Essential Active-Site Lysine of Brain Glutamate Dehydrogenase Isoproteins

Abstract: Two soluble forms of bovine brain glutamate dehydrogenase (GDH) isoproteins were inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff's base formation with amino groups of the enzyme. Sodium borohydride reduction of the pyridoxal 5'-phosphate-inactivated GDH isoproteins produced a stable pyridoxyl enzyme derivative that could not be reactivated by dialysis. The pyridoxyl enzyme was studied through fluorescence spectroscopy. No substrates or coenzymes separately gave complete protection against pyridoxal 5'-phosphate. A combination of 10 m M 2-oxoglutarate with 2 m M NADH, however, gave complete protection against the inactivation. Tryptic peptides of the isoproteins, modified with and without protection, resulted in a selective modification of one lysine. In both GDH isoproteins, the sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other GDH species.     

Stability and Activation of Glutamate Apodecarboxylase from Pig Brain

The stability and activation of glutamate apodecarboxylase was studied with three forms of the enzyme from pig brain (referred to as the alpha, beta, and gamma forms). Apoenzyme was prepared by incubating the holoenzyme with aspartate followed by chromatography on Sephadex G-25. Apoenzyme was much less stable than holoenzyme to inactivation by heat (for beta-glutamate decarboxylase (beta-GAD) at 30 degrees C, t1/2 values of apo- and holoenzyme were 17 and greater than 100 min). ATP protected holoenzyme and apoenzyme against heat inactivation. The kinetics of reactivation of apoenzyme by pyridoxal-P was consistent with a two-step mechanism comprised of a rapid, reversible association of the cofactor with apoenzyme followed by a slow conversion of the complex to active holoenzyme. The reactivation rate constant (kr) and apparent dissociation constant (KD) for the binding of pyridoxal-P to apoenzyme differed substantially among the forms (for alpha-, beta-, and gamma-GAD, kr = 0.032, 0.17, and 0.27 min-1, and KD = 0.014, 0.018, and 0.04 microM). ATP was a strong competitive inhibitor of activation (Ki = 0.45, 0.18, and 0.39 microM for alpha-, beta-, and gamma-GAD). In contrast, Pi stimulated activation at 1-5 mM but inhibited at much higher concentrations. The results suggest that ATP is important in stabilizing the apoenzyme in brain and that ATP, Pi, and other compounds regulate its activation.     

Multiple Forms of Glutamate Decarboxylase in Porcine Brain

Three forms of glutamate decarboxylase from hog brain (termed α-, ß-, and γ-GAD) were separated by hydrophobic interaction chromatography on phenyl-Sepharose, by isoelectric focusing, and by polyacryl-amide gel electrophoresis. When rechromatographed on phenyl-Sepharose, each form migrated as a single entity, indicating that the forms are not readily interconvertible. The three forms are not different-sized aggregates of one form, since all three have the same approximate molecular weight (100,000) as determined by Sephadex G-200 chromatography. The pIs of the three forms separated by phenyl-Sepharose were determined by isoelectric focusing. The values obtained (5.3, 5.5, and 5.8 for α-, ß-, and γ-GAD, respectively) were comparable to the pIs of the three peaks of activity observed upon focusing of enzyme that had been subjected to phenyl-Sepharose chromatography. These results indicate that phenyl-Sepharose chromatography and isoelectric focusing separate the same three components. When synaptosomal extracts were analyzed by phenyl-Sepharose chromatography without intervening purification steps, all three forms were present, but the proportion of ß-GAD was somewhat higher and that of γ-GAD somewhat lower than in the usual preparations.     

