Purification of L-glutamate decarboxylase by affinity chromatography

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.     

Glutamate decarboxylase side reactions catalyzed by the enzyme

A homogeneous glutamate decarboxylase isolated from pig brain contains 0.8 mol of tightly bound pyridoxal 5-phosphate/enzyme dimer. Upon addition of exogenous pyridoxal 5-phosphate (pyridoxal-5-P), the enzyme acquires maximum catalytic activity. Preincubation of the enzyme with L-glutamate (10 mM) brings about changes in the absorption spectrum of bound pyridoxal-5-P with the concomitant formation of succinic semialdehyde. However, the rate of this slow secondary reaction, i.e. decarboxylative transamination, is 10(-4) times the rate of normal decarboxylation. It is postulated that under physiological conditions enzymatically inactive species of glutamate decarboxylase, generated by the process of decarboxylative transamination, are reconstituted by pyridoxal-5-P produced by the cytosolic enzymes pyridoxal kinase and pyridoxine-5-P oxidase. The catalytic activity of resolved glutamate decarboxylase is recovered by preincubation with phospho-pyridoxyl-ethanolamine phosphate. The experimental evidence is consistent with the interpretation that the resolved enzyme binds the P-pyridoxyl analog, reduces the stability of the covalent bond of the phospho-pyridoxyl moiety, and catalyzes the formation of pyridoxal-5-P.     

Glutamate decarboxylase from barley embryos and roots. General properties and the occurrence of three enzymic forms.

Glutamate decarboxylase in extracts of barley has a Km value for L-glutamate of 22 mM and is activated by the addition of pyridoxal phosphate by up to 3.5 times. Sucrose-density-gradient experiments indicate the presence of two enzyme forms with molecular weights 256000 and 120000. The lower-molecular-weight form appears to be relatively inactive and spontaneously associates to the higher-molecular-weight form on storage. The enzyme is inhibited by thiol reagents and the distribution of activity on density gradients is altered in favour of the lower-molecular-weight form by the presence of 2-mercaptoethanol. After removal of the 2-mercaptoethanol spontaneous association to the higher-molecular-weight form occurs. The presence of oxygen in the extraction buffer and in the water during imbibition leads to a relative increase in the higher-molecular-weight form compared with situations where oxygen is excluded. In contrast, glutamate decarboxylase in extracts of 3-day-old barley roots has a Km value for L-glutamate of 3.1 mM and is activated up to 10% by addition of pyridoxal phosphate. The root enzyme occurs as a single species with molecular weight 310000 and this is unaffected by 2-mercaptoethanol although thiol reagents do act as weak inhibitors. The molecular weight is also unaffected by the presence or absence of oxygen in the extraction buffers.     

Turnover and accessibility of a reentrant loop of the Na+-glutamate transporter GltS are modulated by the central cytoplasmic loop

GltS of Escherichia coli is a secondary transporter that catalyzes Na+-glutamate symport. The structural model of GltS shows two homologous domains with inverted membrane topology that are connected by a central loop that resides in the cytoplasm. Each domain contains a reentrant loop structure. Accessibility of the Cys residues in two GltS mutants in which Pro351 and Asn356 in the reentrant loop in the C-terminal domain were replaced by Cys is demonstrated to be sensitive to the catalytic state supporting a role for the reentrant loops in catalysis. Saturating concentrations of the substrate L-glutamate protected both mutants against inactivation by thiol reagents, while the presence of the co-ion Na+ stimulated the inactivation of both mutants. Insertion of the 10 kDa biotin acceptor domain (BAD) of oxaloacetate decarboxylase of Klebsiella pneumoniae in the central cytoplasmic loop blocked the access pathway from the periplasmic side of the membrane to the cysteine residues in mutants P351C and N356C in the reentrant loop. Kinetically, insertion of BAD increased the maximal rate of uptake 2.7-fold while leaving the apparent affinity constants for L-glutamate and Na+ unaltered. The data suggests that insertion of BAD in the central loop results in conformational changes at the translocation site that lower the activation energy of the translocation step without affecting the access pathway from the periplasmic side for substrate and co-ions. It is concluded that changes in the central loop that connects the two domains may have a regulatory function on the activity of the transporter.     

