PHOSPHATE ACTIVATED GLUTAMINASE ACTIVITY AND GLUTAMINE UPTAKE IN PRIMARY CULTURES OF ASTROCYTES

Abstract— Uptake and release of glutamine were measured in primary cultures of astrocytes together with the activity of the phosphate activated glutaminase (EC 3.5.1.2). In contrast to previous findings of an effective, high affinity uptake of other amino acids (e.g. glutamate, GABA) no such uptake of glutamine was observed, though a saturable, concentrative uptake mechanism did exist (K _m = 3.3 ± 0.5 m m ; V _max= 50.2 ± 12.6 nmol ± min⁻¹± mg⁻¹). The phosphate activated glutaminase activity in the astrocytes (6.9 ± 0.9 nmol ± min⁻¹± mg⁻¹) was similar to the activity found in whole brain (5.4 ± 0.7 nmol ± min ^−l± mg⁻¹), which may contrast with previous findings of a higher activity of the glutamine synthetase (EC 6.3.1.2) in astrocytes than in whole brain. The observations are compatible with the hypothesis of an in vivo flow of glutamate (and GABA) from neurons to astrocytes where it is taken up and metabolized, and a compensatory flow of glutamine towards neurons and away from astrocytes although the latter cell type may be more deeply involved in glutamine metabolism than envisaged in the hypothesis.     

Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics

Abstract The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S -transferase, UDP-glucuronosyltransferase, UDP-glucosyltransferase, and N -acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min⁻¹ mg⁻¹), UDP-glucosyltransferase (0.27 nmol min⁻¹ mg⁻¹) and glutathione 5-transferase (20.8 nmol min⁻¹ mg⁻¹). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min⁻¹ mg⁻¹) and N -demethylation (0.17 nmol min⁻¹~' mg⁻¹) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min⁻¹ mg⁻¹) and the microsomes (0.13 nmol min⁻¹ mg⁻¹). N -Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.     

Alternative oxidase from Arum and soybean: Its stabilization during purification

Complete purification of the alternative oxidase from plant mitochondria has not been achieved successfully, because of its instability on solubilization. We report here that the addition of pyruvate to the isolation medium stabilizes the activity of the solubilized enzyme. A procedure is described for the rapid isolation and partial purification of the cyanide-insensitive alternative oxidase from both Arum maculatum and soybean cotyledon ( Glycine max ) mitochondria. The degree of purification was 16- and 74-fold for Arum and soybean enzyme, respectively. The specific activities increased from 1 300 to 20 300 nmol oxygen consumed mg⁻¹ protein min⁻¹ (using duroquinol as substrate) after purification for the Arum erizyme and from 6 to 445 nmol oxygen consumed mg⁻¹ protein min⁻¹ for the soybean enzyme. A turnover for the partially purified Arum enzyme was estimated to be 47 electrons s⁻¹.
The partially purified enzyme from both Arum and soybean cotyledon mitochondria was sensitive to alternative oxidase inhibitors such as salicylhydroxamic acid, n -propyl gallate and octyl gallate, but not to myxottriazol, KCN or antimycin A. The activity of the enzyme could be stimulated by pyruvate, but not by malate and suceinate. The stability of the purified enzyme was also dependent on the continued presence of pyruvate. In the absence of pyruvace, the enzyme activity was lost in a time-dependent manner and the ability of pyruvate to recover the activity was also irreversibly lost.     

