Changes in brown-adipose-tissue mitochondrial processes in streptozotocin-diabetes.

Yeast glucose-6-phosphate dehydrogenase was inhibited by low NADPH concentrations in cell-free extracts, and de-inhibited by GSSG; extensive dialysis of the crude extract did not diminish the GSSG effect. Immunoprecipitation of glutathione reductase abolished the de-inhibition of glucose-6-phosphate dehydrogenase by GSSG. Purified glucose-6-phosphate dehydrogenase was inhibited by NADPH but not de-inhibited by GSSG, and upon addition of pure glutathione reductase GSSG completely de-inhibited the glucose-6-phosphate dehydrogenase.     

Fatty Acyl-CoAs as feedback regulators of hexose monophosphate shunt in rat adipocytes

Summary The high basal glucose utilization through hexose monophosphate shunt found in our experimental conditions were almost completely inhibited by oleate, octanoate and caproate. However, the inhibition of glucose oxidation due to butyrate was about 50% whereas ketone bodies and acetate did not inhibit. The rate of triacylglycerol formation was not significantly modified with the above organic acids except oleate that presented a 5-fold increase on labeling incorporation into lipids. Oleate inhibition of glucose oxidation was completely prevented by the NADPH oxidant menadione. There was no inhibition by octanoate, caproate, butyrate or ketone bodies of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or malic enzyme in adipose tissue homogenates. In contrast, specifically glucose-6-phosphate dehydrogenase was inhibited by oleoyl-CoA. The oleoyl-CoA inhibition was prevented by enzyme preincubation with low NADP concentration. The data lend further support for the hypothesis that fatty acids and NADP fulfill an important role in the modulation of the hexose monophosphate shunt activity.     

Oxidative enzyme systems of the honey bee,Apis mellifera L

1. Oxidative dissimilation has been studied in enzymes from the honey bee. Using mitochondria isolated from the thoraces, complete oxidation of most of the TCA cycle members has been shown. 2. The presence of the acetate-activating enzyme, citrate-condensing enzyme, isocitric dehydrogenase, alpha-ketoglutarate dehydrogenase, glucose-6-phosphate, and 6-phosphogluconic dehydrogenase has been demonstrated and the cofactor requirements established. 3. The oxidation of isocitric acid has been shown to be either non-specific for the D- or L-isomer, or the presence of a racemase is indicated. 4. The presence of the pentose cycle is indicated in the soluble portion of the thoracic homogenate.     

Characterization of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase from hazel cotyledons

NADP reduction was shown to occur in a crude cytosolic extract from the cotyledonary material of hazel seed prior to the addition of erogenous dehydrogenase substrate. This activity interfered with the assay of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities. The inherent NADP reduction was removed by ammonium sulphate fractionation. Subsequent de-salting of the resulting partially-purified fraction permitted assay of G6PDH and 6PGDH. Both enzymes were shown to be NADP specific. Typical Michaelis-Menten kinetics were shown for each enzyme, towards NADP and their respective substrate.     

Enzyme systems in Tetrahymena geleii S. I. anaerobic dehydrogenases concerned with carbohydrate oxidation

The rates of activity of the dehydrogenase systems in Tetrahymena, which are concerned with carbohydrate oxidation, in descending order of activity are: lactic > isocitric > succinic = glucose > glucose-6-phosphate = 6-phosphogluconic = malic > glutamic = cytochrome linked α-glycerophosphate dehydrogenase. No evidence was obtained to indicate the presence of DPN linked α-glycerophosphate dehydrogenase.     

Pentose phosphate metabolism during dormancy breakage in Corylus avellana L.

Commercially obtained fruits of Corylus avellana exhibit the characteristic loss of dormancy of this seed following chilling under moist conditions. The activities of cytosolic and organellar enzymes of pentose phosphate pathway in cotyledonary tissue were assayed throughout stratification and over a similar period in damp vermiculite at 20° C. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconic acid dehydrogenase (6PGDH) were both found in cytosolic extracts in all treatments; only 6PGDH was present in the organellar fraction.The enzyme activities monitored in seeds at 20° C remained relatively constant over the course of the investigation except in the case of cytosolic 6PGDH where it is suggested an inhibitor of the enzyme accumulated. This inhibitor was removed by the partial purification procedure. Increases in the activities of the enzymes occurred during stratification, the major increase coinciding exactly with dormancy breakage but prior to the initiation of germination. The marked increase in G6PDH and 6PGDH concurrent with the change in germination potential of the chilled seed may have considerable biochemical significance in breaking down the dormant state.Abbreviations G6P glucose-6-phosphate - G6PDH glucose-6 phosphate dehydrogenase - NADP nicotinamide adenine dinucleotide phosphate - 6 PGDH 6-phosphogluconic acid dehydrogenase - PPP pentose phosphate pathway     

Glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase of wild oat seeds

The presence of the initial enzymes of the pentose phosphate pathway, namely glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase, has been demonstrated in dormant seed of wild oat. Before a partial characterization of these enzymes was made, an inherent NADP-reducing activity and an enzyme deactivating component, both present in the crude extract, were removed by ammonium sulphate precipitation and subsequent desalting. Both enzymes were then shown to be NADP-specific. Typical Michaelis-Menten kinetics were shown by each enzyme towards NADP and their respective substrates. Soluble cytoplasmic dehydrogenase enzymes were present in both embryo and endosperm extracts.     