D- AND L-STEREOISOMERS OF ALLYLGLYCINE: CONVULSIVE ACTION AND INHIBITION OF BRAIN L-GLUTAMATE DECARBOXYLASE

DL-Allylglycine was resolved into the L- and D-stereoisomers using hog kidney acylase. Both isomers were active as convulsants after administration to mice. The dose of D-allylglycine required to induce convulsions was greater than that of the L-isomer. Studies on the concentration of the two isomers in brain suggest that the lower effectiveness of D-allylglycine is partially due to its slower penetration into the brain through the blood-brain barrier. Both isomers of allylglycine inhibited brain glutamate decarboxylase in vitro to approximately the same extent, however, in vivo L-allylglycine inhibited the enzyme more strongly than the D isomer. Concentrations of allylglycine which caused a significant inhibition of L-glutamate decarboxylase in vivo were ineffective in inhibiting the enzyme in vitro. Oxidation products derived from L- or D-allylglycine by the action of either L- or D-amino acid oxidase caused an almost complete inhibition of the enzyme in vitro. It is suggested that a common intermediate derived from the two isomers (possibly 2-keto-4-pentenoic acid) is responsible for the in vivo inhibition of L-glutamate decarboxylase and possibly also for the induction of convulsions.     

Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride.

Incubation of malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with ethoxyformic anhydride caused the time-dependent loss of its ability to catalyze reactions requiring the nucleotide cofactor NADP+ or NADPH, such as the oxidative decarboxylase, the NADP+ - stimualted oxalacetate decarboxylase, the pyruvate reductase, and the pyruvate-medium proton exchange activities. Similar loss of oxidative decarboxylase and pyruvate reductase activities was affected by photo-oxidation in the presence of rose bengal. The inactivation of oxidative decarboxylase activity by ethoxyformic anhydride was accompanied by the reaction of greater than or equal to 2.3 histidyl residues per enzyme site and was strongly inhibited by NADP+. Ethoxyformylation also impaired the ability of malic enzyme to bind NADP+ or NADPH. These results support the involvement of histidyl residue(s) at the nucleotide binding site of malic enzyme.     

Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution

Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.     

Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli.

Vanadate was used as a substrate analogue to modify and subsequently localize active site serine residues of isocitrate lyase from Escherichia coli. Irradiation of the enzyme on ice with UV light in the presence of vanadate resulted in inactivation. Inactivation was prevented by the substrates glyoxylate or Ds-isocitrate and to a much lesser extent by succinate. Reduction of photoinactivated isocitrate lyase by NaBH4 partially restored enzyme activity. The photomodified enzyme was labeled by reduction with NaB[3H]4 in the presence and absence of the substrates succinate plus glyoxylate. Highly differential labeling of serine residues 319 and 321 in the absence of substrates suggests their importance in the action of isocitrate lyase. These residues are highly conserved in all five known sequences of this enzyme.     

Partial separation of synaptosomes accumulating 4-aminobutyrate or glutamate by zonal centrifugation on a discontinuous sucrose gradient.

Rat cerebral cortex slices were incubated with 1muM [3H]4-aminobutyrate or [3H]L-glutamine. The subcellular distribution of the accumulated labelled substances were determined by fractionating the nuclei-free homogenates on a 5-step discontinuous sucrose density gradient in a B XIV zonal rotor. The gradient was designed to separate the synaptosomes into 3 subpopulations of increasing density. The patterns of distribution of [3H]4-aminobutyrate and [3H-a1L-glutamate in the three synaptosomal peaks were distinctly different. This indicates the presence of separate types of nerve ending accumulating these two potential neurotransmitters, which are known to be metabolically closely linked through the enzyme glutamate decarboxylase. Recentrifugation of the 3 synaptosomal peaks on flat step gradients in a swingout rotor did not result in any further enrichment in transmitter-specific synaptosomes.     

Malonate decarboxylase of Malonomonas rubra, a novel type of biotin-containing acetyl enzyme.

Cell suspensions or crude extracts of Malonomonas rubra grown anaerobically on malonate catalyze the decarboxylation of this substrate at a rate of 1.7-2.5 mumol.min-1.mg protein-1 which is consistent with the malonate degradation rate during growth. After fractionation of the cell extract by ultracentrifugation, neither the soluble nor the particulate fraction alone catalyzed the decarboxylation of malonate, but on recombination of the two fractions 87% of the activity of the unfractionated extract was restored. The decarboxylation pathway did not involve the intermediate formation of malonyl-CoA, but decarboxylation proceeded directly with free malonate. The catalytic activity of the enzyme was completely abolished on incubation with hydroxylamine or NaSCN. Approximately 50-65% of the original decarboxylase activity was restored by incubation of the extract with ATP in the presence of acetate, and the extent of reactivation increased after incubation with dithioerythritol. Reactivation of the enzyme was also obtained by chemical acetylation with acetic anhydride. These results indicate modification of the decarboxylase by deacetylation leading to inactivation and by acetylation of the inactivated enzyme specimens leading to reactivation. It is suggested that the catalytic mechanism involves exchange of the enzyme-bound acetyl residues by malonyl residues and subsequent decarboxylation releasing CO2 and regenerating the acetyl-enzyme. The decarboxylase was inhibited by avidin but not by an avidin-biotin complex indicating that biotin is involved in catalysis. A single biotin-containing 120-kDa polypeptide was present in the extract and is a likely component of malonate decarboxylase.     

