Electrophoresis of glutamic acid decarboxylase (EC 4.1.1.15) from mouse brain in sodium dodecyl sulphate polyacrylamide gels

Electrophoretically and ultracentrifugally homogeneous glutamic acid decarboxylase purified from mouse brain showed multiple protein bands after electrophoresis in SDS polyacrylamide gel. The positions and intensities of the multiple bands were constant despite different treatments of the enzyme with various concentrations of SDS, β-mercaptoethanol, and urea at different temperatures. The major band had an apparent molecular weight of approximately 60,000 daltons and there were three minor bands of molecular weights, about 120,000, 90,000, and 75,000 daltons, respectively. The molecular weights of almost all bands were approximately integral multiples of 15,000. The possible subunit structure of this enzyme has been discussed in the light of the latter data and data previously reported from ultracentrifugation and gel filtration studies. We suggest that this enzyme may be a hexamer consisting of 15,000-dalton sub-units and that dissociation of these sub-units in SDS is accompanied by reassociation into a variety of aggregates, the probability of whose formation is determined by structural features that are more important than the differences encountered under the environmental conditions employed in these studies.     

Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15).

Abstract— The activity of L–glutamate decarboxylase (EC 4.1.1.15) (GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO₂ method, column separation, electro–phoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmol/min/mg protein (100%), followed by heart (7.4%), kidney (6.3%) and liver (1.5%). Measurement of brain GAD using the radiometric CO₂ assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the CO₂ method were about 3–4 times higher than those obtained by the GABA method, suggesting that the CO₂ method does not give a valid measurement of GAD activity in a crude non–neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland, or spleen. GAD from brain, spinal cord, heart, kidney and liver were further compared by double immunodiffusion, enzyme inhibition by antibody, and microcomplement fixation using antibody against GAD purified from mouse brain. GAD from brain and spinal cord appear to be identical as judged from the following results: the immunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti–GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in non–neural tissues is different from that in brain and spinal cord.     

Monoclonal antibodies against rat brain glutamic acid decarboxylase (GAD)

Monoclonal antibodies against rat brain GAD have been produced and immunochemically characterized in comparison with a traditional anti-GAD antiserum (Oertel et al., Neuroscience6, 2689–2700, 1981). An immunopurified fraction in which GAD represented an estimated 5% of the total protein was used as immunogen. Out of 10 mice injected with this fraction, 6 appeared to be immunized: their sera immunoprecipitated quantitatively GAD activity. Three cell fusions were performed between spleen cells of the best immunized mice and SP2/OAg14 myeloma cells. Around 500 hybridoma were generated in each hybridization experiment. The culture medium of 13 hybridoma significantly trapped GAD activity. All immunoprecipitation curves established with the ascitic fluid obtained from the positive hybridoma, showed a lower titer, at least 50-fold, than the titer of the conventional antiserum. None of these ascitic fluids was able to stain directly any protein from a rat high speed supernatant after western blotting. However, the electrophoretical analysis of the proteins immunotrapped by any of the monoclonal antibodies, followed by western blotting and immunolabelling with the anti-GAD antiserum (“cross-immunoblotting”) showed the same two stained monomers. They have the same molecular weight (respectively 59 and 62 kDa ± 2 kDa) as those stained directly by the anti-GAD antiserum from a rat brain supernatant. Although all monoclonal antibodies showed a lower affinity then the conventional antiserum, which prevents them from being used directly in immunoblotting they permit to definitively establish that the two monomers immunolabelled by the conventional antiserum are constitutive subunits of the rat brain GAD.     

Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.     

Activity-dependent cleavage of brain glutamic acid decarboxylase 65 by calpain

Previously, we reported that l-glutamic acid decarboxylase isoform 65 (GAD65) could be cleaved in vitro to release a stable truncated form which lacks amino acid 1-69 from the N-terminus, GAD65(Delta1-69). However, whether such a truncated form is also present under certain physiological conditions remains elusive. In the present study, we showed that, upon sustained neuronal stimulation, GAD65 could be cleaved into a truncated form in a rat synaptosomal preparation. This truncated form had similar electrophoretic mobility to purified recombinant human GAD65(Delta1-69). Furthermore, we demonstrated that this conversion was calcium dependent. Calcium-chelating reagents such as EDTA and 1,2-bis-(o-aminphenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetra-acetoxy-methyl ester prevented the cleavage of GAD65. In addition, our data suggested that calpain, a calcium-dependent cysteine protease, is activated upon neuronal stimulation and could be responsible for the conversion of full-length GAD65 to truncated GAD65 in the brain. Moreover, calpain inhibitors such as calpain inhibitor I or calpastatin could block the cleavage. Results of our in vitro cleavage assay using purified calpain and immunopurified rat GAD65 also supported the idea that GAD65 could be directly cleaved by calpain.     

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.     

