GABA and synaptic inhibition of mouse cerebellum lacking glutamate decarboxylase 67

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter and also presumed to be a neurotrophic factor. GABA is synthesized by glutamate decarboxylase (GAD). A mouse lacking a 67 kDa isoform of GAD (GAD67) has a reduced GABA level in its brain at birth and does not survive postnatally because of cleft palate. In this study, to investigate the functional and developmental roles of GABA in the postnatal cerebellum, selective GAD67 deletion was achieved using a Cre-loxP strategy. In this mouse, GABA level was reduced to 16-44% in the cerebellum but not in the cerebrum. Inhibitory synaptic transmission to Purkinje cells was seriously impaired. However, the morphology of Purkinje cells and the density of synaptic terminals in the cerebellar cortex appeared unaffected, suggesting that GABA does not participate in cerebellar development substantially.     

Reactivation and reconstitution of glutamate decarboxylase upon the interaction of its dimers with pyridoxal phosphate]

The relationship between the reactivation and reconstitution of the hexameric form of glutamate decarboxylase during the interaction of inactive apoenzyme dimers with pyridoxal phosphate (PLP) has been studied. It was shown that the restoration of enzymatic activity, appearance of spectral maximum at 340 nm, and reconstitution of the hexamer depend on the amount of PLP added; this reaction is completed when the PLP concentration reaches that of the initial enzyme. This native hexamer of the holo- and apoenzyme does not practically contain exposed sulfhydryl groups. Ten cysteine residues become available after DS-Na denaturation. The dimer of the apoenzyme contains 8 exposed and 2 buried cysteine residues. The hexamer formation from the dimers is accompanied by the burying of the cysteine residues. When half of the required PLP was added, 7 cysteine residues became buried in experiments with DTNB and six in experiments with 4.4'-DTDP. Further addition of PLP led to the disappearance of the exposed sulfhydryl groups.     

Purification and characterization of glutamate decarboxylase from Solanum tuberosum

Glutamate decarboxylase has been purified from potato tubers. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gel filtration on Sephadex G-200 gave a relative molecular mass Mr, of 91 000 for the native enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a subunit Mr of 43 000. Thus the enzyme appears to be a dimer of identical subunits. It has 2 mol pyridoxal 5'-phosphate/mol protein, which could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. The enzyme has an absorption maximum at 370 nm in sodium phosphate buffer, pH 5.8. Reduction of the enzyme with sodium borohydride abolished the absorption maximum at 370 nm with attendant loss of catalytic activity. The enzyme exhibited pH-dependent spectral changes. The enzyme was specific for L-glutamate and could not decarboxylate other amino acids tested. The enzyme was maximally active at pH 5.8 and a temperature of 37 degrees C. Isoelectric focussing gave a pI of 4.7 Km values for L-glutamate and pyridoxal 5'-phosphate were 5.6 mM and 2 microM respectively. Thiol-directed reagents and heavy metal ions inhibited the enzyme, indicating that an -SH group is required for activity. The nature of the functional groups at the active site of the enzyme was inferred from competitive inhibition studies. L-Glutamate promoted inactivation of the enzyme caused by decarboxylation-dependent transamination was demonstrated. The characteristics of potato enzyme were compared with enzyme from other sources.     

Purification and general properties of glutamate decarboxylase from Clostridium perfringens

1. A seven-step procedure for preparing highly purified glutamate decarboxylase from Clostridium perfringens is described. 2. The homogeneity of the pure enzyme was established by sucrose-density-gradient centrifugation and starch-gel electrophoresis. 3. The isoelectric point of the pure enzyme is about pH4.5 and the molecular weight is 290000. 4. The pH optimum for activity is 4.7. The pure enzyme is specific for l-glutamate; beta-hydroxyglutamate is decarboxylated at a lower rate. 5. Evidence is presented that each mol of enzyme contains 2mol of firmly bound pyridoxal 5-phosphate. 6. Resolution does not occur at acid pH; by dilution with neutral or alkaline buffers the enzyme is inactivated and the coenzyme is released. 7. Reconstitution of active enzyme was obtained by protecting the apoenzyme with thiol compounds.     