Turnover and accessibility of a reentrant loop of the Na+-glutamate transporter GltS are modulated by the central cytoplasmic loop

Abstract

GltS of Escherichia coli is a secondary transporter that catalyzes Na⁺-glutamate symport. The structural model of GltS shows two homologous domains with inverted membrane topology that are connected by a central loop that resides in the cytoplasm. Each domain contains a reentrant loop structure. Accessibility of the Cys residues in two GltS mutants in which Pro351 and Asn356 in the reentrant loop in the C-terminal domain were replaced by Cys is demonstrated to be sensitive to the catalytic state supporting a role for the reentrant loops in catalysis. Saturating concentrations of the substrate L-glutamate protected both mutants against inactivation by thiol reagents, while the presence of the co-ion Na⁺ stimulated the inactivation of both mutants. Insertion of the 10 kDa biotin acceptor domain (BAD) of oxaloacetate decarboxylase of Klebsiella pneumoniae in the central cytoplasmic loop blocked the access pathway from the periplasmic side of the membrane to the cysteine residues in mutants P351C and N356C in the reentrant loop. Kinetically, insertion of BAD increased the maximal rate of uptake 2.7-fold while leaving the apparent affinity constants for L-glutamate and Na⁺ unaltered. The data suggests that insertion of BAD in the central loop results in conformational changes at the translocation site that lower the activation energy of the translocation step without affecting the access pathway from the periplasmic side for substrate and co-ions. It is concluded that changes in the central loop that connects the two domains may have a regulatory function on the activity of the transporter.     

Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.     

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.     

A COMPARATIVE STUDY OF L-GLUTAMATE DECARBOXYLASE FROM MOUSE BRAIN AND BOVINE HEART WITH PURIFIED PREPARATIONS1

Abstract— L-Glutamate decarboxylase (EC 4.1.1.15) (GAD), the enzyme responsible for the formation of GABA, has been purified to homogeneity from mouse brain (Wu et at., 1973) and antibodies specific for neuronal GAD have been obtained (SAITO et al., 1974a). The present report describes the purification of GAD from bovine heart more than 2000-fold over the homogenate by initial solubilization with Triton X-100. subsequent fractionation with ammonium sulfate, column chromatography on DEAE cellulose, calcium phosphate gel, and DEAE-Sephadex, and gel filtration. At least two forms of GAD have been observed in bovine heart preparations; one of them appears as a high molecular weight form (Peak I, MW 360,000) and the other one as a low molecular weight form (Peak II, MW 105,000). Cysteine sulfinic acid and cysteic acid, both precursors of taurine, had no effect on the purified heart enzyme or on neuronal GAD at 10 mM, suggesting that cysteine sulfinic acid and cysteic acid probably are not substrates for any species of GAD described above. The heart enzyme and neuronal GAD differ in several respects. First, they are different immunochemically as judged by the lack of cross reactivity between the purified heart enzyme and the antibody against purified neuronal GAD. Second, they are different biochemically. 5,5′-Dithiobis[2-nitrobenzoic acid] (DTNB). one of the most potent inhibitors of neuronal GAD [K_i= 1.0 × 10^?8M] inhibits the heart enzyme only to a small extent at 1 mM. On the other hand, pyruvic acid, which inhibits the heart enzyme to an extent of 90% at 10 mM, only inhibits the neuronal enzyme slightly. Third, they are different in their substrate specificity. The neuronal enzyme can catalyze α-decarboxylation of both L-glutamate and L-aspartate while the heart enzyme can use only L-glutamate as substrate. Moreover, an unidentified product probably derived from L-glutamate is obtained in the reaction mixture of the heart enzyme but is not observed with the brain enzyme, suggesting that the heart enzyme may catalyze a reaction converting L-glutamate to products other than GABA. It is therefore concluded that heart GAD and neuronal GAD are two different entities. Work is in progress to determine whether the heart enzyme is related to the glial enzyme. Should the antibody against the heart enzyme cross-react with the glial enzyme, the role of the glial enzyme in GABA function can then be studied by immunochemical and immunocytochemical methods.     

Arg305 of Streptomyces L-glutamate oxidase plays a crucial role for substrate recognition

Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using L-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate.     

