0022-3042 79 0701-016080?(M,O
of Neurochrmtstry Vol. 33, pp. 169 to 179 Pergnmon Press Ltd 1979. Printed in Great Britain 0 International Society lor Neurochemirtry Ltd
Journal
PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN Y. Y. THOMAS Su's3, J.-Y. Wuz and DOMINICM. K. LAM' Cullen Eye Institute and
* Department
of Cell Biology, Baylor College of Medicine, Houston, TX 77030, U.S.A.
(Received 20 September 1978. Accepted 29 January 1979) Abstract-L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7-15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 2000 and 90,000 f 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 f 2000, 40,000 k 5000 and 90,000 k 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 pg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 6&70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1 :1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.
ALTHOUGH y-aminobutyric acid (GABA) has been functional architecture of GABA pathways in the censhown to be a major inhibitory neurotransmitter in tral nervous system. both invertebrate and vertebrate nervous systems For the past several years, we have been studying (KUFFLER & EDWARDS, 1958; TAKEUCHI & TAKEUCHI,the GABA pathways in retinas of teleosts and other lower vertebrates using biochemical, autoradio1965; OBATA & TAKEDA, 1969), very few GABA synapses have been precisely localized in the verte- graphic and electrophysiological techniques (LAM, brate central nervous system. The enzyme involved 1972, 1975a, b ; LAM et al., 1978; MARC et al., 1978). in the synthesis of GABA, glutamic acid decarboxy- The immunocytochemical localization of GAD in lase (GAD), has been shown to be concentrated only these retinas is clearly another important method for in GABAergic neurons. Localization of this enzyme the elucidation of GABA pathways. Unfortunately, at cellular and subcellular levels is, therefore, an im- the antibody against GAD from mouse brain does portant step toward identification of GABAergic not cross-react with the GAD from fish brains and neurons. GAD has been purified to homogeneity from retinas (SAITOet al., 1974a). Thus, as a first step to mouse brain and its properties extensively character- obtain an antibody against GAD from teleost brains ized (WU et al., 1973; Wu & ROBERTS, 1974; Wu, and retinas, we have purified G A D from brains of 1976; Wu et al., 1978). Furthermore, a specific anti- the channel catfish, Ictalurus punctatus. In this paper, body against GAD has been obtained and employed the procedures for such purification and for the profor immunochemical studies and immunocytochemiduction of a specific antibody against the purified cal localization of GAD-containing neurons (SAITOet enzyme are presented. al., 1974a, b ; MCLAUCHLIN et al., 1974, 1975; SAITO, 1976). These studies suggest that such an approach may ultimately lead to a detailed description of the MATERIALS AND METHODS To whom correspondence should be addressed. Abbreviations used: GAD, L-glutamic acid decarboxylase; GABA, y-aminobutyric acid; PP, pyridoxal phosphate; AET, 2-aminoethylisothiouroniumbromide hydrobromide. 169
Materials. Brains of channel catfish (Ictalurus puncfatus) were purchased from Adams Fish Farm, Angleton, TX. Pyridoxal phosphate (PP), 2-aminoethylisothiouronium bromide hydrobromide (AET), glutathione (reduced form, GSH), hyamine solution, and standard proteins for molecular weight determination were obtained from Sigma
170
Y. Y. THOMAS Su, J.-Y. Wu and DOMINIC M. K. LAM
Chemical Co., St. Louis, MO. Ultra pure ammonium sulfate was obtained from Schwarz-Mann, Orangeburg, NY: EDTA was from Mallinckrodt, St. Louis, MO; potassium phosphate salts were from Fisher Scientific, Pittsburgh, PA. LKB Ultro-gel was purchased from LKB, Rockville, MD; DEAE Sephacel was from Pharmacia Fine Chemicals, Piscatoway, NJ; calcium phosphate gel and materials for polyacrylamide gel electrophoresis were obtained from Bio-Rad Laboratories, Richmond, CA. Uniformly I4C-labeled glutamic acid was obtained from New England Nuclear, Boston, MA. Glass distilled water was used for the preparation of all solutions. Enzjime ussuy. GAD was assayed by a sensitive and simple radiometric method as previously described (ROBERTS & SIMONSEN.1963; Wu, 1976). Uniformly I4C-labeled glutamic acid was introduced into a disposable culture tube that had been kept in an ice water bath. The tube was sealed with a serum rubber stopper which held a central well containing 0.2ml hyamine solution. The reaction was started by injecting 0.2 ml of enzyme solution in standard buffer (0.05 M-potassium phosphate, pH 7.2, containing protectors 0.1 mM-PP, 1 mM-AET, 1 mM-EDTA and 1 mM-GSH) into the culture tube. The tube was then incubated in a water bath for 30min at 37°C. The reaction was terminated by injecting 0.2 ml of 0.5 N - H , S O ~into the reaction mixture. The reaction mixture was furtber incubated for another 60 min to ensure the complete release of CO, and its absorption into the hyamine base. The I4CO, absorbed in hyamine was then counted in a liquid scintillation counter. Enzyme activity was expressed as pmol I4CO2 formed per min per mg of protein at 37°C. Protein determination. The protein concentration was determined either by a modified Lowry method (LOWRY et al., 1951:MILLFR,1959) or by a more sensitive Coomassie Blue method (BRADFORD, 1976). Bovine serum albumin was used as standard protein.
