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Uptake kinetics and contents of GABA in cultured, normal (i.e. nontransformed) glia cells obtained from the brain hemispheres of newborn mice were measured together with the activity of the GABA transaminase. During three weeks of culturing the activity of the transaminase rose from a low neonatal value toward the level in the adult brain. The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics. Both theKm (40 μM) and theVmax (0.350 nmol×min−1×mg−1 cell protein) were reasonably comparable to the corresponding values in brain slices, and theVmax was much higher than that reported for other glial preparations. The GABA content was low (

Using antibodies against pig brain phosphate activated glutaminase, the enzyme appears to be rather conservative as we have observed immuno- staining in the brain from all species investegated [pig, cow, rabbit, rat, mouse, man, fish (cod and salmon) and bird (chicken)]. In addition, phosphate activated glutaminase from cultured mouse cerebral cortex inter- neurons (mainly GABA-ergic), cerebellar granule cells (glutamatergic) and astrocytes stained in an analogous manner. However, no phosphate activated glutaminase-like immunostaining was found in lobster ganglion. yeast and E. coli. Using the Western blotting technique, phosphate activated glutaminase from dodecyl sulfate treated samples from all the above mentioned preparations revealed a MW close to 64 K d . The MW is similar to the MW of the subunit of phosphate activated glutaminase in a highly purified pig brain preparation, The Western blotting technique seems to be well suited to identify phosphate activated glutaminase-like immunoreactivity in different tissues.

The development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic-oxaloacetic-transaminase (GOT), glutamine synthetase (GS), GABA-transaminase (GABA-T) and ornithine-δ-aminotransferase (Orn-T) was followed in mouse cerebral cortex in vivo and in cultured mouse cerebral cortex interneurons. It was found that GLDH, GOT and Orn-T exhibited an enhanced developmental pattern in the cultured neurons compared to cerebral cortex. The activities of PAG and GABA-T developed in parallel in vivo and in culture but the activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. Compared to cerebral cortex the cultured neurons exhibited higher activities of PAG, GLDH and Orn-T, whereas the activities of GABA-T and GOT were lower in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebral cortex in vivo. It is concluded that neurons from cerebral cortex represent a reliable model system by which the metabolism and function of GABAergic neurons can be conveniently studied in a physiologically meaningful way.

Uptake kinetics for glutamine were studied in several different neuronal preparations (perikarya prepared by gradient centrifugation, cultured cortical neurons, cultured, presumably glutamatergic cerebellar neurons, and brain prisms). In no case were any indications found of a high affinity uptake but a rather efficient low affinity uptake did occur. A similar, equally efficient low affinity uptake is, however, found in astrocytes. Thus, no preferential glutamine uptake occurs into neurons. It is, therefore, not likely that a net flow of glutamine takes place from astrocytes to neurons, compensating for the loss of TCA constituents when glutamate and GABA are released.

A method for preparing highly purified glutaminase (EC 3.5.1.2, l-glutamine amidohydrolase) from pig renal cortex is described. The main steps consist of sodium sulfate fractionation followed by alternative solubilization by dialysis against Tris-HCl buffer and precipitation with phosphate-borate. The final enzyme preparation contains no impurities which could be detected by sucrose gradient centrifugations, sedimentation velocity experiments, immune diffusion, or disc electrophoresis. Glutaminase has been found to exist in different interconvertible molecular forms. A Tris-soluble enzyme form (Tris-HC1 enzyme), obtained by dialysis against Tris-HCl, has an average sedimentation coefficient of 7.3 and an apparent molecular weight of 140,000 to 160,000, as determined by sucrose gradient centrifugation and Sephadex gel filtration. Another enzyme form (phosphate enzyme), obtained by dialysis of the Tris-HCl enzyme against phosphate, has an average sedimentation coefficient of 10.8 and an apparent molecular weight of 250,000 to 290,000. A phosphate-borate-precipitated form (phosphate-borate enzyme), with an average sedimentation coefficient of 50 and an apparent molecular weight of about 2,000,000, is obtained by addition of borate to the phosphate enzyme. Sulfate has an effect on the sedimentation coefficient similar to that of phosphate, and glutamate, citrate, and malonate have an effect similar to that of Tris-HCl. The sedimentation coefficients of both the Tris-HCl enzyme and the phosphate enzyme are increased by borate and the dye bromthymol blue. The sedimentation coefficient of the phosphate enzyme is lowered by chloride and urea. The Tris-HCl enzyme has a pH optimum of about pH 9, whereas no distinct optimum in the pH range 8 to 9 has been shown for the phosphate-borate enzyme. These forms of glutaminase are activated not only by anions, but also by ammonium ions and bromthymol blue, which suggests allosteric properties.

