Immunoaffinity purification of l -glutamate decarboxylase

Neurochem. Int. Vol. 17, No. 3, pp. 449-455, 1990 Printed in Great Britain. All rights reserved

019%0186/9053.00+0.00 Copyright ~ 1990 Pergamon Press plc

I M M U N O A F F I N I T Y PURIFICATION OF L-GLUTAMATE DECARBOXYLASE JANG-YEN WU 1'2'3'*, J. Y. LIU ~, C. T. LIN 1'3, DIANE EVANS j a n d W. H. TSAI 3 ~Neuroscience Program and Department of Physiology and Anatomy, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033, 2Department of Physiology and Cell Biology, University of Kansas, Lawrence, KS 66045-2106, U.S.A. and 31nstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China (Received 13 November 1989; accepted 5 February 1990) A b s t r a c ~ A rapid and efficient immunoaffinity procedure for the purification of a new form of brain Lglutamate decarboxylase (GAD) is described. A well characterized monoclonal antibody against rat brain GAD is used as an affinity ligand. The GAD-anti-GAD complex is dissociated by a relatively gentle condition e.g. 0.2 M acetate buffer, pH 4 or 5. GAD preparations thus obtained are still enzymatically active and have a minimum molecular weight of 67,000 Da. The significance of this new form of GAD is also discussed.

7 - A m i n o b u t y r i c acid ( G A B A ) has been established as a m a j o r inhibitory n e u r o t r a n s m i t t e r in the vertebrate a n d invertebrate nervous system (Kravitz, 1967; Roberts, 1975; Snyder, 1975; Wu, 1983). T h e ratelimiting step in G A B A biosynthesis is the decarboxylation o f L-glutamate by L-glutamate decarboxylase ( G A D ; L-glutamate 1-carboxy-lyase, E C 4.1.1.15) ( R o b e r t s a n d K u r i y a m a , 1968). Several lines of evidence, including developmental a n d kinetic studies, have suggested the existence o f multiple forms o f G A D ( W o n g et al., 1974; Spink et al., 1983 ; D e n n e r and Wu, 1985). Recent advances in h y b r i d o m a techniques have allowed one to develop specific m o n o clonal a n t i b o d y for various related proteins such as isozymes. T h e specific m o n o c l o n a l a n t i b o d y can then be used as a n affinity ligand for a n efficient purification of the respective antigen. In this c o m m u n i c a t i o n we describe the isolation a n d purification o f a new form o f G A D from m o u s e a n d rat b r a i n by the a p p r o a c h described above, namely, immunoaffinity c o l u m n c h r o m a t o g r a p h y using m o n o c l o n a l a n t i - G A D as affinity ligand.

EXPERIMENTAL PROCEDURES

Preparation o f L-glutamate decarboxylase GAD was isolated and purified from rat and mouse brain as previously described (Wu, 1976 ; Denner et al., 1987). The purification procedures included the extraction of GAD by homogenizing brain in H20 containing l mM 2-aminoethylisothiouronium bromide hydrobromide (AET) and 0.2 mM pyridoxal phosphate (PLP), followed by high speed centrifugation (100,000 g) to remove the cell debris and membrane components. The crude extract thus obtained was further purified through a series of column chromatographies including DEAE-cellulose, hydroxylapatite and gel filtration as described (Wu, 1976; Denner et al., 1987). Assay.for L-glutamate decarboxylase GAD was assayed by a radiometric method as previously described (Denner and Wu, 1985). Disposable culture tubes, 15 × 85 mm, containing 10 ~1 L-[1-~4C] glutamic acid (15 /~Ci/ml, 40 mM sodium glutamate in 0.1 M potassium phosphate, pH 7.0) and 90/A of 50 mM potassium phosphate buffer, pH 7.2, containing 1 mM AET, 0.2 mM PLP and 1 mM EDTA (standard GAD buffer) were capped with rubber stoppers holding center wells containing 0.2 ml hyamine hydroxide. The reaction was started by injecting 100 #1 enzyme solution in standard GAD buffer into the tube. After a 30 min incubation at 3WC in a shaking water bath, 0.2 ml of 0.5 N H2SO4 was injected through the stopper to terminate the reaction. Tubes were shaken for additional 60 rain at 37'C to allow a complete evolution and absorption of ~4CO~ in hyamine. Center wells were then transferred to vials containing 5 ml Econofluor (NEN Research Products) and ~4COE was determined in a liquid scintillation counter.