Liver dehydrogenase levels in rainbow trout, Salmo gairdneri, fed cyclopropenoid fatty acids and aflatoxin B1

Cyclopropenoid fatty acids in the diet of rainbow trout caused significant reductions in liver protein and activity of glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase. Changes in total activity were usually accompanied by similar changes in specific activity. The activity of glucose-6-phosphate dehydrogenase appeared to be more sensitive to the ingestion of cyclopropenoid fatty acids than the other dehydrogenases studied. Feeding 20 ppb aflatoxin B(1) to rainbow trout did not significantly change the activity of the dehydrogenases except for a small increase in the activity of glucose-6-phosphate dehydrogenase after 21 days of feeding. Relationships of these changes to the cocarcinogenicity of cyclopropenoid fatty acids and the carcinogenicity of aflatoxin are discussed.     

Changes in enzymatic activities involved in glucose metabolism by acyl-CoAs in Trypanosoma cruzi

This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+ afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.     

Rapid Method for Preparing Cell-Free Extracts of Aspergillus ochraceus

A rapid method for preparing cell-free extracts of Aspergillus ochraceus was developed. Mycelial mats were prefrozen in liquid nitrogen, ground to a fine powder in a cold mortar, and homogenized in an all-glass mechanical homogenizer. This method provided preparations averaging 43.0 mg of protein per g of mycelium (wet weight). The method was fast, efficient, and did not subject the extract to temperatures above 1 C or to heavy metals. The preparation method was suitable for studying a variety of in vitro fungal enzyme systems. Amylase, acid phosphatase, alkaline phosphatase, catalase, fatty acid synthetase, glucose-6-phosphate dehydrogenase, beta-glucosidase, beta fructofuranosidase, and trehalase activities were measurable in the preparations.     

Selective Inhibition of Bacterial Enzymes by Free Fatty Acids

Octanoic acid inhibits, in vitro, the bacterial enzymes glucose-6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, fumarase, lactate dehydrogenase, and the malic enzyme of Arthrobacter crystallopoietes. The free fatty acid appears to act as an inhibitor of lipogenesis, although it does not affect the rate of gluconeogenesis. To demonstrate that this inhibition may be of physiological significance in vivo, those enzymes not involved in lipogenesis, such as fructose-1, 6-diphosphatase, phosphoglucomutase, phosphohexoisomerase, aconitase, nicotinamide adenine dinucleotide phosphate (NADP) isocitrate dehydrogenase, NADP glutamate dehydrogenase, malate dehydrogenase, and isocitrate lyase, were assayed and found not to be inhibited by the free fatty acid.     

Gymnemic acids inhibit rabbit glyceraldehyde-3-phosphate dehydrogenase and induce a smearing of its electrophoretic band and dephosphorylation

Gymnemic acids (GA) inhibited rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Binding of GA to GAPDH was observed by surface plasmon resonance measurement. Incubation of GAPDH with GA induced a smearing of the GAPDH band in SDS-PAGE. The GA-induced smearing was diminished by prior incubation of GA with gamma-cyclodextrin or by GA treatment with NAD. GA treatment did not affect the electrophoretic mobility of glucose-6-phosphate isomerase and dehydrogenase. GA treatment diminished the GAPDH band detected by an antibody to phosphoserine, but did not affect the phosphoserine bands of glucose-6-phosphate isomerase and dehydrogenase. These results indicated that GA specifically induced dephosphorylation of GAPDH.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

Selective denaturation of several yeast enzymes by free fatty acids.

The denaturation of eight purified yeast enzymes, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, alcohol dehydrogenase, beta-fructosidase, hexokinase and glucose-6-phosphate isomerase, promoted under controlled conditions by the free fatty acids myristic and oleic, is selective. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1 oxidoreductase, EC 1.1.1.49) is extremely sensitive to destabilization and was studied in greater detail. Results show that chain length and degree of unsaturation of fatty acids are important to their destabilizing effect, and that ligands of the enzyme can afford protection. The denaturation process results in more than one altered form. These results can be viewed in the perspective of the possibility that amphipathic substances, and in particular free fatty acids, may play a role for enzyme degradation in vivo, by initiating steps of selective denaturation.     

