Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

Human Brain 6-Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.     

Mechanism for Regulating the Distribution of Glucose Carbon Between the Embden-Meyerhof and Hexose-Monophosphate Pathways in Streptococcus faecalis

Glucose-adapted Streptococcus faecalis produced little if any (14)CO(2) from glucose-1-(14)C, although high levels of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1, 6-diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in V(max) and an increase in the apparent K(m) for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that beta-mercaptoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suggested by the observation that this enzyme from several other sources was also sensitive to FDP.     

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.

1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.     

Circadian variations in the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in the liver of control and streptozotocin-induced diabetic rats

The aim of this study was to examine: the 24 h variation of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin-induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light-dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6-phosphogluconate dehydrogenase activity was measured by substituting 6-phosphogluconate as substrate. Glucose-6-phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two-way ANOVA revealed that 6-phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6-phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose-6-phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6-phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one-way ANOVA). Time-dependent variation in glucose-6-phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ-treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH-generating enzymes exhibit 24 h variation, which is not influenced by diabetes.     

Inactivation of G1ucose-6-Phosphate Dehydrogenase Isozymes from Human and Pig Brain by Aluminum

Prolonged intake of low levels of aluminum from the drinking water has been found to increase the aluminum content in rat brain homogenates and to reduce the activity of hexokinase and glucose-6-phosphate dehydrogenase (G6PD). To determine the interaction of G6PD with aluminum in the brain, we have recently purified two isozymes of G6PD (isozymes I and II) from human and pig brain. Unlike isozyme I, isozyme II also had 6-phosphogluconate dehydrogenase (6-PGD) activity. We report here that G6PD isozymes I and II from human and pig brain purified to apparent homogeneity are inactivated by aluminum. Aluminum did not affect the 6-PGD activity of isozyme II. The aluminum-inactivated enzyme contained 1 mol of aluminum/mol of enzyme subunit. The protein-bound metal ion was not dissociated by exhaustive dialysis at 4 degrees C against 10 mM Tris-HCl (pH 7.0) containing 0.2 mM EDTA. Preincubation of aluminum with citrate, NADP+, EDTA, NaF, ATP, and apotransferrin protected the G6PD isozymes against aluminum inactivation. However, when the G6PD isozymes were completely inactivated by aluminum, only citrate, NaF, and apotransferrin restored the enzyme activity. The dissociation constants for the enzyme-aluminum complex of the isozymes varied from 2 to 4 microM, as measured by using NaF, a known chelator for aluminum. Inhibition of G6PD by low levels of aluminum further strengthens the suggested role of aluminum toxicity in the energy metabolism of the brain.     

Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in Escherichia coli

The fungus, Cunninghamella elegans has been widely used in bioremediation and microbial models of mammalian studies in many laboratories. Using the polymerase chain reaction to randomly amplify the insert directly from the single non-blue plaques of a C. elegans cDNA library, then partly sequencing and comparing with GenBank sequences, we have identified a clone which contains C. elegans 6-phosphogluconate dehydrogenase gene. The polymerase chain reaction product was cloned into a plasmid, pGEM-T Easy vector for full insert DNA sequencing. The 6-phosphogluconate dehydrogenase gene (1458 bases) and the deduced protein sequence were determined from the insert DNA sequence. The gene was found by open reading frame analysis and confirmed by the alignment of the deduced protein sequence with other published 6-phosphogluconate dehydrogenase sequences. Several highly conserved regions were found for the 6-phosphogluconate dehydrogenase sequences. The 6-phosphogluconate dehydrogenase gene was subcloned and over-expressed in a plasmid–E. coli system (pQE30). The cell lysate of this clone has a very high 6-phosphogluconate dehydrogenase enzyme activity. Most of the recombinant protein in this system was formed as insoluble inclusion bodies, but soluble in high concentration of urea-buffer. Ni-NTA resin was used to purify the recombinant protein which showed 6-phosphogluconate dehydrogenase enzyme activity. The recombinant protein has a predicted molecular size correlating with that revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The C. elegans 6-phosphogluconate dehydrogenase was in a cluster with yeast' 6-phosphogluconate dehydrogenase in the phylogenetic tree. Bacterial 6-phosphogluconate dehydrogenase and higher organisms' 6-phosphogluconate dehydrogenase were found in different clusters.     

Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland.

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.     

