucose 6-phosphate dehydrogenase (G6PD) variant, G6PD Thessaloniki, which was found in the red blood cells of a 70-year-old woman who had idiopathic myelofibrosis, is described. G6PD Thessaloniki had a low Michaelis constant (Km) for G6P (20 microM), high Km for NADP (10.1 microM), normal pH optimum, reduced heat stability, decreased electrophoretic mobility (96-98% of the normal), increased 2-deoxy-G6P and decreased galactose 6-phosphate utilization. Several other enzymatic activities measured in the patient's red blood cells were normal. Studies of red blood cell survival and glucose utilization gave evidence of haemolysis caused by defective glucose utilization by the pentose phosphate pathway. The only son of the patient had normal G6PD in his red blood cells. In an attempt to investigate the origin of G6PD Thessaloniki, heat stability tests of G6PD extracted from the patient's skin have been performed.     

Partial purification and some kinetic properties of glucose-6-phosphate dehydrogenase from Phycomyces blakesleeanus

Glucose-6-phosphate dehydrogenase from sporangiophores of Phycomyces blakesleeanus NRRL 1555 (-) was partially purified. The enzyme showed a molecular weight of 85 700 as determined by gel-filtration. NADP+ protected the enzyme from inactivation. Magnesium ions did not affect the enzyme activity. Glucose-6-phosphate dehydrogenase was specific for NADP+ as coenzyme. The reaction rates were hyperbolic functions of substrate and coenzyme concentrations. The Km values for NADP+ and glucose 6-phosphate were 39.8 and 154.4 microM, respectively. The kinetic patterns, with respect to coenzyme and substrate, indicated a sequential mechanism. NADPH was a competitive inhibitor with respect to NADP+ (Ki = 45.5 microM) and a non-competitive inhibitor with respect to glucose 6-phosphate. ATP inhibited the activity of glucose-6-phosphate dehydrogenase. The inhibition was of the linear-mixed type with respect to NADP+, the dissociation constant of the enzyme-ATP complex being 2.6 mM, and the enzyme-NADP+-ATP dissociation constant 12.8 mM.     

Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus subtilis spores.

Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent Km values of the enzyme were 5.7 X 10(-4) M for glucose-6-phosphate and 2.4 X 10(-4) M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg++ or Ca++. The inactive glucose-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Insulin-like effect of vanadate on malic enzyme and glucose-6-phosphate dehydrogenase activities in streptozotocin-induced diabetic rat liver

The effect of oral administration of sodium orthovanadate on hepatic malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activities was investigated in nondiabetic and diabetic rats. Streptozotocin-induced diabetic rats were characterized by 4.7-fold increase in plasma glucose and 82% decrease in plasma insulin levels. The activities of hepatic malic enzyme and glucose-6-phosphate dehydrogenase were also diminished (P less than 0.001). Vanadate treatment in diabetic rats led to a significant decrease (P less than 0.001) in plasma glucose levels and to the normalization of enzyme activities, but it did not alter plasma insulin levels. In nondiabetic rats vanadate decreased the plasma insulin level by 64% without altering the enzyme activities. Significant correlation was observed between plasma insulin and hepatic lipogenic enzyme activities in untreated and vanadate-treated rats. Vanadate administration caused a shift to left in this correlation suggesting improvement in insulin sensitivity.     

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.     

Immunological studies on glucose 6-phosphate dehydrogenase of rat liver

Glucose 6-phosphate dehydrogenase (G6PD) was purified from the supernatant fraction of rat liver to a homogeneous preparation by a specific elution with substrate. A specific antibody against the purified enzyme was prepared in rabbits and was shown to completely inhibit the enzyme activity and precipitate the enzyme protein of liver supernatant. With this antiserum, liver supernatants with varying specific G6PD activities obtained under several experimental conditions and supernatants from other tissues examined all formed single precipitin lines, which fused with each other in the Ouchterlony double-diffusion system. Three interconvertible microheterogeneous forms of G6PD in liver, supernatant were immunologically indistinguishable from each other. The G6PDs in participate fractions of liver were, however, distinct from the supernatant enzyme both in inhibition of the enzyme activity and in formation of precipitation by the specific antiserum. Liver supernatant G6PD, which was inactivated with various reagents or by heating, showed a simultaneous loss of ability to form precipitin line. Aggregation and disaggregation of the dehydrogenase to the tetramer and monomer, respectively, also resulted in loss of immunological reactivity. The increase in G6PD activity in the cytoplasm of carbon tetrachloride-treated or glucose casein-refed rat liver was accompanied by a proportional increase in the quantity of immunologically reactive G6PD protein.     

A new improved electrophoretic method of glucose-6-phosphate dehydrogenase with discontinuous buffer and gel system

A new improved electrophoretic method of glucose-6-phosphate dehydrogenase (G6PD) was devised by using a slab disc electrophoresis with a discontinuous gel of 1-mm thickness. This was suitable for electrophoresis of thermolabile G6PD variants with low activities. Using this system, sharp G6PD bands and acceptable separations of G6PD Konan, G6PD Tokyo, and G6PD Yokohama, were obtained and compared with those of starch gel electrophoresis.     

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.