THE CONTROL OF PYRUVATE DEHYDROGENASE IN ISOLATED BRAIN MITOCHONDRIA

Abstract— The activity and control of the pyruvate dehydrogenase complex in isolated rat brain mitochondria has been studied. The activity of this complex in mitochondria as isolated from normal fed rats was 78 ± 10nmol.min⁻¹ mg mitochondrial protein⁻¹ (n = 18) which represented 70% of the total pyruvate dehydrogenase activity. The pyruvate dehydrogenase in isolated brain mitochondria could be inactivated by incubation in the presence of ATP, oligomycin and NaF. The rate of inactivation was dependent upon the added ATP concentration but inactivation below approx 30% of the total pyruvate dehydrogenase activity could not be achieved. The inactivation of pyruvate dehydrogenase in brain mitochondria was inhibited by pre-incubation with pyruvate. Reactivation of inactivated pyruvate dehydrogenase in rat brain mitochondria was incomplete in the incubation medium unless 10mM-Mg²⁺+ 1 mM-Ca²⁺ were added; NaF, however, prevented any reactivation (Fig. 4). It is concluded that the pyruvate dehydrogenase complex in rat brain mitochondria is controlled in a manner similar to that in other tissues, and that pyruvate protection of pyruvate dehydrogenase activity may be important in maintaining brain energy metabolism.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

Copper uptake by free and immobilized cyanobacterium

Abstract Copper uptake in free and immobilized cells of the cyanobacterium Nostoc calcicola has been examined. The immobilized cells invariably maintained a higher profile of Cu intake rate (12.7 nmol mg⁻¹ protein min⁻¹) over the free cells (6.0 nmol mg⁻¹ protein min⁻¹). The total Cu uptake in immobilized cells was almost two and a half-times more than their free cell counterpart under identical experimental conditions. Also, the immobilized cells showed a stronger positive correlation between Cu adsorption and uptake. The results have been discussed in terms of improved metabolic efficiency of immobilized cells.     

Characterization of Neuronal Amino Acid Transporters: Uptake of Nitric Oxide Synthase Inhibitors and Implication for Their Biological Effects

Abstract: In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors N ^G-methyl- l -arginine and N ^G-nitro- l -arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108-15. Uptake of N ^G-methyl- l -arginine was characterized by biphasic kinetics ( K _m1 = 8 µmol/L, V _max1 = 0.09 nmol × mg⁻¹× min⁻¹; K _m2 = 229 µmol/L, V _max2 = 2.9 nmol × mg⁻¹× min⁻¹) and was inhibited by basic but not by neutral amino acids. Uptake of N ^G-nitro- l -arginine followed Michaelis-Menten kinetics ( K _m = 265 µmol/L, V _max = 12.8 ± 0.86 nmol × mg⁻¹× min⁻¹) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of N ^G-methyl- l -arginine is mediated by system y⁺, whereas systems L and T account for the transport of N ^G-nitro- l -arginine. In agreement with these data on uptake of the inhibitors, l -lysine and l -ornithine antagonized the inhibitory effects of N ^G-methyl- l -arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas l -tryptophan, l -phenylalanine, and l -leucine interfered with the effects of N ^G-nitro- l -arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors.     

Mycorrhiza formation on Norway spruce (Picea abies) roots affects the pathway of anaplerotic CO2 fixation

Activities of carboxylation enzymes were analyzed in the mycelium of the mycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker, in non-mycorrhizal short roots of Norway spruce ( Picea abies [L.] Karst.) and in myconhizas of these two partners. While pyruvale carboxylase (PC, EC 6.4.1.1) and phosphoenolpyruvate carboxykinase activities (PEPCK.EC 4.1.1.49) could be detected in the mycelium of A. muscaria , phosphoenolpyruvate carboxyknase (PEPC, EC 4.1.1.31) was only active in root tissue. In A. muscaria , PC activity was generally low (around 10 nmol mg^−tprotein min⁻) but PEPCK activity was above 250 nmol mg⁻¹ protein min⁻¹. Mycorrhizal development on short roots decreased PEPC activity by more than 75%, although dilution by the fungal biomass in mycorrhizas was only 35%. This reduction in activity was paralleled by a decreased content of PEPC protein. By means of micro-analytical methods it was shown that PEPC activity was lowest in the central zones of the mycorrhizas, Whereas PEPC activity was highest in the corresponding central sections in non-mycorrhizal short roots. ¹⁴CO₂ labelling, on the other hand, revealed that in vivo CO₂ fixation was higher in mycorrhizas compared to non-mycorrhizal short roots. It is concluded that fungal carboxylases (probably PEPCK) are important for anaplerotic CO₂ fixation during nitrogen assimilation in mycorrhizas of Norway spruce.     