Use of a cloned cDNA sequence to measure changes in 6-phosphogluconate dehydrogenase mRNA levels caused by thyroid hormone and dietary carbohydrate

A cDNA clone containing sequences complementary to the mRNA coding for rat hepatic 6-phosphogluconate dehydrogenase has been isolated and used to measure changes in specific mRNA levels during dietary and hormonal regulation of this enzyme. Hepatic mRNA was fractionated by sucrose gradient centrifugation to enrich for 6-phosphogluconate dehydrogenase mRNA sequences. A cDNA library was prepared from the fraction with maximal activity and then screened by differential colony hybridization using probes synthesized either from 6-phosphogluconate dehydrogenase mRNA enriched by polysome immunoadsorption or from unenriched hepatic mRNA. A single colony giving an appropriate differential signal was confirmed to contain sequences encoding 6-phosphogluconate dehydrogenase by specific immunoprecipitation of hybrid-selected translational products. 6-Phosphogluconate dehydrogenase mRNA contains about 2400 bases. The cloned cDNA comprises about 880 bases, or 35% of the mRNA. Southern analysis of restriction endonuclease digests of genomic DNA suggests that the major 6-phosphogluconate dehydrogenase gene is probably present in a single copy in the rat genome. Feeding a fat-free, high carbohydrate diet and administration of thyroid hormone increased the concentration of hybridizable 6-phosphogluconate dehydrogenase mRNA in liver. Thus, both dietary and hormonal regulation of 6-phosphogluconate dehydrogenase synthesis occurs at a pretranslational level.     

Effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study

The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2',5' ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1 h, 2 h, and 3 h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.     

Development of a quantitative histochemical method for determination of succinate dehydrogenase activity in autonomic neurons and its application to the study of aging in the autonomic nervous system

An accurately validated method was developed for quantitative determination of succinate dehydrogenase (EC 1.3.99.1; SDH) activity in individual sympathetic neuron perikarya by microdensitometric measurement of an SDH-nitroblue tetrazolium-derived formazan final reaction product. Optimal incubation medium and reaction conditions were determined for measurement of reaction product in cryostat sections of rat superior cervical and celiac-superior mesenteric ganglia. The Beer-Lambert laws were verified for the ganglion tissue, and microdensitometric measurements (expressed as mean cell density readings; MCDR/min-1), characteristic of the Michaelis-Menten equation, enabled the results to be used for enzyme kinetic determinations of SDH activity. Km and Vmax values were obtained following Hans linear transformation of the readings. Between the ages of 6-24 months no significant variations in Km values were recorded, indicating an unchanged structure for SDH (overall mean Km = 0.083 +/- 0.055 mM). However, in both ganglia there were significant decreases (ranging from 43-54%) in Vmax values for SDH at 24 months. The overall mean Vmax value at 6 months was 4.01 +/- 0.61 (MCDR) and at 24 months was 2.07 +/- 0.76 (MCDR). This suggests that an overall decrease in metabolic activity takes place with age in sympathetic neurons of the rat superior cervical and celiac-superior mesenteric ganglia.     

Enzymatic and Immunologic Identification of Succinic Semialdehyde Dehydrogenase in Rat and Human Neural and Nonneural Tissues

Abstract: We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.     

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.     

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.     

Purification and Characterization of the Two 6-Phosphogluconate Dehydrogenase Species from Pseudomonas multivorans

The two species of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) from Pseudomonas multivorans were resolved from extracts of gluconate-grown bacteria and purified to homogeneity. Each enzyme comprised between 0.1 and 0.2% of the total cellular protein. Separation of the two enzymes, one which is specific for nicotinamide adenine dinucleotide phosphate and the other which is active with nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was facilitated by the marked difference in their respective isoelectric points, which were at pH 5.0 and 6.9. Comparison of the subunit compositions of the two enzymes indicated that they do not share common peptide chains. The enzyme active with nicotinamide adenine dinucleotide was composed of two subunits of about 40,000 molecular weight, and the nicotinamide adenine dinucleotide phosphate-specific enzyme was composed of two subunits of about 60,000 molecular weight. Immunological studies indicated that the two enzymes do not share common antigenic determinants. Reduced nicotinamide adenine dinucleotide phosphate strongly inhibited the 6-phosphogluconate dehydrogenase active with nicotinamide adenine dinucleotide by decreasing its affinity for 6-phosphogluconate. Guanosine-5'-triphosphate had a similar influence on the nicotinamide adenine dinucleotide phosphate-specific 6-phosphogluconate dehydrogenase. These results in conjunction with other data indicating that reduced nicotinamide adenine dinucleotide phosphate stimulates the conversion of 6-phosphogluconate to pyruvate by crude bacterial extracts suggest that in P. multivorans, the relative distribution of 6-phosphogluconate into the pentose phosphate and Entner-Doudoroff pathways might be determined by the intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate and purine nucleotides.     