Influence of stereoisomers of 4-fluoroglutamate on rat brain glutamate decarboxylase

Inhibition of rat brain glutamate decarboxylase (GAD, EC 4.1.1.15) by individual stereoisomers of 4-fluoroglutamate (4-F-Glu) and 2-fluoro-4-aminobutyrate (2-F-GABA) was studied. All stereoisomers of 4-F-Glu inhibited decarboxylation of L-glutamate catalysed by the enzyme preparation. At 1 x 10(-2) M concentration, the most potent inhibitor of GAD was D-erythro-4-F-Glu with about 70% inhibition in the presence of 1.23 x 10(-2)M L-glutamate. The inhibition by all stereoisomers was of the competitive type. Ki values ranged from 2 x 10(-3)M for the D-erythro isomer to 1.1 x 10(-2)M for the D-threo and L-erythro isomers. The influence of all stereoisomers was reversible as shown by dialysis except for a small amount in the case of the D-erythro isomer. The inhibition was independent of external pyridoxal-5'-phosphate added. No inhibition of rat brain GAD was found with 2-fluoro-4-aminobutyrate stereoisomers.     

Purification of L-glutamate-dependent citrate lyase from Clostridium sphenoides and electron microscopic analysis of citrate lyase isolated from Rhodopseudomonas gelatinosa, Streptococcus diacetilactis and C. sphenoides

Citrate lyase from Clostridium sphenoides was purified 72-fold with a yield of 11%. In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of L-glutamate. The purified enzyme was only stable in the presence of 150 mM L-glutamate or 7 mM L-glutamate plus glycerol, sucrose or bovine serum albumin. Changes of the L-glutamate pool and of enzyme activity in growing cells of C. sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular L-glutamate concentration. Citrate lyase isolated from C. sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique. Three different projections of enzyme molecules were observed: 'star' form, 'ring' form and 'triangle' form. In samples from R. gelatinosa and S. diacetilactis, star and ring forms occurred in a ratio of about 1:9. Using the enzyme from S. diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate. The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form. In lyase samples from C. sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of L-glutamate.     

Lipoic acid residues in a take-over mechanism for the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The pyruvate dehydrogenase complex of Escherichia coli contains two lipoic acid residues per dihydrolipoamide acetyltransferase chain, and these are known to engage in the part-reactions of the enzyme. The enzyme complex was treated with trypsin at pH 7.0, and a partly proteolysed complex was obtained that had lost almost 60% of its lipoic acid residues although it retained 80% of its pyruvate dehydrogenase-complex activity. When this complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, the rate of modification of the remaining S-acetyldihydrolipoic acid residues was approximately equal to the accompanying rate of loss of enzymic activity. This is in contrast with the native pyruvate dehydrogenase complex, where under the same conditions modification proceeds appreciably faster than the loss of enzymic activity. The native pyruvate dehydrogenase complex was also treated with lipoamidase prepared from Streptococcus faecalis. The release of lipoic acid from the complex followed zero-order kinetics for most of the reaction, whereas the accompanying loss of pyruvate dehydrogenase-complex activity lagged substantially behind. These results eliminate a model for the enzyme mechanism in which specifically one of the two lipoic acid residues on each dihydrolipoamide acetyltransferase chain is essential for the reaction. They are consistent with a model in which the dihydrolipoamide acetyltransferase component contains more lipoic acid residues than are required to serve the pyruvate decarboxylase subunits under conditions of saturating substrates, enabling the function of an excised or inactivated lipoic acid residue to be taken over by another one. Unusual structural properties of the enzyme complex might permit this novel feature of the enzyme mechanism.     

Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB

Escherichia coli and other enterobacteria exploit the H+ -consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5'-phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl- exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.     

Regulation of acetylated tubulin/Na+,K+-ATPase interaction by L-glutamate in non-neural cells: involvement of microtubules

A subpopulation of membrane tubulin consisting mainly of the acetylated isotype is associated with Na+,K+-ATPase and inhibits the enzyme activity. We found recently that treatment of cultured astrocytes with L-glutamate induces dissociation of the acetylated tubulin/Na+,K+-ATPase complex, resulting in increased enzyme activity. We now report occurrence of this phenomenon in non-neural cells. As in the case of astrocytes, the effect of L-glutamate is mediated by its transporters and not by specific receptors. In COS cells, the effect of L-glutamate was reversed by its elimination from culture medium, provided that d-glucose was present. The effect of L-glutamate was not observed when Na+ was replaced by K+ in the incubation medium. The ionophore monensin, in the presence of Na+, had the same effect as L-glutamate. Treatment of cells with taxol prevented the dissociating effect of L-glutamate or monensin. Nocodazole treatment of intact cells or isolated membranes dissociated the acetylated tubulin/Na+,K+-ATPase complex. The dissociating effect of nocodazol does not require Na+. These results indicate a close functional relationship among Na+,K+-ATPase, microtubules, and L-glutamate transporters, and a possible role in cell signaling pathways.