Prepurution of crude extract. Catfish brains were first weighed and then homogenized in about 10 times volume of ice-cold, double distilled water containing 1 mM-AET, 0.1 mM-PP, 1 mM-EDTA and 1 mM-GSH, pH 7.2. About 2 ml of this homogenate were saved and the rest was centrifuged at 100,000g for 45 min. Concentrated potassium phosphate buffer and protector solution, pH 7.2, were added to the supernatant (crude extract) to give each component a final concentration the same as that of the standard buffer. About 2ml of this solution were saved and the rest served as the starting material for further purification. All the operations were carried out at 4°C unless otherwise mentioned. Ammonium sulfate fractionation. Solid ammonium sulfate was added gradually to the well-stirred crude extract solution to give approx 40% saturation (243g/l.). The pH of the solution was maintained at 7.2 by gradual addition of 0.1 N-NH,OH during the addition of ammonium sulfate. After the addition of ammonium sulfate was completed, the solution was stirred for an additional 20 min and then centrifuged at 1 3 ,2 0 0 ~for 30 min. The pellet was saved and more ammonium sulfate was added to the supernatant to give 75% of saturation (245 g/l.). The solution was centrifuged as before and the pellets thus obtained were dissolved in a minimal volume of standard buffer. The solutions were dialyzed against a large volume of standard buffer to remove ammonium sulfate. All enzyme preparations were stored at -20°C unless otherwise mentioned. First geljltration chromatography. Ultro-gel AcA 34 was equilibrated with standard buffer and packed into a column of 2.5 x 100cm. About 15 ml of enzyme solution obtained from the 4&75% ammonium sulfate fractionation were applied to the column. The column was eluted with standard buffer at a flow rate of 26 ml,h and approx 12 ml per fraction (Fig. I ) were collected. Two peaks with GAD
FRACTION NUMBER
FIG. 1. Gel filtration of GAD from ammonium sulfate fractionation. Fractions of approx 12 ml were was in mg/ml, and enzyme activity (@-a was ) in collected. Protein concentration (A-A) pmol/min.
FRACTION NUMBER FIG. 2. Calcium phosphate gel chromatography of GAD from gel filtration. The column WQS first equilibrated and washed with 0.01 M-phosphate buffer (fractions 1-7) and then washed with 0.05 ~ . p h o s phate buffer (fractions 8-15). Fractions of 12 ml were collected during this washing period. A linear gradient from 0.05 M to 0.3 M-phosphate buffer was applied (fractions 16-76). When the gradient reached 0.3 M, the column was further washed with 0.3 M-phosphate buffer. Fractions of 6 ml were collected. Protein concentration (A-A) was in mg/ml and enzyme activity (&--o) was in pmol/min.