谷氨酸脱羧酶研究进展

谷氨酸脱羧酶(glutamic acid decarboxylase,GAD,EC4.1.1.15)在生物体内广泛存在,其催化产物γ-氨基丁酸(γ-aminobutyric acid,GABA)是哺乳动物体内一种重要的抑制性神经递质。在对自身免疫性疾病以及糖尿病研究中,特别是1型糖尿病,GAD、GABA以及谷氨酸脱羧酶抗体(glutamic acid decarboxylase-antibody,GAD-Ab)等的水平作为病理分析、疾病诊断、免疫治疗的重要参数,历来备受研究者关注。本文就GAD及其催化产物GABA的研究进展进行了综述,为更好地研究自身免疫性疾病的发病机理,探索更加有效安全的治疗方法提供参考。     

Inhibitors of crayfish glutamic acid decarboxylase

Crayfish glutamic acid decarboxylase (GAD), like the homologous enzymes from other species, is inhibited by carbonyl-trapping agents (e.g. aminooxyacetic acid; AOAA) and sulfhydryl reagents (e.g. 5,5-dithiobis-(2-nitrobenzoic acid); DTNB). It also is inhibited by the product GABA, many anions (e.g. SCN^– and Cl^–), and some cations (e.g. Zn⁺²). The inhibition by AOAA, but not that by DTNB, was prevented by increasing the concentration of the pyridoxal phosphate (PLP) coenzyme. GABA blocked the effects of PLP on enzyme activity. The inhibition by AOAA, DTNB, GABA, and chloride all were competitive with substrate. The effect of GABA occurs at physiological concentrations and may contribute to the regulation of GAD activity in vivo. The quantitative effect of anions is dependent on the cation with which they are administered. ATP stimulated GAD activity in homogenates prepared with potassium phosphate or Tris-acetate buffer, even when no exogenous PLP was provided.     

The activity of glutamic acid decarboxylase from intact wheat roots

Experiments with isolated wheat roots and with intact wheat plants showed that glutamic acid decarboxylase from the roots takes part in the transformation of substance in the medium and that its activity is influenced by the medium. Glutamic acid decarboxylase is thus a factor taking part in the formation of substances excreted by the plants roots.     

Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications

Previously, we reported that protein phosphorylation plays an important role in regulating soluble l-glutamic acid decarboxylase (GAD) [Bao, J. (1995) J. Biol. Chem. 270, 6464-6467] and membrane-associated GAD activity [Hsu, C. C. (1999) J. Biol. Chem. 274, 24366-24371]. Here, we report the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrate that protein kinase A (PKA) and protein kinase C isoform epsilon are the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. Direct phosphorylation of GAD65 and GAD67 was demonstrated by incorporation of [(32)P] from [gamma-(32)P]ATP into purified GAD65 and GAD67 and immunoblotting assay using anti-phosphoserine/threonine antibodies. We have identified one specific phosphorylation site, threonine 91 (T91), in hGAD67 that can be phosphorylated by PKA using MALDI-TOF. Site-directed mutation of T91 to alanine abolished PKA-mediated phosphorylation and inhibition of GAD activity. Furthermore, mutation of T91 to aspartic acid or glutamic acid mimics the effect of phosphorylation. A model depicting the effect of phosphorylation on GAD activity upon neuronal stimulation is also proposed.     

Cloning and partial nucleotide sequence of human glutamic acid decarboxylase cDNA from brain and pancreatic islets

We report partial nucleotide sequences of the human enzyme glutamic acid decarboxylase (GAD) from brain and pancreatic islets which encode the middle 180 amino acids of GAD. The brain and islet GAD sequences display a high degree of sequence homology with the equivalent region of other mammalian brain GAD cDNAs. Alignment of the brain and islet GAD sequences showed that there were 45 nucleotide differences which, at the translational level, would result in seven amino acid substitutions. These results which suggest that different isomeric forms of human GAD exist in brain and pancreas may be relevant to the pathogenesis of stiff man syndrome (SMS) and insulin-dependent diabetes mellitus (IDDM), respectively, two distinct but associated clinical disorders in which GAD is the target of autoantibodies.     

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mouse-hamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 (Gad-1) and that an apparent pseudogene locus is on chromosome 10 (Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.     

谷氨酸脱羧酶若干研究进展

谷氨酸脱羧酶是γ－氨基丁酸的合成酶,主要存在脑和胰岛中。因体内存在多种形成的谷氨酸脱羧酶,现无获得各种均一的谷氨酸脱羧酶的 统一方法。谷氨酸脱羧酶的克隆和表达,既弄清了谷氨酸脱羧酶的基因结构与定位,又为谷氨酸脱酶的大规模应用奠定了基础。目前认为谷氨酸脱羧酶是Ⅰ型糖尿病的始动靶抗原,体内注入谷氨酸脱羧酶可预防或延缓ＮＯＤ（ｎｏｎｏｂｅｓｅ ｄｉａｂｅｔｉｃ）小鼠Ⅰ型糖尿病的发生。