*Address all correspondence and reprint requests to: Dr Jang-Yen Wu, Department of Physiology and Cell Biology, University of Kansas, 3038 Haworth, Lawrence, KS 66045-2106, U.S.A. Abbreviations: GAD, L-glutamate decarboxylase ; AET, 2- Protein assay aminoethylisothiouronium bromide hydrobromide; Protein was assayed by the protein~zlye binding method PLP, pyridoxal phosphate ; GABA, 7-aminobutyric acid ; (Bradford, 1976). Standards containing 1 10/~g of bovine DEAE, diethylaminoethyl. serum albumin (BSA) were included in each determination. 449

450

JANG-YEN Wu el ~11.

Ten itg gave an absorbance change of approx. 0.21 O.D. units at 595 nm. Monoc/onal antiho~tl' production Monoclonal antibody was prepared using the same procedures as previously described (K6hler and Milstein, 1975 : Wu el al., 1986). A n t i - G A D producing clones were identified by ELISA test as well as immunoprecipitation test. Only the clones which were positive in both tests were subcloned and the anti-GAD producing single clone was then injected into the peritoneal cavity of immunosuppressed mice for the production of aseites fluid. The ascites fluid was further screened by ELISA, immunoprecipitation and Western immunoblotting tests as previously described (Wu el al., 1986). Enz)'me-lfl~ked intmunoadsorbant assal ( E L I S A ) Aliquots of 50/d of partially purified G A D preparations (after DEAE cellulose, hydroxylapatite and gel filtration column, purity ~ 15%) containing 100 ng of G A D were added to microtiter wells and incubated with constant agitation at room temperature overnight (Wu et al., 1986). Wells were washed three times with 200 #1 of buffer which consisted o f t 5 m M sodium phosphate, 140 m M sodium chloride, pH 7.4 (PBS), 0.05% Tween-20 and 0.1% BSA. Fifty td of either culture medium, or I : 100 diluted ascites fluid was added and incubated for 2 h. The wells were washed three times. After an additional 2 h incubation with peroxidase conjugated rabbit anti-mouse lgG (Miles Yeda) diluted I : 200 with the same wash buffer, wells were washed three more times and treated with 100 itl ofsubstrate [0.05% 2,2'-azino-di-(3-ethylbenzothiazolin-sulfonate) (ABTS), 0.1 M citric acid, pH 4.2, 0.02% hydrogen peroxide] for 15 min at room temperature. The reaction was terminated by adding 40 /~1 0.0006% sodium azide. ~Teslern #7,tmunoblotting test About 20 #g of crude G A D preparation was applied to a 10% SDS polyacrylamide gel (SDS~PAGE), electrophoresed for 4 h at 20 mA, and then transferred from SDS P A G E to nitrocellulose sheet as previously described (Towbin et al., 1979: Wu e/ al., 1986). The nitrocellulose sheet was then treated with 3% BSA followed by rinsing in 0.01 M PBS containing 0.2% BSA and 0.05% Tween 20. The nitrocellulose strips were then immersed in 0.02 mg/ml o f m o n o clonal anti-GAD lgG for 2 h at room temperature. For the control experiments, the same a m o u n t of lgG isolated from control ascites fluid was used instead of anti-GAD lgG. After brief rinsing in the same buffer, the strips were further incubated with biotinylated goat anti-mouse IgG and ABC solution (Avidin biotin complex) as described (Hsu et al., 1981). followed by a brief incubation with peroxidase substrate, diaminobenzidine and H_~O> Protein markers included phosphorylase b, 94,000 Da : BSA, 67,000 Da ; ovalbumin, 43,000 Da ; carbonic anhydrase, 30,000 Da ; soybean trypsin inhibitor, 20,000 Da and :~-lactalbumin, [4,000 Da. hHmt#7oprecipitu/ion test G A D anti-GAD complexes were precipitated using the heat-killed, formalin-fixed Staphylococcus aureus Cowan Type I (SAC) (Bethesda Res. Lab.) procedure (Kessler, 1975). Unless stated otherwise, all dilutions were made with, and pellets resuspended in, PBS containing 1 m M AET, 0.2 mM PLP, I m M EDTA, pH 7.0. Eighty /~1 of partially purified G A D preparation was incubated for 60 h at 4 ' C