activity were obtained. The first peak appeared in the void volume, suggesting a high molecular species. Five batches of second peak fractions were pooled. Ammonium sulfate fractionation was carried out as before and the precipitate formed with the 4&75% of saturation was dissolved in and dialyzed against the standard buffer. Chromatography of calcium phosphate gel. The enzyme solution from the preceding step was dialyzed against 10 mM-potassium phosphate buffer, pH 7.2, containing 0.1 mM-PP, 1 mM-AET and 1 mM-GSH. The dialyzed enzyme solution was applied to a calcium phosphate gel column (2.5 x 15cm) which had been equilibrated with 1 mM-potassium phosphate buffer, pH 7.2, containing 0.1 mM-PP, 1 mM-AET and 1 mM-GSH. After the application of the sample, the column was washed with 1 column volume of 10 mM-potassium phosphate, followed by 1 column volume of 50 mM-potassium phosphate buffer. A linear gradient made from 140ml of 50mM and 140ml of 300 mM-potassium phosphate buffer, pH 7.2, was then applied to the column (Fig. 2). Three peaks with GAD activity were obtained. Since excess calcium phosphate was used, the enzyme activity started to appear at about 0.22 M and the peak fraction appeared at 0 . 2 5 ~ Fractions . with enzyme activity higher than 3.4 pmol/min were pooled and fractionated with ammonium sulfate as before for further purification. Second gel filtration chromatography. The enzyme solution from the preceding step was applied to and eluted from the same Ultro-gel column as described in Fig. 1. The elution profile is shown in Fig. 3. Only one peak with GAD activity was obtained. Fractions with enzyme activity higher than 7 pmol/min were pooled and concentrated with (NH,),SO, as described before. Polyacrylarnide gel electrophoresis and enzyme assay of gel sfices. Five per cent polyacrylamide slab gels, 0.127 x 15 x 15cm or 0.127 x 15 x I O c m , were pre-
pared. The electrophoresis running buffer contained 0.025 M-Tris, 0.192 M-glycine, 1 mM-AET, 1 mM-EDTA and 0.1 mM-PP, pH 8.3. Before application of the sample, the gel was prerun for at least 30min at a current of 1.0 mA/cm of gel. The running buffer used for the prerun contained 0.5 mM-mercaptoethanol in order to remove ammonium persulfate and other ions. Samples consisting of 50-250 pg of protein were prepared in 10% glycerol containing bromophenol blue to mark the front. Electrophoresis was carried out at 4°C at a constant current of 1.5 mA/cm of gel for about 5 h. After electrophoresis, the gel for protein staining was first fixed in 50% trichloracetic acid overnight (usually 40 min-2 h is enough) and then stained with 1% Coomassie Brilliant Blue in 7% acetic acid for 2 h. Destaining was carried out by diffusion in 7% acetic acid. The unstained gel for the enzyme assay was cut into 1 cm slices. Each slice was chopped into small pieces and incubated overnight in 0.2 ml standard buffer, pH 6.2. Enzyme activities were measured as described before. For preparative polyacrylamide gel electrophoresis, the procedures were exactly the same as those of analytical gel electrophoresis with the exception that a thicker gel, 0.38 cm, was used. The enzyme solution obtained from the second gel filtration step was applied to a preparative polyacrylamide gel. Electrophoresis was carried out at a constant current of 50 mA. After electrophoresis the gel slices containing GAD were chopped into small pieces and then incubated overnight in standard buffer at -20°C. The enzyme was extracted several times from the gel by homogenizing the gel in the standard buffer. The gel was removed by a brief centrifugation. Gradient polyacrylamide gel electrophoresis. A 3.7-1 5% polyacrylamide gel was prepared according to the method of LORENTZ(1976). The procedures were the same as those described for the regular polyacrylamide gel electrophoresis except that the running buffer consisted of
Y. Y. THOMAS Su, J.-Y. Wu and DOMINIC M. K. LAM
172
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0
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28
36
FRACTlON NUMBER FIG.3. Gel filtration of GAD from calcium phosphate gel. Fractions of 12 ml were collected. Protein concentration (A-A) was in mg/ml, and enzyme activity (@--O) was in pmol/min.