Regulation of γ-Aminobutyric Acid Synthesis in the Brain

Abstract: γ-Aminobutyric acid (GABA) is synthesized in brain in at least two compartments, commonly called the transmitter and metabolic compartments, and because reglatory processes must serve the physiologic function of each compartment, the regulation of GABA synthesis presents a complex problem. Brain contains at least two molecular forms of glutamate decarboxylase (GAD), the principal synthetic enzyme for GABA. Two forms, termed GAD₆₅ and GAD₆₇, are the products of two genes and differ in sequence, molecular weight, interaction with the cofactor, pyridoxal 5′-phosphate (pyridoxal-P), and level of expression among brain regions. GAD₆₅ appears to be localized in nerve terminals to a greater degree than GAD₆₇, which appears to be more uniformly distributed throughout the cell. The interaction of GAD with pyridoxal-P is a major factor in the short-term regulation of GAD activity. At least 50% of GAD is present in brain as apoenzyme (GAD without bound cofactor; apoGAD), which serves as a reservoir of inactive GAD that can be drawn on when additional GABA synthesis is needed. A substantial majority of apoGAD in brain is accounted for by GAD₆₅, but GAD₆₇ also contributes to the pool of apoGAD. The apparent localization of GAD₆₅ in nerve terminals and the large reserve of apo-GAD₆₅ suggest that GAD₆₅ is specialized to respond to short-term changes in demand for transmitter GABA. The levels of apoGAD and the holoenzyme of GAD (holoGAD) are controlled by a cycle of reactions that is regulated by physiologically relevant concentrations of ATP and other polyanions and by inorganic phosphate, and it appears possible that GAD activity is linked to neuronal activity through energy metabolism. GAD is not saturated by glutamate in synaptosomes or cortical slices, but there is no evidence that GABA synthesis in vivo is regulated physiologically by the availability of glutamate. GABA competitively inhibits GAD and converts holo- to apoGAD, but it is not clear if intracellular GABA levels are high enough to regulate GAD. There is no evidence of short-term regulation by second messengers. The syntheses of GAD₆₅ and GAD₆₇ proteins are regulated separately. GAD₆₇ regulation is complex; it not only is present as apoGAD₆₇, but the expression of GAD₆₇ protein is regulated by two mechanisms: (a) by control of mRNA levels and (b) at the level of translation or protein stability. The latter mechanism appears to be mediated by intracellular GABA levels.     

Brain Glutamate Decarboxylase: Properties of Its Calcium-Dependent Binding to Liposomes and Kinetics of the Bound and the Free Enzyme

Abstract: In the present work we describe several properties of the Ca²⁺-dependent binding of glutamate decarboxylase (GAD) to phosphatidylcholine-phosphatidylserine liposomes. The binding occurs very rapidly, is dependent on temperature in the range 23–37°C, is inhibited up to 35% by K⁺ in a concentration-dependent manner and is slightly increased when the dielectric constant of the medium is decreased by 3% ethanol. The association of GAD and liposomes is very firm, since EGTA displaces only 40% of the bound enzyme and Triton X-100 about 55%. Since apparently only part of the total GAD is able to bind to the liposomes and in a previous study two forms of GAD activity have been identified kinetically, we compared the activations by pyridoxal 5'-phosphate (PLP) of the soluble and the bound GAD, as well as their inhibition by PLP oxime- O -acetic acid. The bound GAD was activated 150–265% by 10⁻⁶ to 10⁻⁴ m -PLP, whereas the activation of GAD that remained soluble was only 65–110% in the same PLP concentration range. In the absence of PLP, the bound GAD was less inhibited by the PLP oxime- O -acetic acid than the soluble GAD, but the inhibition was similar when 0.1 m m -PLP was added. In contrast, activity of both the soluble and the bound GAD was totally blocked by aminooxyacetic acid. Endogenous PLP did not bind to liposomes under the experimental conditions inducing GAD binding. We conclude that the binding of GAD to negatively charged liposomes is primarily ionic. Furthermore, the GAD molecules that bind to the liposomes seem to be deficient in free PLP and therefore, are probably more susceptible to regulation by the coenzyme. These conclusions may be relevant to the hypothesis of a coupling between synthesis and release of GABA in inhibitory nerve endings.     

Binding of ATP to Brain Glutamate Decarboxylase as Studied by Affinity Chromatography

Abstract: The interactions of two forms of porcine brain glutamate decarboxylase (β-GAD and γ-GAD) with the effector ATP were studied by affinity chromatography. A third form, γk-GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP > ADP > AMP. GAD was also eluted by its cofactor, pyridoxal 5'-phosphate, and this was enhanced by 1 mM P_i In contrast, a high concentration (140 mM) of P_i by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.     