Ribonuclease activities in rat sympathetic ganglia: Evidence for the presence of an endogenous inhibitor of alkaline ribonuclease

Using³H-labeled rat brain mature RNA as substrate, substantial ribonuclease activity was detected in homogenates of rat superior cervical ganglia with acidic (pH 5.5) and neutral (pH 7.0-7.5) optima. Very little activity could be measured at greater than pH 8. The acidic and neutral activities differed in the optimal conditions required for assay, and showed differential sensitivity to the sulfhydryl blocking agent, N-ethylmaleimide. Only the neutral activity was stimulated, optimally by 2 mM N-ethylmaleimide, and the magnitude of stimulation indicated that the contributing ribonucleases exist largely in a latent form in the ganglion. Ribonucleases in other tissues with neutral pH dependence, known usually as alkaline ribonucleases, are subject to an N-ethylmaleimide-sensitive endogenous inhibitor protein. The existence of a similar inhibitor in rat superior cervical ganglia was indicated by the latency of neutral ribonuclease activity and confirmed by observing the effect of a soluble fraction from the ganglia on the activity of pancreatic ribonuclease A.     

Activity of aspartate aminotransferase in the hippocampal formation of the rat during post-natal development and after lesion of the hippocampal Schaffer's collaterals: A quantitative histochemical study

The activity of histochemically demonstrable aspartate aminotransferase was measured histophotometrically in the hippocampal formation, in dorsal root ganglia, and in superior cervical ganglia during postnatal development of the rat. The enzyme activity showed a marked (up to 15 times) increase in the dendritic layers of the hippocampal formation during the first weeks of life. Just as previous biochemical findings, the present data coincide with parameters of maturation of glutamatergic/aspartatergic structures in the hippocampal formation and other brain regions. For comparison, aspartate aminotransferase activities in both superior cervical ganglia and dorsal root ganglia were remarkably stable in the period studied.A nearly total transection of the CA3 pyramidal cell axon collaterals (Schaffer's collaterals) resulted in a striking reduction in aspartate aminotransferase activity within their target areas of the CA1 region (by 51.4% in stratum oriens, and by 60.2% in stratum radiatum).Our histochemical and histophotometrical data strongly account for the assumption that aspartate aminotransferase, beyond its numerous functions in general amino acid and nitrogen metabolism, is involved in the synthesis and/or catabolism of glutamate and aspartate used as excitatory transmitters.     

Regulation of p-nitroanisole O-demethylation in perfused rat liver. Adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase.

Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and "malic" enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for "malic" enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes.     

THE PURIFICATION AND PROPERTIES OF RAT BRAIN GLUTAMATE DEHYDROGENASE

—A method is described for the preparation of glutamate dehydrogenase in a highly purified form from rat brain. Only one protein band was detected when the enzyme was subjected to electrophoresis on SDS polyacrylamide gels. The rat brain enzyme was essentially identical to the rat liver enzyme with respect to electrophoresis on SDS polyacrylamide gels, immunochemical properties and most kinetic parameters. However, the brain enzyme was much less reactive with glutamate, was more sensitive to inhibition by haloperidol, and was considerably more stable than the liver enzyme.     

Utilization of Glucose by Clostridium thermocellum: Presence of Glucokinase and Other Glycolytic Enzymes in Cell Extracts

Clostridium thermocellum was shown to ferment glucose in a medium containing salts and 0.5% yeast extract. An active glucokinase was obtained with improved conditions for growth, assay, and preparation of cell extracts. Cell extracts appear to contain a glucokinase inhibitor that interferes with the assays at high protein concentrations. Glucokinase activity is stimulated about 60% by pretreatment with dithiothreitol. Little or no fructokinase or mannokinase activity was detected in cell extracts. The absence of glucokinase in mannitol-grown cells, the increase in glucokinase activity upon incubation of cell suspensions with glucose, and the lack of increase in activity when chloramphenical is added are evidence that glucokinase is an inducible enzyme. The following enzymes were detected in cell extracts (the enzyme activities are shown in parentheses are micromoles per minute per milligram or protein at 27 C): glucokinase (0.48), phosphoglucose isomerase (0.73), fructose 6-phosphate kinase (0.24), fructose diphosphate aldolase (0.59), glyceraldehyde 3-phosphate dehydrogenase (0.53), triose phosphate isomerase (0.13), phosphoglycerate kinase (0.20), phosphoglycerate mutase (0.20), enolase (0.28), pyruvic kinase (0.13), and lactic dehydrogenase (0.13). Glucose 6-phosphate dehydrogenase activity was absent or very low (0.0002) and 6-phosphogluconate dehydrogenase activity also was relatively low (0.015). From these data, it is proposed that carbohydrate metabolism in C. thermocellum proceeds by the Embden-Meyerhof pathway.