Regional Development of Glutamate Dehydrogenase in the at Brain

The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (greater than 2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons greater than midbrain = hypothalamus greater than cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90-day-old adult value, but not in other regions. The enzyme-specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90-day-old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.     

Regional differences in the turnover of neuronal histamine in the rat brain

The turnover rate of histamine (HA) and the half-life of neuronal HA were estimated in 9 regions of the rat brain following pargyline-induced accumulation of tele-methylhistamine (t-MH). The turnover rate was the highest in the hypothalamus (108.7 ng/g/hr). The striatum also showed a high turnover rate (80.2 ng/g/hr) despite much lower levels of HA and t-MH, as compared with the levels in the hypothalamus. The turnover rate was relatively high in the thalamus, cerebral cortex, amygdala and midbrain, but it was very low in the cerebellum. t-MH accumulation in the spinal cord was nil. The HA levels were reduced to various degrees (from nil to less than 40% of the control) by (S)-alpha-fluoromethylhistidine, depending on the regions studied. The neuronal HA content of each brain region was subsequently estimated, and the half-life of neuronal HA in each region was calculated. The half-life of neuronal HA was the shortest (7.7 min) in the striatum, while it was long (about 50 min) in the hypothalamus and thalamus. Half-life values of about 20 min were obtained in other regions. These results show the high levels of histaminergic activity in some parts of the telencephalon, thalamus and midbrain as well as the hypothalamus.     

Branched chain amino acid transaminases in brain in methionine sulphoximine (MSI) toxicity

The effect of intraperitoneal administration of L-methionine-DL-sulphoximine (MSI) was studied on branched-chain amino acid transaminases (BCAA-T) in different regions of rat brain and in liver. Administration of an acute dose of MSI (300 mg/kg body weight) resulted in a significant decrease in leucine aminotransferase activity in cerebral cortex, cerebellum, and brain-stem, while the activity of isoleucine aminotransferase was enhanced in hippocampus, corpus striatum, brain stem, and midbrain. Activities of both these enzymes changed marginally or remained unaltered in other regions of the brain. Valine aminotransferase showed a significant decrease in all the regions of the brain except in cerebellum. Following the administration of a sub-acute dose of MSI (150 mg/kg body wt.), the activities of the three BCAA aminotransferases were found to be enhanced in all regions of the brain. The results are discussed in relation to the utilization of BCAA for the production of glutamate and glutamine in hyperammonemia.     

Influence of Ca2+ and substance P on the (Ca Mg) ATPase (EC 3.6.1.3) activity of synaptosomes from different areas of rat brain

The influence of SP in vitro on the (Ca Mg) ATPase activity of synaptosomes from cerebral cortex, hippocampus, midbrain and thalamus with hypothalamus of rats was studied. It was confirmed that SP increases the activity of the enzyme from hippocampus, midbrain and thalamus with hypothalamus. A condition of this stimulation is the maintenance of Ca2+ concentration between 1 and 2 X 10(-4) M. Ca2+ concentration above and below the optimal (0.2 mM), reduced the SP stimulation of (Ca Mg) ATPase activity.     

Regional distribution of the enzymes of haem biosynthesis and the inhibition of 5-aminolaevulinate synthase by manganese in the rat brain

The activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, is differentially distributed in various regions of the rat brain. The cerebellum possessed the highest enzyme activity of the eight regions studied. The cerebral cortex and the midbrain also exhibited high 5-aminolaevulinate synthase activity; the septum, hypothalamus, thalamus, amygdala and the hippocampus possessed much lower enzyme activity. However, the total porphyrin and haem contents of the different brain segments did not vary greatly. Mn²⁺, when administered subcutaneously to rats, effectively inhibited the activity of 5-aminolaevulinate synthase in the cerebellum, midbrain and cerebral cortex; however, repeated injections of the metal ion neither decreased the haem and porphyrin contents of the brain nor induced haem oxygenase activity. Mn²⁺ was not an effective inhibitor of 5-aminolaevulinate synthase activity in vitro. On the other hand, studies carried out with the liver in vivo suggested that Mn²⁺ may alter the turnover rate of cellular haem and haemoproteins. In that event, it is likely that the inhibition of 5-aminolaevulinate synthase by Mn²⁺ was in part a result of the inhibition of protein synthesis by the metal ion. It is postulated that the haem and porphyrin contents of the brain are maintained at a steady-state level, due in part to the refractoriness to inducers of the regulatory mechanism for haem catabolic enzymes and in part to the ability of the organ to utilize haem precursors derived from extraneuronal sources.     