Activation of Glutamate Apodecarboxylase by Succinic Semialdehyde and Pyridoxamine 5''-Phosphate

Glutamate apodecarboxylase was activated by incubation with succinic semialdehyde and pyridoxamine 5'-phosphate. Activation required both compounds and was highly selective for succinic semialdehyde. Of 18 analogs tested, only glyoxylate, pyruvate, oxaloacetate, and 2-oxoglutarate activated the apoenzyme significantly, but much higher concentrations of these compounds than of succinic semialdehyde were required. In the presence of pyridoxamine 5'-phosphate, the concentration of succinic semialdehyde giving half-maximal activation of apoenzyme was 7 microM. In contrast, the Ki for succinic semialdehyde as a competitive inhibitor of glutamate decarboxylation was 1.2 mM, indicating that apoenzyme with bound pyridoxamine 5'-phosphate has a much higher affinity for succinic semialdehyde than does holoenzyme. The concentration of pyridoxamine 5'-phosphate giving half-maximal activation was 17 microM, which is more than an order of magnitude greater than the corresponding value for pyridoxal 5'-phosphate.     

Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate

In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.     

Two Forms of Rat Brain Glutamic Acid Decarboxylase Differ in Their Dependence on Free Pyridoxal Phosphate

There are two forms of glutamate decarboxylase (GAD) found in the rat brain. One form (form A) does not require exogenous pyridoxal-5'-phosphate (PLP) for activity whereas another form (form B) requires exogenous PLP for activity. These two forms differ greatly in temperature sensitivity, inactivation, and reactivation by the removal and readdition of PLP, electrophoretic mobility, and regional distribution. For instance, forms A and B are inactivated to an extent of 91% and 10%, respectively, by the treatment at 45 degrees C for 30 min; form A is greatly inactivated (77%) by the removal of PLP by aminooxyacetic acid and the readdition of PLP, whereas form B is only slightly inactivated (7%). Forms A and B can be clearly separated by 5% polyacrylamide gel electrophoresis in which form A migrates faster than form B. In all 10 brain regions studied, form A is present in smaller amounts than form B. This difference is greatest in the superior colliculus (the ratio of B to A is about 5), while in the locus coeruleus and cerebellum, forms A and B are present in nearly equal proportion. Forms A and B are similar with respect to relative abundance in hypotonic, isotonic, and hypertonic preparations, inhibition of catalytic activity by a carbonyl-trapping agent, immunochemical properties, and chromatographic patterns in a variety of systems. The significance of forms A and B and PLP in the regulation of gamma-amino-butyric acid (GABA) level is also discussed.     

Characterization of Recombinant Human Aromatic l-Amino Acid Decarboxylase Expressed in COS Cells

The expression vector containing the full-length cDNA of human aromatic L-amino acid decarboxylase (EC 4.1.1.28) was transfected in COS cells by a modified calcium phosphate coprecipitation method. The cells transfected with plasmids that had a true direction of the cDNA gave a major immunoreactive band at 50 kDa. This expressed enzyme catalyzed the decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA), L-5-hydroxytryptophan (L-5-HTP) and L-threo-3,4-dihydroxyphenylserine. The optimal pH of the enzyme activity with L-DOPA as a substrate was 6.5, whereas the enzyme had a broad pH optimum when L-5-HTP was used as a substrate. Addition of pyridoxal phosphate to the incubation mixture greatly enhanced the activity for both L-DOPA and L-5-HTP.     

Effect of Nucleotides and Other Inhibitors on the Inactivation of Glutamate Decarboxylase