— A time-dependent activation of the tris-HC1 enzyme form of pig brain glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) by phosphate, phosphate-borate and carboxylic acids is described. This time-dependent activation increases with increased protein concentration and also with temperature. The sedimentation behaviour of the various activated enzyme preparations is described. The Arrhenius plot for the triq-HC1 enzyme is curved, indicating a reversible equilibrium between two enzyme forms, whereas the plot obtained for the phosphate-borate enzyme was a straight line. The apparent activationenergy has been determined.

The kinetics and other properties of phosphate-activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37 degrees C. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate-activated glutaminase-like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate-treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double-inverse plots of enzyme activity against phosphate are concave-upward, and more so in the presence of an inhibitor. The inhibition by glutamate appears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate-activated glutaminase from pig brain and pig kidney.

Glutaminase (EC 3.5.1.2 L-glutamine amidohydrolase) catalyses the hydrolysis of L-glutamine to glutamate and ammonia. As this reaction is practically irreversible, glutaminase is likely to be a key enzyme which, directly or indirectly, regulates the level of glutamate, an excitatory transmitter candidate in the brain. Moreover, glutamate is the substrate for the decarboxylase reaction which leads to the formation of GABA, an inhibitory transmitter candidate. However, the contribution by glutaminase to the synthesis of these essential metabolites, glutamate and GABA, is poorly understood. Other aspects of the glutaminase reaction should also be mentioned. Since the amide group of glutamine is a major contributor to the pool of labile amide groups (Weil-Malherbe and Gordon, 1971), glutaminase may be a major regulator of this pool, thus making ammonium ions available for various synthetic reactions, e.g. the important synthesis of amino acids and proteins from carbohydrates in the brain. For these reasons, knowledge of the regulation of glutaminase will probably be required for the proper understanding of the essential metabolic reactions in the brain.

Uptake kinetics and contents of GABA in cultured, normal (i.e. nontransformed) glia cells obtained from the brain hemispheres of newborn mice were measured together with the activity of the GABA transaminase. During three weeks of culturing the activity of the transaminase rose from a low neonatal value toward the level in the adult brain. The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics. Both theK m (40 μM) and theV max (0.350 nmol×min(-1)×mg(-1) cell protein) were reasonably comparable to the corresponding values in brain slices, and theV max was much higher than that reported for other glial preparations. The GABA content was low (5 nmol/mg cell protein), which is in agreement with the high activity of the GABA transaminase.

Distribution patterns and developmental profiles of phosphate activated glutaminase (PAG) in the cerebellar cortex of the rat were demonstrated by enzyme activity staining (tetrazolium salt technique) and immunolabeling. Histochemical evaluation of enzyme activity stained sections revealed in the molecular and granular layer (i.e. premigratory zone and external germinal zone in neonate rats) an increase from postnatal day 2 to day 50 by 350 and 400%, respectively. The smallest elevation was found in Purkinje cell bodies (140%). Maximum rise of PAG-activity was observed for all of the areas examined between day 12 and 15. The immunocytochemical visualisation of PAG-like immunoreactivity resulted in spatial and developmental patterns which differed from those of PAG-activity staining and displayed, to some extent, dependency on the way of tissue preparation, especially the fixation procedure.

— 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 (Km= 3.3 ± 0.5 mm; Vmax= 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.