with an equal volume of either anti-GAD lgG or control mouse lgG fractions (10 mg/ml). Fifteen Ill of SAC was added to each microfuge tube and samples were incubated at room temperature with agitation for 30 min, followed by brief centrifugation. Supernatants and pellets were then assayed for G A D activity. Prepar~llio~l ~/ inlmuHoaffTnil )' ~tel About 160 mg of monoclonal anti-GAD IgG ~ purified by Affi-Gel Protein A (Bio-Rad, Richmond, Calif.) affinity column chromatography in 12 ml 0.1 M morpholinopropanesulfonic acid (MOPS), pH 7.5 was combined with I0 ml of Affi-Gel 10 (Bio-Rad). The slurry was gently mixed at 4 C for 2 h, followed by extensive washing with 7 M urea containing 1 M NaCI to remove uncoupled proteins and linally equilibrated with standard G A D buffer prior to the addition of crude G A D sample.

hTtmunoq[~'nily pur(/ieation q~ L-,qlutamate decarho.vy/ase About 3 ml of concentrated partially purified G A D preparation (20 mg/ml) was added to the well equilibrated AffiGel 10 anti-GAD lgG suspension and the mixture was incubated for 96 h at 4 C with gentle mixing. The entire mixture was then packed to a column 1,5 × 15 cm. The column was first washed with standard G A D bufl'er until the absorbance of the elute had reached the baseline. The column was further eluted with 0.2 M acetate buffer, pH 4, until no more protein was eluted out.

RESULTS ELISA

test

In the initial s c r e e n i n g , a m o n g 960 wells 48 wells s h o w e d a p o s i t i v e r e a c t i o n in E L I S A test u s i n g p a r tially purified G A D as a n t i g e n ( d a t a n o t s h o w n ) . F o u r o f t h e s e revealed p o s i t i v e r e a c t i o n s in b o t h E L I S A a n d i m m u n o p r e c i p i t a t i o n tests (see below) a n d were f u r t h e r c l o n e d by l i m i t i n g d i l u t i o n . F o l l o w i n g s c r e e n i n g a n d s u b c l o n i n g , t w o o f t h e single c l o n e s # 6 a n d # 12 were u s e d for ascites fluid p r o d u c t i o n . T h e ascites fluids t h u s o b t a i n e d were f u r t h e r c h a r a c t e r i z e d by i m m u n o p r e c i p i t a t i o n a n d i m m u n o b l o t t i n g tests as d e s c r i b e d below. hnmunoprecipitation

test

When the amount of GAD, anti-GAD lgG and S A C w a s k e p t c o n s t a n t , G A D activity in t h e s u p e r n a t a n t ( A ) d e c r e a s e d as a f u n c t i o n o f i n c u b a t i o n t i m e with a c o n c o m m i t a n t i n c r e a s e in G A D activity in t h e pcllct ( O ) (Fig. I). N o c h a n g e o f G A D activity in t h e pellet w a s o b s e r v e d a f t e r 60 h o f i n c u b a t i o n . A b o u t 4 5 % o f G A D activity w a s r e c o v e r e d in t h e f o r m o f GAD anti-GAD complex. 14"estern i m m u n o b l o t t i n . q test W h e n a c r u d e rat b r a i n G A D p r e p a r a t i o n w a s a n a l y z e d by S D S P A G E , m a n y p r o t e i n b a n d s were

Immunoaffinity purification of GAD

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Fig. 1. Immunoprecipitation of GAD with anti-GAD IgG. Eighty #1 of partially purified GAD (about 10% pure, 2 mg/ml) was incubated for 60 h at 4~'C with an equal volume of anti-GAD IgG. The immune complexes were then precipitated with SAC as described in Experimental Procedures. GAD activity after immunoprecipitation was measured in the supernatant (A) and the pellet (O). GAD activity expressed as % of control which was the GAD activity in the presence of normal mouse IgG was plotted as a function of incubation time.

stained with Amido Black (Fig. 2, lanes 2 and 3). A similar protein pattern was obtained after the gel was transferred to nitrocellulose sheet. However, only one protein band corresponding to a molecular weight of 67,000 Da was stained with monoclonal a n t i - G A D IgG (Fig. 2, lane 5). A similar result was obtained with mouse brain G A D preparation (Fig. 2, lane 6) suggesting a high degree of specificity of monoclonal anti-GAD. N o protein band was stained in the control experiments where monoclonal a n t i - G A D IgG was replaced by the same amount of normal mouse IgG.