Abstract: The effects of 17 nucleotides and nucleotide analogs and 11 other compounds on the glutamate-promoted inactivation of brain glutamate decarboxylase were examined. Among the nucleotides, the major determinant of potency was the polyphosphate chain, Glutamate-promoted inactivation was strongly enhanced by low concentrations (<100 μM) of adenosine tetraphosphate and all eight nucleoside triphosphates tested. Nucleoside diphosphates enhanced inactivation, but were much less effective than the nucleoside triphosphates; nucleoside monophosphates were not effective. Modification of the polyphosphate chain of the nucleoside triphosphates also affected potency; adenylylimidodiphosphate and α,β-methylene ATP were about as effective as nucleoside diphosphates, but α,β-methylene ATP was nearly as effective as ATP. The nucleoside base had only a small effect on potency; purine nucleotides were more potent than pyrimidine nucleotides, and one nucleotide with a tricyclic base, 1, N⁶-etheno ATP, was as effective as the purine nucleoside triphosphates. The 2'-hydroxyl group of ribose was unimportant, since deoxy ATP was as effective as ATP. Three nonnucleotide polyanions were strong promoters of inactivation; inositol hexasulfate and 5-phosphorylribose 1-pyrophosphate were at least as effective as ATP; inositol hexaphosphate (phytate) was as effective as the nucleoside diphosphates. These results suggest that a major determinant of potency was a strong negative charge on the molecule. Negative charge was not sufficient, however, since fructose 1,6-bisphosphate did not promote inactivation. Inactivation by all of these compounds was slow, requiring more than 20 min for full effect. Two competitive inhibitors, chloride and glutarate, acted immediately and also reduced rather than enhanced glutamate-promoted inactivation.     

Glutamate Decarboxylase Activities in Single Vertebrate Neurons

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.     

Glutamate Decarboxylase Distribution in Discrete Motor Nuclei in the Cat Brain

Abstract: The distribution of activity of glutamate decarboxylase (GAD), the enzyme synthesising γ-aminobutyric acid (GABA), was measured in the cat brain by means of microdissection of the structures from frozen slices and a radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. GAD presented an uneven distribution among these areas. Highest activities were found in the basal ganglia, particularly in the substantia nigra and in the globus pallidus, and to a lesser extent in the cerebellum. Relatively low levels of the enzyme were found in the thalamus and in the cerebral motor cortex. Special detailed studies were made in the caudate nucleus, the substantia nigra, and in the red nucleus for the purpose of defining the intranuclear distribution of their GABAergic innervation. There were only small differences in the rostro-caudal distribution of the enzyme in the head of the caudate nucleus but GAD activity was higher in the ventral than in the dorsal part of the structure. In the substantia nigra, GAD activity was high in both the medial and intermediate thirds of the structure. The GAD activity decreased from the caudal to the rostral part of the nucleus. GAD levels were lower in the caudal part of the red nucleus than in the rostral part. These results indicate that GABA would be present as a putative neurotransmitter in many motor nuclei of the cat brain. In view of the general inhibitory action of this amino acid, this could be related to the presence of inhibitory responses widely distributed in these nuclei as identified by mean of electrophysiological studies. The origin of these GABAergic innervations in many cases remains to be determined.     

Resolution and Brain Regional Distribution of Cysteine Sulfinate Decarboxylase Isoenzymes from Hog Brain

Abstract: Cysteine sulfinate decarboxylase (CSD; EC 4.1.1.29) activity from porcine brain was resolved into three peaks by hydroxylapatite chromatography. The first two peaks (I and II) did not decarboxylate and were not inhibited by glutamate. The third peak (III) cochromatographed with glutamate decarboxylase (GAD; EC 4.1.1.15) activity. The K_m values of cysteine sulfinate for peaks I, II, and III were 5.5 × 10⁻⁴ m , 1.3 × 10⁻⁴ m , and 4.5 × 10⁻³ m , respectively. The possibility that the same enzyme was responsible for peak III CSD and GAD activities was suggested by several findings: (1) Mutual competitive inhibition was observed between glutamate and cysteine sulfinate for these activities. (2) Similar first-order heat-inactivation curves were obtained for peak III CSD and GAD when incubated at 55xBOC. (3) Both activities were inhibited similarily by ATP and chloride ion. High concentrations of glutamate (0. l m ) inhibited peak III CSD activity more than 90% but had no effect on either peak I or II CSD activities. This difference in sensitivity of the isoenzymes to inhibition by glutamate was used to examine the relative regional distributions and the relative contributions to total activity of the glutamate-sensitive (peak III CSD, GAD) and glutamate-insensitive (peaks I and II CSD) isoenzymes. Glutamate-insensitive CSD activity contributed only part of the total activity in all brain regions tested (ranging from 23% in the superior colliculus to 64% in the pons). However, the specific activity of glutamate-insensitive CSD was more constant than the total or glutamate-sensitive specific activities among the brain regions tested. The results indicate that GAD is responsible for a significant proportion of the total CSD activity in porcine brain.     