The excretion in urine after single oral therapeutic doses of morphine derivatives was analysed with radioimmunoassay (RIA) and homogeneous enzyme immunoassay (EMIT) for opiates. In contrast to the rapid excretion of ethylmorphine and codeine, pholcodine showed positive results for opiates 2–6 weeks after intake when the urines were analysed with the RIA-method. When analysed with the EMIT-method, positive results were obtained for pholcodine for approximately 10 days. As pholcodine is a common component in cough mixtures, its prolonged excretion could represent a hazard in interpreting the results from drug analyses of urines.

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in cultured neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.

Glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) catalyzes the cleavage of L-glutamine to yield ammonia and L-glutamate. The brain type of glutaminase, first described by Krebs (1935), was characterized as phosphate-activated glutaminase I by Errera and Greenstein (1949), to distinguish this enzyme from the liver type, glutaminase II, which is identical with a glutamine-α-oxoacid transaminase (Greenstein and Carter, 1947). In addition, other glutaminases designated phosphate-independent glutaminases have been described by Katanuma et al. (1967) in various tissues including brain. Phosphate-independent glutaminase has been purified from liver and kidney (Katanuma et al., 1968), but not from brain.

Calcium stimulates the hydrolysis of glutamine in synaptosomes prepared from rat brain both by the sucrose- (12) and the Ficoll/sucrose-gradient techniques (13). The calcium activation is phosphate-dependent and maximal effect is obtained at a calcium concentration of 0.5–1.0 mM. It is reduced by increasing the numbers of synaptosomes in the incubation mixture, and abolished by the product inhibitors of glutaminase, glutamate and ammonia, but unaffected by the uncoupler 2,4-dinitrophenol which inhibits the mitochondrial proton pump. Moreover, since the hydrolysis of glutamine is mediated by glutaminase (EC 3.5.1.2), and calcium does not activate the purified enzyme, an indirect phosphate-dependent effect of calcium on glutaminase is most likely. Calcium activates preferentially the N-ethylmaleimide insensitive fraction of glutaminase. The calcium activation is not dependent on synaptosomal membranes as it is found in synaptosomes subject to previous freezing. It is also found in isolated synaptosomal mitochondria and is thus a property of nerve endings. The calcium activation of glutaminase is unaffected by potassium in depolarizing concentrations, and may not be directly involved in the neurotransmission processes, but possibly in replenishing depleted stores of transmitter glutamate.

The specific activity profiles of the glutamate synthesizing enzymes, phosphate activated glutaminase (EC 3.5.1.2), aspartate aminotransferase (EC 2.6.1.1), glutamate dehydrogenase (EC 1.4.1.2) and ornithine aminotransferase (EC 2.6.1.13) have been followed postnatally for 28 days in mouse hippocampus and compared to corresponding profiles in cerebellum and cerebral cortex (cf. refs 10 and 18). Phosphate activated glutaminase and glutamate dehydrogenase showed activity patterns similar to those found for cerebellum and glutamatergic granula cells cultured from cerebellum, whereas the aspartate aminotransferase activity pattern was found to be more similar to that previously observed for cerebral cortex as well as cultured cerebral interneurons which are likely to be GABAergic. The specific activity of ornithine aminotransferase was essentially unaltered during postnatal development, which is similar to what has been found for cerebellum and cerebral cortex.

Publisher Summary This chapter provides an overview of glutaminase from mammalian tissues. The mitochondrial phosphate activated glutaminase (PAG), which is the most important glutaminase in mammalian tissues, is described. Measurement of the reaction products ammonia or glutamate may be used for assay of PAG. Measurement of ammonia suffers from two disadvantages—the great water solubility of this compound makes it difficult to keep the background values sufficiently low and some undissociated ammonia may escape, particularly at prolonged incubations at 37° when the pH exceeds 8.0. Ammonia can be monitored, for example, with Nessler's reagent after microdistillation, by the ammonia electrode, or by coupling to the glutamate dehydrogenase (GDH) reaction, whereby NADH oxidation is measured. The two latter methods permit measurement of enzyme rates. The assay of PAG in isolated mitochondria, synaptosomes, cultured neurons, granulocytes, and astrocytes is reviewed in the chapter. Glutamate formed in a prefixed time is assayed following incubation of the cellular material with L-glutamine (preferably in the physiological concentration range), two concentrations of phosphate (to ensure that PAG is assayed), and inhibitors to prevent the metabolism of glutamate.