lmmunoaffinity purification of L-9lutamate decarboxylase by monochmal anti-GAD affinity column chromatoyraphy Since an excess amount of G A D was applied to the affinity column, a large G A D and protein peak appeared in the early wash which represents the excess and unbound G A D . G A D which was specifically bound to a n t i - G A D was dissociated from G A D - a n t i G A D complex by 0.2 M acetate buffer, p H 4, as shown in Fig. 3. Although the exposure of G A D to pH 4 condition was very brief, only 5-10 min, substantial inactivation of G A D activity did occur resulting in a poor recovery ( < 30%) of G A D activity. G A D antiG A D complex could also be dissociated by a milder condition such as 0.2 M acetate buffer, pH 5. U n d e r this mild condition, about 50 % of G A D activity could be recovered (Fig. 4). Similar results were obtained for both mouse and rat brain preparations. The immunoaffinity purified G A D still retained G A D activity

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Fig. 2. Immunoblotting test with monoclonal anti-GAD. About 10(~300 #g of crude GAD preparation was applied to 10% SDS PAGE and electrophoretically transferred to the nitrocellulose sheet. Lanes 1~, were stained with Amido Black and lanes 5 and 6 were treated with monoclonal antiGAD IgG followed by biotinylated goat anti-mouse IgG, ABC solution (Avidin biotin complex) and peroxidase substrates, diamino-benzidine (DAB) and H202. Lane 1, standard protein markers, phosphorylase B (94 kDa); bovine serum albumin (BSA) (67 kDa); ovalbumin (43 kDa) and carbonic anhydrase (30 kDa); lane 2, 300/~g of crude rat brain high speed (100,000 g) supernatant solution; lane 3, 100 /~g of rat brain GAD preparation after DE-cellulose column ; lane 4, 20 #g of BSA ; lane 5, the same as lane 3 except that the amount of protein applied was 50/~g ; lane 6, the same as lane 5 except that the protein applied was prepared from mouse brain.

and appeared to be homogenous as judged from SDS~ P A G E in which only a simple protein band corresponding to a molecular weight of 67,000 Da was obtained (Figs 5 and 6). The purification of G A D by immunoaffinity column procedure is summarized in Table 1. DISCUSSION Although G A D has been purified or highly purified from several species including mouse (Wu et al., 1973 ; Wu, 1976), rat (Blinderman et al., 1978; Denner et al., 1987), bovine (Wu, 1982), human (Blinderman et al., 1978) and catfish (Su et al., 1979), the purification procedures used in the present studies are different from those described previously. It is based on the

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Fig. 3. lmmunoafflnity purification of rat brain G A D by monoclonal antibody. About 3 ml of partially purified G A D from rat or mouse brain which had been purified through the DE cellulose column was incubated with about 10 ml of monoclonal anti-GAD IgG Affl-Gel 10 immunoafflnity gel for 4 days at 4 C with constant shaking. The incubation mixture was then packed to a 1.5 × 15 cm column and the effluent was collected. The column was eluted with 0.05 M potassium phosphate buffer containing l mM AET and 0.2 mM pyridoxal phosphate, pH 7.2 (standard buffer). The arrow indicates the start of elution with 0.2 M acetate buffer, pH 4. About 3 ml per fraction was collected in a tube containing l ml of 0.2 M potassium phosphate, pH 7.5 standard buffer to neutralize acetate buffer. Each fraction was assayed for G A D activity ( O - - - - O ) and OD at 280 nm ( A A).

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Fig. 4. Immunoaffinity purification of mouse brain G A D by monoclonal antibody. The conditions were the same as those described in Fig. 3 with the exception that mouse brain G A D instead of rat brain G A D was used and the specific elution was with 0.2 M acetate buffer, pH 5, instead of 0.2 M acetate buffel, pH 4. 452

Immunoaffinity purification of G A D

A

B

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A

Fig. 5. SDS polyacrylamide gel electrophoresis of immunoaffinity purified rat brain GAD. (A) About 50 ng of the purified rat brain G A D eluted by 0.2 M acetate buffer, pH 4 (see Fig. 3) was treated with 4% SDS and 0.1% 2-mercaptoethanol at 100°C for 3 min and then applied to 10% SDS-PAGE. The gel was stained with silver-staining solution. (B) The same as (A) except that the sample was the purified rat brain G A D eluted by 0.2 M acetate buffer, pH 5. (C) The original partially purified rat brain G A D sample that was applied to the immunoaffinity column. (D) Standard protein markers: phosphorylase b, 94 kDa; BSA, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa; ~-lactalbumin, 14 kDa.

