Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain

The relationship between the susceptibility to convulsions, the content of pyridoxal 5′-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-σ-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5′-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5′-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5′-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5′-phosphate levels. The correlation between pyridoxal 5′-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain.

The relationship between the susceptibility to convulsions, the content of pyridoxal 5'-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-gamma-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5'-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5'-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5'-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5'-phosphate levels. The correlation between pyridoxal 5'-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

Stereochemical action of mouse brain glutamate decarboxylase

4-[4-²H]Aminobutyrate was prepared by incubation in ²H₂O of glutamate with a partially purified glutamate decarboxylase from mouse brain. The 4R configuration was assigned to the compound on the basis of ¹H nmr analysis of the ω-camphanoylamide of its methyl ester in the presence of Eu(dpm)₃. Moreover 4-[4(S)4-³H,U-¹⁴C]aminobutyrate was shown to be formed from [2(S)2-³H,U-¹⁴C]glutamate by the same enzyme fraction. It is therefore demonstrated that glutamate decarboxylation catalyzed by this enzyme preparation occurs with retention of configuration.     

Pyridoxal phosphate-sensitized photoinactivation of glutamate decarboxylase from Clostridium perfringens

1. l-Glutamate decarboxylase (EC 4.1.1.15) from Clostridium perfringens was inactivated by exposure to visible light at pH6.2. 2. Inactivation does not occur at pH4.6 or in the absence of bound pyridoxal phosphate. 3. On prolonged photo-oxidation six histidine residues per molecule of enzyme were destroyed. 4. The loss of six cysteine residues per molecule occurred both in irradiated samples and in controls oxygenated in the dark. 5. This dark-oxidation of cysteine residues is apparently required before the photo-oxidation process. 6. The absorbance, fluorescence and circular-dichroism properties of the enzyme as well as its elution volume during Sephadex gel-filtration were unaffected by prolonged irradiation. 7. However, an apparently homogeneous product of photo-oxidation could be separated from the control enzyme by ion-exchange chromatography. 8. The K(m) for l-glutamate was unchanged in an irradiated sample retaining 22% of control activity. 9. These data and the catalytic role of imidazole residues at the active sites of amino acid decarboxylases are discussed.     

The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain

1. The subcellular distributions of glutamate decarboxylase and aspartate transaminase were studied in rat and guinea-pig brain. 2. Glutamate decarboxylase is localized in the synaptosome fraction. The mean density of the particles containing the enzyme is slightly greater than those derived from cholinergic neurones, though overlap is substantial. 3. The enzyme is readily released from synaptosomes by hypo-osmotic treatment, but in the presence of Ca(2+), Na(+) and K(+) it sediments with particulate material. 4. The release and binding of the enzyme to membrane fractions by Ca(2+) were investigated. 5. Aspartate transaminase is present in brain as two isoenzymes with different kinetic properties. One isoenzyme is associated with the cytoplasm and the other with mitochondria.     

Changes in glycolipid glycosyltransferases and glutamate decarboxylase and their relationship to differentiation in neuroblastoma cells

Glycolipid glycosyltransferase activities involved in the biosynthesis $\text{in vitro}$ of neutral and acidic glycosphingolipids were measured in C-1300 tumors and cloned cells derived therefrom. An adrenergic clone (NIE-115) was grown in tissue culture in the presence of dibutyryl cyclic AMP and the levels of glycosyltransferases were measured before and after differentiation. Increased activities of galactosyltransferases and sialyl-transferases with a concomitant increase in glutamate decarboxylase activity (the enzyme that catalyzes the synthesis of an inhibitory neurotransmitter, γ-aminobutyric acid) were observed.     