0.046 M-Tris, 0.192 M-glycine and protectors. The electrophoresis was carried out at 4°C at a constant voltage of 7 V/cm of gel for 15 h. Sodium dodecyl suqate (SDS) polyacrylamide gel electrophoresis. 12% SDS-polyacrylamide gel electrophoresis was carried out on 1.5 mm thick slab gels at 1.5 mA/cm, as described by LAEMMLI (1970). The samples of GAD or marker proteins were incubated at 100°C for 3min in 0.06 M-Tris buffer, pH 6.7, containing 0.104 SDS and 0.1% 2-mercaptoethanol. Electrophoresis was carried out at 4°C at 1.5 mA/cm for 3 h in a buffer containing 0.046 M-Tris, 0.192~-glycineand 0.1% SDS. Staining and destaining of the gel was as described above. Estimation of molecular weight. The molecular weight of GAD was estimated by gel filtration according to the method of ANDREWS(1964) and WHITAKER (1963) and by gradient polyacrylamide gel electrophoresis as described by LORENTZ (1976). The molecular weight of subunits was estimated by SDS-polyacrylamide gel electrophoresis as reported by LAEMMLI (1970). Ferritin, aldolase, ovalbumin, pepsin and bovine serum albumin were included as marker proteins. In gel electrophoresis, the relative mobility of each protein band was calculated in the following manner: Relative mobility
=
distance of protein migration length of gel after staining length of gel before staining distance of bromophenol blue migration
Production of G A D antiserum. The electrophoretically homogeneous enzyme was employed as an antigen. Antiserum was produced in a rabbit by biweekly injections into the infrascapular muscles of 20pg of the enzyme in complete Freund's adjuvant. A total of 80 pg of the purified GAD was used. The rabbit was bled through the ear vein
I week after the fourth injection. IgG from the GAD antiserum (anti-GAD IgG) and serum from the unimmunized rabbits (normal IgG) were prepared by DEAE-Sephacel column chromatography as described earlier (FAHEY, 1967). The anti-GAD IgG and normal IgG were stored at -20°C in small batches and thawed before use. Inhibition studies. The studies of the inhibition of enzyme activity by GAD antiserum were carried out with previously described procedures (SAITOet al., 19740). Inhibition of the enzyme by antiserum at different time intervals was measured as follows: 12 pg of GAD in 100 pl of the standard buffer were incubated at 0°C with 240 ml of antiserum (19.2 pg protein). The enzymatic activities of portions of the mixtures were measured after 18, 24, 48, 72, 96, 120, 144 and 168 h incubation. Inhibition of the enzyme by increasing amounts of antiserum was measured as follows: 100 pl of GAD (12 pg of protein) were incubated at 0°C for 4 days with 20 p1 (1.64 pg) to 240 pl (19.2 pg) of the antiserum. The standard buffer was added to make a final volume of 5002. In control experiments the same amount of unimmunized rabbit serum was used in place of the antiserum. In addition to the incubation mixture, the supernatant fluid and the precipitate obtained from the mixture by centrifugation at lOOOg for 30min were also used for the enzyme assay. The precipitates formed between the enzyme and the antiserum were washed 3 times with the standard buffer and then suspended in the same buffer before the assay.
RESULTS
The successive steps in the purification of G A D from 600 catfish brains are summarized in Table 1. The enzyme was partially inactivated during electrophoresis. The extent of enzyme inactivation was deter-
GEL SLICE NUMBER
2
0
I
I
I
4 I
I
I
10
8
6 1
1
I
1
I
I
FIG.4. 7.57;, polyacrylamide gel electrophoresis of G A D from gel filtration and G A D from preparative gel electrophoresis. The protein pattern of G A D from gel filtration is at the bottom and that extracted from preparative gel electrophoresis is at the center. About 5 0 ~ gof each sample were applied to the gel. Migration was from left to right. The enzyme activity measured in slices of a parallel gel was in terms of c.p.m./gel slice ( I cm).
173
DISTANCE MIGRATED
0
-
(cm)
2
4
6
8
I
I
I
I
FIG. 5. 3.7-15”/, gradient polyacrylamide gel electrophoresis of G A D from gel filtration and G A D from preparative gel electrophoresis. The protein pattern from gel filtration is at the top and that extracted from gel electrophoresis at the bottom. About 40pg of each sample were applied to the gel. Migration was from left to right. The enzyme activity measured in slices of B parallel gel was in terms of c.p.m./gel slice ( I cm). 174
MW
90000----
40000- - - -
22000- - - -
FIG.
6. SDS-polyacrylarnide gel electrophoresis of purified GAD. About 15 jig of purified GAD were
electrophoresed on a 12.5% SDS-polyacrylarnide gel. See text for details.
175
FIG.7. Ouchterlony double diffusion test of anti-GAD IgG from rabbit. Enzyme crude extract was placed in the center well. and wells a and f contained undiluted anti-GAD I& and unimmunized rabbit IgG. respectively. Wells b, c. d and e contained anti-GAD IgG at 2. 4. 8 and 16-fold dilutions. respectively.