The ontogenetic development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic-oxaloacetic-transaminase (GOT), glutamine synthetase (GS), and ornithine-delta-aminotransferase (Orn-T) was followed in cerebellum in vivo and in cultured cerebellar granule cells. It was found that PAG, GLDH, and GOT exhibited similar developmental patterns in the cultured neurons compared to cerebellum. PAG showed, however, a more pronounced phosphate activation in the cultured granule cells compared to in vivo. The activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. On the other hand Orn-T exhibited an increase in its specific activity in the cultured cells as a function of time in culture in contrast to the non-changing activity of this enzyme in vivo. Compared to cerebellum the cultured neurons exhibited higher activities of GLDH, GOT, and Orn-T whereas the activity of PAG was only slightly higher in the cultured cells. The activity of GS in the cultured neurons was only 5-10% of the activity in cerebellum in vivo. It is concluded that cultured cerebellar granule cells represent a reliable model system by which the metabolism and function of glutamatergic neurons can be conveniently studied in a physiologically meaningful way.

Phosphate activated glutaminase comprises two kinetically distinguishable enzyme forms in cultures of cerebellar granule cells, of cortical neurons and of astrocytes. Specific activity of glutaminase is higher in cultured neurons compared with astrocytes. Glutaminase is activated by phosphate in all cell types investigated, however, glutaminase in astrocytes reguires a much higher concentration of phosphate for half maximal activation. One of the products, glutamate, inhibits the enzyme strongly, whereas the other product ammonia has only a slight inhibitory action on the enzyme.

— A procedure for preparing highly purified phosphate-activated glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) from pig brain is described. The main steps consist of extraction with acetone, followed by sodium sulphate fractionation of the solubilized acetone powder. Thereafter, solubilization by dialysis against a buffer containing tris-HC1, mercaptoethanol, and EDTA, followed by precipitation with phosphate-borate, is repeated twice. The final preparation contains no impurities which can be detected by polyacrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. By the latter method, molecular weight is determined to be 187,000. By polyacrylamide gel electrophoresis in sodium dodecyl sulphate, one protein band with molecular weight 64,000 is found.

Highly purified pig renal glutaminase has been subjected to electron microscopy. It is shown that the polymers formed after addition of phosphate-borate to enzyme dissolved in Tris-HCl buffer consist of long, double-stranded helices. Addition of phosphate to the Tris-HCl form of the enzyme probably induces the formation of dimers of the Tris-HCl units. It is further demonstrated that the rate of formation of the phosphate form and phosphate-borate form from Tris-HCl units, closely follows the increase in the catalytic activity of the enzyme.

Kinetic properties of two enzyme forms of purified pig kidney glutaminase, the phosphateborate and Tris-Cl enzymes, have been investigated. Plots of velocity versus phosphate concentration are sigmoidal for both forms. However, with α-ketoglutarate present, double reciprocal plots of velocity versus phosphate are linear for the phosphate-borate enzyme, and parabolic for the Tris-Cl enzyme. Inhibition by glutamate is non-competitive for the phosphate-borate enzyme, and competitive for the Tris-Cl enzyme. Moreover, with α-ketoglutarate present, phosphate activates the phosphate-borate enzyme only slightly, but it counteracts the inhibitory effect of glutamate strongly. When glutamine is the variable substrate, phosphate decreases the apparent Michaelis constant and increases maximal velocity for both enzyme forms. Using glutamate as the fixed variable and glutamine as the variable substrate, double reciprocal plots are linear, showing non-competitive inhibition between glutamine and glutamate for both enzyme forms. Replots of slopes versus glutamate are non-linear and of intercepts versus glutamate, linear. The experimental data are explained by assuming an ordered mechanism in which glutamate is the first product to be released.

Pig brain glutaminase (EC 3.5.1.2, l-glutamine amidohydrolase) is activated by certain anions (e.g. phosphate, fluoride, carboxylic acids) and inhibited by others (e.g. chloride, bromide, iodide and glutamate). The only cation which has been found to activate the enzyme is the ammonium ion. This applies to both the tris-HCl form and the phosphate-borate form of glutaminase.