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.     

Resolution and reconstitution of anion exchange reactions

To illustrate the emerging class of anion exchange proteins in bacteria, this article discusses the biochemical and physiological properties of phosphate (Pi)-linked antiporters that accept glucose 6-phosphate (G6P) as their primary substrate. These systems have a bifunctional active site that binds a pair of negative charges, whether presented as a single divalent anion or a pair of monovalent substrates. Exchange stoichiometry therefore moves between the limits of 2:1 and 2:2 according to the ratio of mono- and divalent substrates at either membrane surface. This predicts an interesting reaction sequence in vivo because internal pH is more alkaline than external pH; one expects an asymmetric exchange as a pair of monovalent G6P anions moves against a single divalent G6P, and in this way an otherwise futile self-exchange of G6P can result in a net inward flux driven (indirectly) by the pH gradient. Despite their biochemical complexity, at a molecular level the Pi-linked antiporters resemble other secondary carriers. Indeed, the current listing of nearly two dozen such proteins suggests a structural theme in which the minimal functional unit has two sets of six transmembrane alpha helices separated by a central hydrophilic loop. Presently described examples show that this topology can derive from either a single protein or from pairs of identical subunits. The finding of this common structure makes it possible to begin building more detailed structural models that have more general implications.     

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.     

Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product gamma-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 degrees C, the isotope effect is k14/k15 = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is k14/k15 = 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction [O'Leary, M.H., Yamada, H., & Yapp, C.J. (1981) Biochemistry 20, 1476] shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.     

Gene expression and characterization of thermostable glutamate decarboxylase from Pyrococcus furiosus

Gene encoding for a putative glutamate decarboxylase (GAD: EC 4.1.1.15) from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (PF1159) from P. furiosus showed some identity with other group II decarboxylases from an archaea and bacteria. The GAD from P. furiosus (PfGAD) was expressed in Escherichia coli, and the recombinant protein has a molecular mass of 41 kDa, determined by SDS-PAGE. The optimum temperature and pH for GAD activity were 75°C and 6.0, respectively. The half-life of heat inactivation was approximately 60 min at 90°C. The GAD activity was found to be dependent on various salts, such as CaCl₂, NaCl, KCl, and NaBr, with an optimum concentration of 400 mM, but not (NH₄)₂SO₄. PfGAD demonstrated activity against various substrates, such as l-glutamate, l-aspartate, and l-tyrosine. The results of the kinetics experiment indicated that l-aspartate was a better substrate of PfGAD than l-glutamate and Ltyrosine.     

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca²⁺/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.     

Isolation and functional reconstitution of the aspartate/glutamate carrier from mitochondria

The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%.     

An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues.     

Resolution and reconstitution of H+ -ATPase complex from beef heart mitochondria

Mitochondrial H+ -ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a "membrane" (NaBr-F0) and a "soluble" fraction by treatment with 3.5 M sodium bromide. The NaBr-F0 fraction is completely devoid of beta, delta, and epsilon subunits of the F, ATPase and largely devoid of alpha and gamma subunits of F1, where F0 is used to denote the membrane fraction and F1, coupling factor 1. This is confirmed by complete loss of ATPase and Pi-ATP exchange activities. The addition of F1 (400 micrograms X mg-1 F0) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F1-F0 complex consequent to sodium bromide extraction. Restoration of Pi-ATP exchange and H+ -pumping activities require coupling factor B in addition to F1-ATPase. The oligomycin-sensitive ATPase and 32Pi-ATP exchange activities in reconstituted F1-F0 have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F1-F0 complex. The data suggest that the altered properties of NaBr-F0 observed in other laboratories are probably inherent to their F1-F0 preparations rather than to sodium bromide treatment itself. The H+ -ATPase (F1-F0) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35-37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F1, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler- and oligomycin-insensitive. The membrane-bound hydrophobic component F0, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and Pi-ATP exchange activities on binding to F1-ATPase (33). The purest preparations of bovine heart mitochondrial F0 show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial F0 is not known. The F0 preparations from bovine heart reported so far have been derived from H+ -ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37).(ABSTRACT TRUNCATED AT 400 WORDS)