176
L-Glutamic acid decarboxylase from catfish brain
177
TABLE1
Homogenate Supernatant 40-75%* Ultro-gel (4&75%)* Calcium phosphate (40-750/:,)* Ultro-gel (0-75%)* Gel extraction
Total volume (ml)
Total protein (mg)
600 550 60 22
6750 3052.5 976.2 264
12 6 8
Specific enzyme activity (pmol/mg/min) 103
23.6 10.8 1.12
Total enzyme activity (pmol/min)
4 5.8 29.6 64.6
27.0 17.7 17.6 17.0
454 826 2415t 4830$
9.1 8.9 2.7 5.4
* Fractionation with ammonium sulfate at per cent of saturation as indicated. t Specific activity actually obtained from gel extract without any correction. $ Specific activity calculated with the correction for the inactivation of the enzyme during electrophoresis.
mined to be about 50% by comparing the enzyme activity of equal amounts of GAD before and after electrophoresis. If the specific activity of the enzyme extracted from the gel was corrected for the inactivation, it represents about a 1200-fold purification over the original homogenate. The purified enzyme protected with AET, pyridoxal phosphate and reduced glutathione is stable at - 20°C in the dark for several months. Thawing and freezing cause a slight decrease in activity.
as demonstrated in imrnunodiffusion experiments in which a sharp, single precipitin band was obtained with GAD antiserum and crude enzyme preparations (Fig. 7). Furthermore, the precipitin band also contained GAD activity, while a comparable piece of gel did not show any enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex.
Criteria of purity
by
(a) Polyacrylamide gel electrophoresis of 50 pg of the purified enzyme revealed a single protein band with the location of the enzyme activity corresponding to the location of the protein band (Fig. 4). (b) On 3.7 to 15% gradient polyacrylamide gel electrophoresis, the purified enzyme preparation also migrated as a sharp, single protein band which contained all the enzyme activity (Fig. 5).
Inhibition test
The activity of the enzyme was partially inhibited incubating with the GAD antisera
loo
I
4t IW
Molecular weights of the native enzymes
The molecular weight of GAD was estimated by gel filtration to be 84,000 2000 and by gradient polyacrylamide gel electrophoresis as 90,000 4000, assuming that GAD was similar to the marker proteins-ferritin, aldolase, ovalbumin, pepsin and bovine serum albumin-as a globular protein.
+
Molecular weight of the subunits
GAD was treated with SDS and 2-mercaptoethanol prior to electrophoresis on the SDS-polyacrylamide gel system. Three major protein bands were obtained which correspond to molecular weights of 22,000 F 2000, 40,000 5000 and 90,000 5 6000 respectively (Fig. 6). Ouchterlony double immunodiflusion tests with G A D antiserum
Antibodies against the purified GAD appeared to be specific only to GAD, even in crude preparations,
TIME ( D A Y S ) FIG.8. Inhibition of GAD by the antiserum as a function of incubation time at 0°C. Twelve micrograms of GAD were incubated with 19.2 pg of antiserum. GAD activities were assayed after the incubation period. The percentage of inhibition was obtained by comparing the activity of the sample and that of control (GAD incubated with unimmunized rabbit serum).
178
Y. Y. THOMAS Su, J.-Y. Wu and DOMINIC M. K. LAM
homogenizing medium; secondly, the use of preparative gel electrophoresis as the last step of purification. In hypotonic medium, most of the soluble proteins were liberated into the supernatant. About 70% of GAD activity was obtained in the supernatant compared to about 25% obtained from the crude mitochondrial fraction in the mouse brain preparation using isotonic medium. A similar result with mouse brain has also been reported from this laboratory using hypotonic medium (Wu et al., 1976). Hence, the use of hypotonic solution provided a general and efficient method for extracting enzyme from brain. Preparative polyacrylamide gel electrophoresis provides a simple and easy method for protein purification. Two drawbacks are the partial enzyme inactivation during electrophoresis and low recovery of enzyme from the gel. However, this method is particularly useful when a small quantity of homogeneous 0 100 200 3 3 protein is needed, for instance, as an antigen for antiANTISERUM (11) body production. The enzyme preparation obtained FIG.9. Inhibition of GAD by increasing amounts of anti- by a combination of various column chromatoserum after 4 days incubation at 0°C. Twelve micrograms graphies and preparative gel electrophoresis appears to of GAD were incubated with 1.64-19.2pg of antiserum; be homogeneous. This is based on the following A-A, reaction mixture; 0---0, supernatant from observations: first, in polyacrylamide gel electroreaction mixture; M, precipitate obtained from the phoresis, which separates proteins according to their reaction mixture by brief centrifugation. charge and size, the purified enzyme preparation migrates as a single protein band which contains all GADj19.2pg GAD antiserum) at 0°C for varying