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

Polysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 10⁴ daltons and the smaller with a molecular weight of 3.65 ± 10⁴ daltons. Specific activity of the enzyme is 526 μmols min⁻¹mg⁻¹ for the smaller protein a nd 263 μmols min ⁻¹ mg⁻¹ for the la rger protein. The Michaelis-Menten constant (K_m) is 72.8 μM ± 17.15 and the v_max is 1.62 μmols min⁻¹± 0.928 for the 92.6-kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane, dithiobis-(2-nitrobenzoate), or N -ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme .     

Carbon monoxide dehydrogenase and acetate thiokinase in Methanothrix soehngenii

Abstract In Methanothrix soehngenii acetate is first activated by an acetate thiokinase rather than a phosphotransacetylase. The specific activity of the acetate thiokinase was 5.29 μmol acetate activated min⁻¹ mg⁻¹ protein with a half maximum rate at 0.74 mM acetate and at 0.047 mM CoA. In cell-free extracts a CO-dehydrogenase activity was measured of 3.02 μmol min⁻¹ mg⁻¹ protein with a half maximum rate at 0.44 mM CO and at 0.18 mM methylviologen. NADP and NAD could not replace methylviologen. F₄₂₀ showed only low activity as electron acceptor.     

Demonstration of HOQNO and antimycin A sensitive coupling of NADH oxidation and APS and sulfite reduction in a marine Desulfovibrio strain

Abstract In cell suspensions of the marine sulfate-reducing bacterium Desulfovibrio 20020 (DSM 3099) permeabilized with formaldehyde or Triton X-100, sulfite-dependent NADH oxidation activities of 0.05 μmol · min⁻¹· mg⁻¹ protein were detected. NADH oxidation coupled to APS, thiosulfate and fumarate reduction was also demonstrated. All the activities were subject to inhibition by HOQNO and antimycin A. The rate of NADH oxidation coupled to the reduction of sulfite was extremely low in cell-free extracts. The physiological function and possible mechanism of the NADH oxidation coupled to the reduction of various electron acceptors are discussed.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Monoamine oxidase activity in kidney and heart of Piaractus mesopotamicus (Holmberg)

The values of Michaelis–Menten constant (K_M) and maximum velocity (V_MAX) for kidney and heart monoamine oxidase (MAO) from pacu Piaractus mesopotamicus were determined. The mean ± s . e . K_M values were 17·28 ± 2·27 μM for kidney and 15·38 ± 1·86 μM for heart. MAO activities were 111·60 ± 3·25 and 15·12 ± 0·30 nmols min⁻¹ g⁻¹ of wet tissue for kidney and heart, respectively. In addition, MAO inhibitory studies in these two tissues indicate that this enzyme may be a different isoform of MAO.     

Isolation and characterization of two distinct membrane fractions from the Gram-positive nucleotide producer Brevibacterium ammoniagenes ATCC 6872

Abstract Bouyant density gradient centifugation of 'crude membranes' of the coryneform bacterium Brevibacterium ammoniagenes yielded two distinct membrane fractions which differed significantly in equilibrium densities (1.15 and 1.18 g/cm³), NADH dehydrogenase activity (0.29 and 0.09 μmol·min⁻¹·mg⁻¹), protein composition and association of ribosomes. As determined by one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) proteins unique to either the free membrane (FM) fraction or the denser ribosome-containing complexed membrane (CM) fraction were identified.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Influence of blue light on the activity of phosphofructokinase in Chlorella kessleri