Changes in the rates of synthesis and messenger RNA levels of hepatic glucose-6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone

The lipogenic capacity of rat liver is increased in animals fed a high carbohydrate, fat-free diet or by the administration of 2,2',5'-triiodo-L-thyronine. Underlying this change is a generalized induction of the enzymes involved in lipogenesis, including glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme, which together serve to generate the additional NADPH required for increased fatty acid synthesis. This report presents evidence indicating that induction of the hexose-shunt dehydrogenases involves increased enzyme synthesis secondary to elevated enzyme specific mRNA levels, as has previously been shown for malic enzyme. Activities of specific mRNAs, estimated by cell-free translation of hepatic poly(A)-containing RNA in the mRNA dependent rabbit reticulocyte lysate, were compared with enzyme specific activities and relative rates of specific enzyme synthesis. The 2-fold increase in glucose-6-phosphate dehydrogenase specific activity in hyperthyroid rats and the 13-fold increase in rats fed a high carbohydrate, fat-free diet, relative to euthyroid, chow-fed controls were paralleled by comparable increases in the synthetic rates and mRNA levels of this enzyme. Similarly, consonant changes in the rate of enzyme synthesis and concentration of 6-phosphogluconate dehydrogenase mRNA accompanied the 2.5- and 3-fold increases in specific activity of this enzyme observed in response to hormonal and dietary induction, respectively. Thus, both thyroid hormone and carbohydrate feeding appear to induce glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase primarily by increasing the effective cellular concentrations of their respective mRNAs and, consequently, their rates of synthesis.     

Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

Multiple Forms of Pseudomonas multivorans Glucose-6-Phosphate and 6-Phosphogluconate Dehydrogenases: Differences in Size, Pyridine Nucleotide Specificity, and Susceptibility to Inhibition by Adenosine 5′-Triphosphate

Two major species of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) differing in size, pyridine nucleotide specificity, and susceptibility to inhibition by adenosine 5'-triphosphate (ATP) were detected in extracts of Pseudomonas multivorans (which has recently been shown to be synonymous with the species Pseudomonas cepacia) ATCC 17616. The large species (molecular weight ca. 230,000) was active with nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) and was markedly inhibited by ATP, which decreased its affinity for glucose-6-phosphate and for pyridine nucleotides. This form of the enzyme exhibited homotropic effects for glucose-6-phosphate. The small species (molecular weight ca. 96,000) was active with NADP but not with NAD, was not inhibited by ATP, and exhibited no homotropic effects for glucose-6-phosphate. Under certain conditions multiplicity of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) activities was also noted. One form of the enzyme (80,000 molecular weight) was active with either NAD or NADP and was inhibited by ATP, which decreased its affinity for 6-phosphogluconate. The other form (120,000 molecular weight) was highly specific for NADP and was not susceptible to inhibition by ATP. Neither form of the enzyme exhibited homotropic effects for 6-phosphogluconate. The possible relationships between the different species of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are discussed.     

Carbohydrate and lipid metabolism in swine skeletal muscle in the pre- and neonatal periods

Studies have been made on the activity of hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase, as well as on the intensity of in vitro oxidation of [U-14C]-glucose and [U-14C]-palmitate (together with in vivo lipid synthesis from these compounds) in porcine skeletal muscles during pre- and postnatal periods of life. It was shown that active utilization of glucose in oxidative metabolism and lipid synthesis is possible during the transition from prenatal to neonatal period. The increase in the rate of oxidation of fatty acids in skeletal muscles of piglets, in contrast to other animals, does not inhibit carbohydrate utilization.     

Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver

The activity and mRNA level of hepatic enzymes in fatty acid oxidation and synthesis were compared in rats fed diets containing either 15% saturated fat (palm oil), safflower oil rich in linoleic acid, perilla oil rich in α-linolenic acid or fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) for 15 days. The mitochondrial fatty acid oxidation rate was 50% higher in rats fed perilla and fish oils than in the other groups. Perilla and fish oils compared to palm and safflower oils approximately doubled and more than tripled, respectively, peroxisomal fatty acid oxidation rate. Compared to palm and safflower oil, both perilla and fish oils caused a 50% increase in carnitine palmitoyltransferase I activity. Dietary fats rich in n-3 fatty acids also increased the activity of other fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. The extent of the increase was greater with fish oil than with perilla oil. Interestingly, both perilla and fish oils decreased the activity of 3-hydroxyacyl-CoA dehydrogenase measured using short- and medium-chain substrates. Compared to palm and safflower oils, perilla and fish oils increased the mRNA level of many mitochondrial and peroxisomal enzymes. Increases were generally greater with fish oil than with perilla oil. Fatty acid synthase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activity and mRNA level were higher in rats fed palm oil than in the other groups. Among rats fed polyunsaturated fats, activities and mRNA levels of these enzymes were lower in rats fed fish oil than in the animals fed perilla and safflower oils. The values were comparable between the latter two groups. Safflower and fish oils but not perilla oil, compared to palm oil, also decreased malic enzyme activity and mRNA level. Examination of the fatty acid composition of hepatic phospholipid indicated that dietary α-linolenic acid is effectively desaturated and elongated to form EPA and DHA. Dietary perilla oil and fish oil therefore exert similar physiological activity in modulating hepatic fatty acid oxidation, but these dietary fats considerably differ in affecting fatty acid synthesis.