小麦谷氨酸脱羧酶的纯化及部分性质研究

谷氨酸脱羧酶（ｇｌｕｔａｍａｔｅｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）催化谷氨酸脱羧生成γ－氨基丁酸（γ－ａｍｉｎｏｂｕｔｙｒａｔｅ,ＢＡ）,植物中已从南瓜［１］、马铃薯和林生山黧豆［２］纯化了ＧＡＤ．ＧＡＤ活性在禾本科作物中作为...     

Heteromers of Glutamate Decarboxylase Isoforms Occur in Rat Cerebellum

Abstract: The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD₆₅ and GAD₆₇). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising <3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD₆₅ and GAD₆₇, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₇ was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₅ remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD₆₅ and GAD₆₇ even after extensive washing. These results are consistent with the presence of heteromultimers of GAD₆₅ and GAD₆₇ in cerebellum in addition to homomers of each form.     

Taurine Biosynthesis in Rat Brain In Vivo: Lack of Relationship with Cysteine Sulfinate Decarboxylase Glutamate Decarboxylase-Associated Activity (GAD/CSDII)

Two distinct forms of cysteine sulfinate decarboxylase (CSD), respectively, CSDI and CSDII, have already been separated in rat brain. One of them, CSDII, appeared to be closely associated with glutamate decarboxylase (GAD). We have investigated whether the taurine concentration in brain was dependent on CSDII activity in vivo. CSDI and CSDII activities were specifically measured in crude brain extracts after selective immunotrapping. After 4 days of chronic treatment of mice with gamma-acetylenic gamma-aminobutyric acid, a drastic and identical decrease in CSDII and GAD activities was observed in the brain. Taurine concentration and CSDI activities were not significantly altered. Following striato-nigral pathway lesioning in the rat brain, GAD and CSDII show an identical 80% decrease in the substantia nigra. In contrast, CSDI activity and taurine concentration in the substantia nigra were similarly but only slightly affected with an about 30% decrease. Our results provide further evidence that GAD and CSDII are indeed the same enzyme. They show that CSDII does not play any role in the biosynthesis of taurine in vivo. Our findings suggest that CSDI might be the biosynthetic enzyme for taurine in vivo and that there might be some endings projecting into the substantia nigra that contain CSDI and taurine.     

Inhibition of Brain Glutamate Decarboxylase Activity Is Related to Febrile Seizures in Rat Pups

Because previous work showed that in the newborn brain, but not in the adult brain, glutamate decarboxylase (GAD) is notably susceptible to heat, we have studied the possible involvement of GAD inhibition in febrile convulsions and the related changes in gamma-aminobutyric acid (GABA) content. Rats of different ages were subjected to hyperthermia, and GAD activity was determined in brain homogenates by measuring the release of 14CO2 from labeled glutamate and by measuring the formation of GABA. The latter method gave considerably lower values than the former in the youngest rats, and was considered more reliable. With this method, we found a 37-48% inhibition of GAD activity in rat pups 2-5 days old, which showed febrile seizures at progressively higher body temperatures, whereas in 10- and 15-day-old animals, which did not show convulsions, GAD activity was not affected by hyperthermia. Whole-brain GABA levels, however, did not change at any age. In contrast to GAD, choline acetyltransferase and lactic dehydrogenase activities were not altered by hyperthermia at any of the ages studied. These results suggest that a decreased efficiency of the inhibitory neurotransmission mediated by GABA, consequent to the inhibition of GAD activity, may be a factor related to febrile convulsions.     

Distribution of γ-Aminobutyric Acid and Glutamate Decarboxylase in the Layers of Rat Oviduct

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20-microns thick freeze-dried sections (0.2-1.5 micrograms dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10-fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.     

High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate

We examined the effect of the pyridoxal 5'-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40 degrees C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40 degrees C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60 degrees C.