Alanine aminotransferase and some other enzymes in different populations of free brain cortex mitochondria

The activity of certain key enzymes involved in glutamic acid metabolism was studied in purified brain mitochondria and in mitochondrial subfractions separated in a discontinuous 1.2--1.6 mol/l sucrose gradient. Alanine aminotransferase and glutamate dehydrogenase were found to be matrix enzymes and aspartate aminotransferase to be associated with the inner mitochondrial membranes. After the purified mitochondria had been separated into 5 subfractions, aspartate aminotransferase and NAD+-dependent isocitrate dehydrogenase were found to be bound to the lighter mitochondrial subfractions settling at the 1.4--1.5 mol/l sucrose boundary while alanine aminotransferase, 4-aminobutyrate transaminase and glutamate dehydrogenase were associated with the heavier subfractions settling below 2.4 mol/l sucrose. The highest specific activity of the given enzymes was found in the subfraction settling at the 1.4--1.5 mol/l sucrose boundary, the only exception being alanine aminotransferase activity, whose maximum was found in the subfractions settling in 1.5 and 1.6 mol/l sucrose. It was concluded that alanine aminotransferase, in conjunction with glutamate dehydrogenase, is linked to NH3 binding and to the oxidation of reduced adenine nucleotides; in addition, alanine aminotransferase is presumed to have the function of transporting glutamate from the mitochondria to the extramitochondrial space.     

Distribution and characterization of the glutamate receptors in the CNS of ataxic mutant mouse

In a search for neurochemical involvement in cerebellar ataxia, Rolling Mouse Nagoya (C3Hf/Nem-rol), which shows only hypoplasia of the cerebellum but no pathological configuration of the cerebellar structure, was used to study glutamate receptors in the CNS. Kainic acid binding sites were significantly decreased in the thalamus, hypothalamus, pons, and cerebellum, and in the frontal cortex of both the ataxic mutant mouse and the non-ataxic heterozygote. Only spinal cord and midbrain of the ataxic mutant mouse showed decreased distribution of kainate binding sites in the membrane fraction.Among enzymes responsible for supplying glutamate to the receptor, GDH showed higher activity in the spinal cord of the ataxic mutanat mouse.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Regional distribution and relative amounts of glutamate decarboxylase isoforms in rat and mouse brain.

The levels of the two isoforms of glutamate decarboxylase (GAD) were measured in 12 regions of adult rat brain and three regions of mouse brain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting with an antiserum that recognizes the identical C-terminal sequence in both isoforms from both species. In rat brain the amount of smaller isoform, GAD65, was greater than that of the larger isoform, GAD67, in all twelve regions. GAD65 ranged from 77-89% of total GAD in frontal cortex, hippocampus, hypothalamus, midbrain, olfactory bulb, periaqueductal gray matter, substantia nigra, striatum, thalamus and the ventral tegmental area. The proportion of GAD65 was lower in amygdala and cerebellum but still greater than half of the total. There was a strong correlation between total GAD protein and GAD activity. In the three mouse brain regions analysed (cerebellum, cerebral cortex and hippocampus) the proportion of GAD65 (35,47, and 51% of total GAD) was significantly lower than in the corresponding rat-brain regions. The amount of GAD67 was greater than the amount of GAD65 in mouse cerebellum and was approximately equal to the amount of GAD65 in mouse cerebral cortex and hippocampus.     