453

B

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Fig. 6. SDS polyacrylamide gel electrophoresis of immunoaffinity purified mouse brain GAD. (A) The same as Fig. 5(A) except that the sample was prepared from mouse brain (see Fig. 4) instead of rat brain. (B) The original crude mouse brain G A D preparation that was applied to the immunoaffinity column. (C) The same standard proteins as in Fig. 5.

high degree o f specificity o f a n t i g e n - a n t i b o d y interaction, resulting in o n e - s t e p p u r i f i c a t i o n o f G A D . O n e o f the difficulties i n v o l v e d in i m m u n o a f f i n i t y purification o f G A D is to find a c o n d i t i o n t h a t is s t r o n g

Table 1. lmmunoal~nity purification of L-glutamate decarboxylase from rat brain

Sample (1) (2) (3) (4) (5)

Homogenate Supernatant DEAE cellulose Immunoaffinityt lmmunoaffinity~

Total activity (units)*

Total protein (mg)

4.0 2.7 0.8 0.21 0.38

2100 1050 54 0.12 0.12

Specific activity (units/rag) × 103 1.9 2.6 14.8 1750 3166

Purification of GAD was from 10 brains. * One unit ~ I /~molof product formed per min at 37"C under standard conditions. t 0.2 M acetate buffer, pH 4.0 was used to dissociate GAD anti-GAD. $ 0.2 M acetate buffer, pH 5.0 was used to dissociate GAD-anti-GAD.

Yield (%)

Purification (-fold)

100 68 20 5.2 9.5

l 1.4 7.8 921 1666

454

JANG-YEN WU el ~tl.

enough to dissociate antigen a n t i b o d y complex, yet is gentle enough to preserve G A D activity. This difficulty is minimized by using m o n o c l o n a l a n t i b o d y since the binding between antigen a n d m o n o c l o n a l a n t i b o d y is not as strong as those between antigen and polyclonal antibodies a n d hence the complex can be dissociated using relatively mild conditions such as 0.2 M sodium acetate at pH 4 or 5. A n o t h e r a d v a n t a g e of using m o n o c l o n a l a n t i b o d y as an affinity ligand is to separate closely related proteins such as isozymes. It has been s h o w n that G A D exists in multiple forms which differ in either molecular weight (Blinderman et al., 1978 ; W u et al., 1981 : D e n n e r et al., 1987), i m m u n o c h e m i c a l properties ( W o n g et al., 1974), h y d r o p h o b i c properties (Spink et a/.. 1983) or kinetic properties ( D e n n e r and Wu, 1985). G A D reported in this c o m m u n i c a t i o n differs from that we purified previously ( D e n n e r et al., 1987) in its molecular weight. The former has a m i n i m u m molecular weight of 67,000 Da whereas the latter appears to be consisted of two subunits of 40,000 and 80,000 Da. These results m a y suggest that the 67,000 D a species of G A D is unstable u n d e r the electrophoretic conditions used and hence is not detected. This is supported from the observation that less than 30% of G A D activity is actually recovered from the preparative gel ( D e n n e r et al., 1987). W h e t h e r this new form of G A D is related to the ~, [4 a n d ",, forms of G A D as reported by Spink et al. (1983) or the A and B forms as reported by D e n n e r a n d W u (1985) remains to be determined. Since G A B A receptor has also been shown to be present in multiple forms in the m a m malian brain (Levitan et al., 1988: M o n t p i e d et al., 1988), it will be of interest to determine the relation of various forms of G A D a n d G A B A receptors and to elucidate their roles in G A B A e r g i c transmission. Aeknowledgements-This work is supported by grants NS20978, EY05385 and NS20922 from the National Institutes of Health, U.S.A. and grant BNS-8820581 from the National Science Foundation, U.S.A. and by a grant from the National Science Council, Taiwan, Republic of China. REFERENCES

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