In crude extracts of Chlorella kessleri Fott and Novákóva cells grown autotrophically in white light the activity of phosphofructokinase (PFK, EC 2.7.1.11) is 62.9 ± 1.5 nmol (mg protein)⁻¹ min⁻¹ under optimized test conditions. It is greatly increased in red [88.3 ± 1.8 nmol (mg protein)⁻¹ min⁻¹], but somewhat decreased [57.0 ± 0.5 nmol (mg protein)⁻¹ min⁻¹] in blue light of equal productivity. Mixtures of blue and red light yield the low activity as long as blue light represents at least 35% of the total quantum fluence rate. The rough wavelength dependence of the counteracting effect of short wavelength light on the increasing effect of red light exhibits a broad peak at 460 nm, reminiscent of action spectra of the blue/UV photoreceptors(s). Upon transfer of red light-grown cells to blue light, the decrease develops slowly within 72 h; it cannot be prevented by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). Since there is less carbohydrate in blue than in red light-exposed cells, correlations between biosynthesis of PFK and level of carbohydrate are discussed, based on the assumption that red light decreases and/or blue light increases the transport of metabolites across the chloroplast envelope.     

Xylanolytic activity of commercial juice-processing enzyme preparations

Of 22 commercial juice-processing enzyme preparations investigated, Clarex ML was found to exhibit the highest xylanase activity. The xylanase from Clarex ML was most active at 50–60°C and pH 5·0–5·5. The K _m and V _max values of the enzyme with oat-spelt xylan as the substrate were 8·6 mg ml⁻¹ and 42 μmol xylose l⁻¹ min⁻¹, respectively. Xylobiose was the main product of enzymatic hydrolysis of xylan.     

Bioassay of insulin activity of pancreatic tissue in active and aestivated African lungfish, Protopterus annectens (Owen)

The insulin activity of the pancreatic tissues of free-swimming and aestivated African lungfish, P. annectens (Owen), was bioassayed against standard insulin in rabbits. The hepatic glycogen content and plasma glucose concentration in free-swimming and aestivated lungfish were also estimated. The liver glycogen content was 64·47 ± 6·32 and 7·19 ± 2·68 mgg⁻¹ net wt and the plasma glucose concentration was 37·62 ± 4·22 and 6·39 ± 1·29% in free-swimming and aestivated lungfish, respectively.
Extracts of pancreatic tissue of the lungfish produced a dose-dependent decrease in the plasma glucose concentration in rabbits. The magnitude of the decrease in plasma glucose concentration produced by extracts from aestivated specimens was significantly smaller (about 26%) compared to that produced by pancreatic extracts from free-swimming active lungfish (about 37%). The insulin activity of the pancreatic tissue was 0·82 ± 0·02 and 1·27 ± 0·25 iu mg⁻¹ dry pancreatic tissue weight in aestivated and free-swimming lungfish, respectively.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

Peroxidase isoenzymes in the defense response of Capsicum annuum to Phytophthora capsici

Quantitative and qualitative changes in isoperoxidase patterns from stems of three cultivars of pepper ( Capsicum annuum L.). one susceptible, one intermediate and one resistant, were found upon inoculation with Phytophthora capsici using a decapitation method. The peroxidase activity was determined in the intercellular fluid as well as in the cytosolic fraction of the necrotic, healthy and intermediate zones of stems of the three cultivars, 6 days after inoculation. In the intercellular fluid, peroxidase activity of the susceptible cv. Yolo Wonder increased somewhat from 4.7 (healthy zone) to 12.9 (intermediate zone) μmol mg⁻¹ protein min⁻¹, whereas in the intermediate cv. Americano, the peroxidase activity decreased from 123 (healthy zone) to 78 (intermediate zone) μmol mg⁻¹ protein min⁻¹. The most dramatic increase (5.7 to 662 μmol mg⁻¹ protein min⁻¹) in intercellular peroxidase activity was found in the resistant cv. Smith-5. This, in conjunction with the appearance of an additional acidic isoperoxidase (pI 4.4) specific for the cv. Smith-5, could be the reason for the resistance of this cultivar against the fungus attack. The release of peroxidase into the intercellular space as a defense reaction was confirmed by histochemical analysis, showing that peroxidase activity occurred in the intercellular spaces of those stems of the resistant cultivar that had not yet been invaded by the fungus, but was detected neither in the other cultivars nor in the intercellular spaces of such stems of the intermediate and susceptible cultivars that contained growing mycelium of P. capsici. The lack of staining in the intercellular spaces of the susceptible cultivars could be attributed to their low content in peroxidase.