Reactivation of substrate-inactivated brain glutamate decarboxylase

The effects of ATP and inorganic phosphate (Pi) on the reactivation of glutamate apodecarboxylase by its cofactor pyridoxal-5'-phosphate (pyridoxal-P) was studied. Apoenzyme was prepared by preincubation with glutamate. Apoenzyme prepared with glutamate alone was reactivated slowly and incompletely by adding a saturating concentration of pyridoxal-P (20 microM). Reactivation was slightly enhanced by 1-10 mM Pi. Reactivation by pyridoxal-P plus Pi was greatly enhanced by the presence of low concentrations (less than 100 microM) of ATP during the preparation of apoenzyme with glutamate. Reactivation was much lower if Pi was omitted. Enhancement of reactivation by ATP was due to its effect during apoenzyme formation, since ATP did not enhance reactivation if added only during reactivation and since the enhancing effect persisted after the removal of free ATP by chromatography on Sephadex G-25 after apoenzyme preparation and before reactivation. Reactivation was inhibited by high concentrations of ATP (greater than 100 microM), possibly by competition of ATP for the cofactor binding site. Four factors (glutamate, pyridoxal-P, ATP, and Pi) control a cycle of inactivation and reactivation that appears to be important in the regulation of brain glutamate decarboxylase.     

Post-mortem degradation of brain glutamate decarboxylase

The post-mortem stability of the GABA synthesizing enzyme glutamate decarboxylase (GAD) was studied by using SDS–PAGE and quantitative immunoblotting to measure the rates of degradation of GAD in the cerebral cortex, hippocampus, and cerebellum of rats and mice as a function of time after death. The intact 65- and 67-kDa isoforms of GAD (GAD₆₅ and GAD₆₇) disappeared gradually over a 24-h period. In both rats and mice, the degraded GAD appeared as a band with an apparent molecular mass of 55–57 kDa; no significant amounts of smaller forms were observed. The 55–57 kDa band reacted with antiserum W887, which recognizes a shared epitope at the carboxyl-terminal end of both GADs, indicating that GAD was cleaved near the amino-terminal end of the molecule. GAD₆₇ was cleaved at a site between the amino-terminus and the epitope for antiserum W883 (located within residues 79–93 of GAD₆₇), as antiserum W883 stained a 56-kDa band on the blots. The appearance of degraded GAD paralleled the loss of total GAD (GAD₆₅+GAD₆₇), and after 24 h the 55–57 kDa band accounted for 97, 88, and 59% of the intact GAD lost from rat cerebellum, cerebral cortex and hippocampus. On a percentage basis, GAD₆₇ was degraded more rapidly than was GAD₆₅ in all brain regions studied. The loss of GAD activity was greater in rat than mouse brain, even though the percent loss of intact GAD protein was similar.     

Regulatory properties of brain glutamate decarboxylase

1. Glutamate decarboxylase is a focal point for controlling gamma-aminobutyric acid (GABA) synthesis in brain. Several factors that appear to be important in the regulation of GABA synthesis have been identified by relating studies of purified glutamate decarboxylase to conditions in vivo. 2. The interaction of glutamate decarboxylase with its cofactor, pyridoxal 5'-phosphate, is a regulated process and appears to be one of the major means of controlling enzyme activity. The enzyme is present in brain predominantly as apoenzyme (inactive enzyme without bound cofactor). Studies with purified enzyme indicate that the relative amounts of apo- and holoenzyme are determined by the balance in a cycle that continuously interconverts the two. 3. The cycle that interconverts apo- and holoenzyme is part of the normal catalytic mechanism of the enzyme and is strongly affected by several probable regulatory compounds including pyridoxal 5'-phosphate, ATP, inorganic phosphate, and the amino acids glutamate, GABA, and aspartate. ATP and the amino acids promote apoenzyme formation and pyridoxal 5'-phosphate and inorganic phosphate promote holoenzyme formation. 4. Numerous studies indicate that brain contains multiple molecular forms of glutamate decarboxylase. Multiple forms that differ markedly in kinetic properties including their interactions with the cofactor have been isolated and characterized. The kinetic differences among the forms suggest that they play a significant role in the regulation of GABA synthesis.     

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 by adenosine-5'-triphosphate of glutamic decarboxylase from a subcellular fraction of mouse brain.

Glutamate decarboxylase from a mouse brain P2 fraction undergoes a twofold activation in the presence of 0.5 mM ATP. No such stimulation by ATP occurs if the enzyme is assayed in the presence of excess pyridoxal phosphate as cofactor. The ATP-induced stimulation is almost completely eliminated if the enzyme is dialysed before its assay. [lambda-32P]ATP present during the enzyme measurement is converted to [32P]pyridoxal phosphate. These results demonstrate that the activation produced by ATP is the result of the generation of cofactor during the course of the assay. This phenomenon may be a reflection of a control mechanism of glutamate decarboxylase activity.     