Activation of branched-chain alpha-ketoacid dehydrogenase in isolated hepatocytes by branched-chain alpha-ketoacids

The effects of branched-chain alpha-ketoacids on flux through and activity state of the branched-chain alpha-ketoacid dehydrogenase complex were studied in hepatocytes prepared from chow-fed, starved, and low-protein-diet-fed rats. Very low concentrations of alpha-ketoisocaproate caused a dramatic stimulation (50% activation at 20 microM) of alpha-ketoisovalerate decarboxylation in hepatocytes from low-protein-fed rats. alpha-Keto-beta-methylvalerate was also effective, but less so than alpha-ketoisocaproate. alpha-Ketoisocaproate did not stimulate alpha-ketoisovalerate decarboxylation by hepatocytes from chow-fed or starved rats. To a smaller degree, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate stimulated alpha-ketoisocaproate decarboxylation by hepatocytes from low-protein-fed rats. The implied order of potency of stimulation of flux through branched-chain alpha-ketoacid dehydrogenase was alpha-ketoisocaproate greater than alpha-keto-beta-methylvalerate greater than alpha-ketoisovalerate, i.e., the same order of potency of these compounds as branched-chain alpha-ketoacid dehydrogenase kinase inhibitors. Fluoride, known to inhibit branched-chain alpha-ketoacid dehydrogenase phosphatase, largely prevented alpha-ketoisocaproate and alpha-chloroisocaproate activation of flux through the branched-chain alpha-ketoacid dehydrogenase. Assay of the branched-chain alpha-ketoacid complex in cell-free extracts of hepatocytes isolated from low-protein-diet-fed rats confirmed that alpha-ketoacids affected the activity state of the complex. Branched-chain alpha-ketoacids failed to activate flux in hepatocytes prepared from chow-fed and starved rats because essentially all of the complex was already in the dephosphorylated, active state. These findings indicate that inhibition of branched-chain alpha-ketoacid dehydrogenase kinase activity by branched-chain alpha-ketoacids is important for regulation of the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase.     

Regional distribution of the histamine metabolite, tele-methylimidazoleacetic acid, in rat brain: effects of pargyline and probenecid

Abstract: tele -Methylimidazoleacetic acid (t-MIAA), a major brain histamine metabolite, was measured in nine rat brain regions by a gas chromatography-mass spectrometric method that also measures the precursor amine, tele -methylhistamine (t-MH). The t-MIAA concentration of cerebellum, medulla-pons, midbrain, caudate nucleus, hypothalamus, frontal cortex, hippocampus, and thalamus varied 15-fold, hypothalamus showing the highest level (2.21 nmol/g) and cerebellum the lowest (0.15 nmol/ g). The concentrations of t-MIAA and t-MH were significantly correlated in all regions except midbrain, which had relatively more t-MIAA. Probenecid did not alter whole-brain t-MIAA levels. Treatment with pargyline, an inhibitor of monoamine oxidase, lowered the t-MIAA levels in all regions.     

Distribution of 3α-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans

3α-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3α-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3α-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3α-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activity was found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3α-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. We used the rat cDNA as the probe to screen a human liver λ gt11 cDNA library. A total of four different cDNAs were identified and sequenced. One of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5′-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3α-hydroxysteroid dehydrogenase and human chlordecone reductase. We are currently investigating the functional relationship between the enzymes encoded by these human cDNAs and 3α-hydroxysteroid dehydrogenase.     

Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain.

The subcellular localizations of gamma-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. gamma-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and gamma-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of gamma-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.     

Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver

Glutamine synthetase (EC 6.3.1.2) was localized within the matrix compartment of avian liver mitochondria. The submitochondrial localization of this enzyme was determined by the digitonin-Lubrol method of Schnaitman and Greenawalt (35). The matrix fraction contained over 74% of the glutamine synthetase activity and the major proportion of the matirx marker enzymes, malate dehydrogenase (71%), NADP-dependent isocitrate dehydrogenase (83%), and glutamate dehydrogenase (57%). The highest specific activities of these enzymes were also found in the matrix compartment. Oxidation of glutamine by avian liver mitochondria was substantially less than that of glutamate. Bromofuroate, an inhibitor of glutamate dehydrogenase, blocked oxidation of glutamate and of glutamine whereas aminoxyacetate, a transaminase inhibitor, had little or no effect with either substrate. These results indicate that glutamine metabolism is probably initiated by the conversion of glutamine to glutamate rather than to an alpha-keto acid. The localization of a glutaminase activity within avian liver mitochondria plus the absence of an active mitochondrial glutamine transaminase is consistent with the differential effects of the transaminase and glutamate dehydrogenase inhibitors. The high glutamine synthetase activity (40:1) suggests that mitochondrial catabolism of glutamine is minimal, freeing most of the glutamine synthesized for purine (uric acid) biosynthesis.     

Chronic metabolic effects of ammonia in mouse brain.

Chronic ammonia toxicity in experimental mice was induced by exposing them for 2 and 5 days to 5 % (v/v) ammonia solution. The enzymes concerned with glutamate metabolism (aspartate-, alanine- and tyrosine aminotransferases, glutamate dehydrogenase and glutamine synthetase) and (Na+ + K+)-ATPase were estimated in the three regions of brain (cerebellum, cerebral cortex and brain stem) and in liver. Glutamate, aspartate, alanine, glutamine and GABA, RNA and protein were also estimated in the three regions of brain and liver. A significant rise in the activity of (Na+ + K+)-ATPase in all the three regions of brain along with a fall in the activity of alanine aminotransferase was noticed. Changes in the activities of other enzymes were also observed. A significant increase in alanine and a decrease in glutamic acid was observed while no change was observed in the content of other amino acids belonging to the glutamate family. As a result of this, changes in the ratios of glutamate/glutamine and glutamate + aspartate/GABA was observed. The results indicated that the brain was in a state of more depression and less of excitation. Under these conditions the liver tissue was showing a profound rise in the activity of the enzymes of glutamate metabolism. The results are further discussed.     

Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.     

Ethanol and oleate inhibition of alpha-ketoisovalerate and 3-hydroxyisobutyrate metabolism by isolated hepatocytes.

Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.     

Effect of starvation or streptozotocin-diabetes on phosphate-activated glutaminase of different rat brain regions

Phosphate-activated glutaminase (PAG) was assayed in homogenates of brain cerebellum, hippocampus or striatum from normal, starved for 48 h or 120 h or streptozotocin-diabetic rats. Only the hippocampal enzyme was increased (47%) by diabetes. Starvation had no effect in any of the regions studied. PAG of synaptosomes or of non-synaptosomal mitochondria from the hippocampus was also increased by 48% and 22% respectively in diabetes. PAG of synaptosomes from the cortex, the cerebellum, or the striatum or of the non-synaptosomal mitochondria from the cortex were not affected by diabetes or prolonged (120h) starvation. A suggestion is presented that peripheral insulin, indirectly, may regulate PAG activity in a specific region of the rat brain.Abbreviations used PAG phosphate-activated glutaminase - LDH lactate dehydrogenase - s.a. specific activity     

Purification and partial characterization of branched-chain alpha-ketoacid dehydrogenase kinase from rat liver and rat heart

Branched-chain alpha-ketoacid dehydrogenase kinase was purified to homogeneity from rat liver and rat heart. The initial step was the purification of rat liver and heart branched-chain alpha-ketoacid dehydrogenase complex with high kinase activity by a modification of a method described previously. Preservation of high kinase activity during purification of the complex required the presence of fresh dithiothreitol throughout the procedure. The kinase was released from the complex by oxidation of dithiothreitol with potassium ferricyanide and purified by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography. Both kinase preparations gave only one polypeptide band with a molecular weight of 44,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex by the purified kinase was inhibited by alpha-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex. The kinase did not exhibit autophosphorylation and does not correspond to the same protein as pyruvate dehydrogenase kinase. The kinase phosphorylated histone (type II-S), but this reaction was slow relative to the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex and was not inhibited by alpha-chloroisocaproate.     

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids, established inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by branched-chain alpha-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain alpha-ketoacid dehydrogenase activity state.