Development of [3H]muscimol binding to subcellular particles of a culture of mouse brain

Specific binding of [³H]muscimol (11.5 nM), related to GABA receptors, occurred in subcellular particles prepared from 4-, 8-, and 12-day cultures of embryonic (14-to 15-day-old) mouse cerebrum, but not to particles prepared from 20-to 45-day cultures. Phase-contrast microscopy revealed that the presence of neuronal elements (neuroblasts, neurites) paralleled the binding of [³H]muscimol. Further evidence is thus provided for using high-affinity [³H]muscimol binding as a neuronal marker.Supported by a UNESCO/IBRO fellowship     

Cofactor and tryptophan accessibility and unfolding of brain glutamate decarboxylase

Cofactor and tryptophan accessibility of the 65-kDa form of rat brain glutamate decarboxylase (GAD) was investigated by fluorescence quenching measurements using acrylamide, I-, and Cs+ as the quenchers. Trp residues were partially exposed to solvent. I- was less able and Cs+ was more able to quench the fluorescence of Trp residues in the holoenzyme of GAD (holoGAD) than the apoenzyme (apoGAD). The fraction of exposed Trp residues were in the range of 30-49%. In contrast, pyridoxal-P bound to the active site of GAD was exposed to solvent. I- was more able and Cs+ was less able to quench the fluorescence of pyridoxal-P in holoGAD. The cofactor was present in a positively charged microenvironment, making it accessible for interactions with anions. A difference in the exposure of Trp residues and pyridoxal-P to these charged quenchers suggested that the exposed Trp residues were essentially located outside of the active site. Changes in the accessibility of Trp residues upon pyridoxal-P binding strongly supported a significant conformational change in GAD. Fluorescence intensity measurements were also carried out to investigate the unfolding of GAD using guanidine hydrochloride (GdnHCl) as the denaturant. At 0.8-1.5 M GdnHCl, an intermediate step was observed during the unfolding of GAD from the native to the denatured state, and was not found during the refolding of GAD from the denatured to native state, indicating that this intermediate step was not a reversible process. However, at >1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transition leading to the denatured state was reversible. It was suggested that the intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the alpha-helix content of GAD from 36 to 28%. The unfolding pattern suggested that GAD may consist of at least two unfolding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfolding of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD.     

A comparison of the kinetic properties of mouse brain glutamate decarboxylase activities in the mitochondrial and supernatant fractions

Several properties of the glutamate decarboxylase activities in the high-speed supernatant and water-washed mitochondrial fractions were compared. No significant differences in the values of Km for glutamate, the inhibitory effects of chloride ion, the proportion of pyridoxal-P independent activity remaining after exhaustive dialysis, the effects of added pyridoxal-P or the inhibitory effects of ATP were observed between the two fractions. The water-washed mitochondrial fraction was found to contain about 4 percent of the initial activity in the homogenate. Aside from the fact the small amount of glutamate decarboxylase found in the mitochondrial fraction cannot be released from the particles by washing or other mild treatments, there are presently no well documented differences between the glutamate decarboxylases found in the mitochrondrial and supernatant fractions.     

Differences in some properties of newborn and adult brain glutamate decarboxylase

Some properties of glutamate decarboxylase (EC 4.1.1.15) activity in brain of newborn and adult mouse were studied comparatively. It was found that glutamate decarboxylase of the newborn brain was strongly inactivated by homogenization in hypotonic medium, centrifugation of isotonic sucrose homogenates, preincubation at 37°C or the addition of Triton-X-100, whereas the adult brain enzyme was practically unaffected by any of these conditions. It was also found that the newborn glutamate decarboxylase was less activated by pyridoxal 5′-phosphate and less inhibited by pyridoxal 5′-phosphate oxime-O-acetic acid, than the adult enzyme. These differences do not exist for brain dihydroxyphenylalanine decarboxylase (EC 4.1.1.26) and are not due to the release of inhibitors from the newborn brain. On the basis of the results obtained it is postulated that two forms of glutamate decarboxylase exist in brain: a newborn form, which is unstable and has high affinity for pyridoxal 5′-phosphate, and an adult form, which is much more stable and has low affinity for pyridoxal 5′-phosphate. The possible implications of these findings in the establishment of the σ-aminobutyric acid dependent synaptic inhibitory mechanisms during development are discussed.