the enzyme activity; secondly, in gradient polyacrylperiods of time (Fig. 8). The maximum inhibition, amide gel electrophoresis, which separates proteins 64%, was obtained after 4 days incubation. Inhibition according to their size only, the purified enzyme soluwas expressed as percentage of the activity of the tion also migrates as a single protein band which is enzyme which was incubated with the same amount coincident with the enzyme activity ; thirdly, the antiof serum from unimmunized rabbits. The enzyme ac- body against the purified enzyme preparation forms tivity was stable at 0°C during the course of incuba- a single and sharp precipitin band when it is tested tion. Serum from the unimmunized rabbits inhibited aganst a crude enzyme preparation in immunodiffuthe enzyme activity slightly (1&20%). The degree of sion tests. Furthermore, the precipitin band obtained inhibition of enzyme activity also varied as a function in immunodiffusion tests or the precipitate obtained of the amount of GAD antisera which were incubated in the enzyme inhibition study also contains GAD with a constant amount of GAD protein at 0°C for activity, suggesting that the complex is indeed a 4 days (Fig. 9). The maximum inhibition, 62%, which GAD-anti-GAD complex. It also suggests that the was similar to the results obtained in Fig. 8, was antibody is monospecific to GAD and the antigenobtained at a ratio of GAD protein/GAD antiserum purified GAD preparation-used for antibody proprotein of about 1 :1.6. When the enzyme activity was duction is homogeneous. The molecular weight of measured after the incubation mixtures had been cen- GAD estimated from gradient polyacrylamide gel trifuged, it was found that the activity in the superna- electrophoresis is somewhat higher than the one tant fluid decreased with the increase of GAD anti- obtained from gel filtration, possibly due to a partial sera and the opposite was true for the activity in the denaturation of protein molecules during electroprecipitate. In the presence of 19.2pg of GAD anti- phoresis as reflected in the partial inactivation of the serum, all the activity in the mixture was recovered enzyme activity. SDS-polyacrylamide gel electroin the precipitate. phoresis revealed three major proteins with molecular weights of 22,000 f 2000, 40,000 5000 and 90,000 6000. Since the native enzyme has a molecular weight around 80,0G+90,000, these three proteins DISCUSSION may represent a monomer, dimer, and tetramer if the The methods described in this paper for the purifi- smallest subunit has a molecular weight around cation of GAD from catfish brain are somewhat dif- 20,000. It is interesting that the mouse brain enzyme ferent from those previously employed for the purifi- also showed multiple protein bands on SDS-polyacrylcation of GAD from mouse brain (Wu et al., 1973; amide gel electrophoresis representing monomer, Wu, 1976). Two major modifications were made: first, dimer and higher degrees of aggregation. However, the use of hypotonic instead of isotonic solutions as more vigorous physical and chemicaI studies are
L-Glutamic acid decarboxylase from catfish brain
needed before the precise subunit structure of GAD can be established. Another interesting similarity between the mouse brain enzyme and catfish brain enzyme is that the enzyme preparations from the hypotonic medium show two enzyme activity peaks o n gel filtration column corresponding to molecular weights of higher than 200,000 and 84,000, respectively (Wu et al., 1976). The nature of the high molecular weight GAD, whether it is merely an aggregated form of the low mo1:cular weight GAD, a complex of the low molecular weight GAD and other macromolecules, or a new type of GAD, is now under invest iga t i on. Acknowledgements-This investigation was supported in part by the Retina Research Foundation (Houston), NIH grants EY 02423 and NS 13224 and a grant from Huntington's Chorea Foundation in memory of Mrs. RUTH BtRMAN.
REFERENCES ANDnEws P. (1964) Estimation of the molecular weight of proteins by Sephadex gel filtration. Biochem. J . 91, 222-233. BRADFORD M. M. (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Analyt. Biochern. 72, 248--254. FAHEYJ. L. (1967) Chromatographic separation of immunoglobulins. in Methods in Immunology and ImmunoC. A. & CHASEM. W., eds.), Vol chemistry (WILLIAMS I . pp. 321-332. Academic Press, New York. KUFFLER S. W. & EDWARDSc . (1958) Mechanism Of y-aminobutyric acid action and its relation t o synaptic inhibition. J . Neurophysiol. 21, 589-610. LAEMMLI U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. LAMD. M. K. (1972) Biosynthesis and content of y-aminobutyric acid in goldfish retina. J . Cell Biol. 54, 225-231. LAMD. M. K. (1975~)Biosynthesis of y-aminobutyric acid by isolated axons of cone horizontal cells in goldfish retina. Nature, Lond. 254, 345-347. LAMD. M. K. (1975b) Synaptic chemistry of identified cells in vertebrate retina. Cold Spring Harh. Symp. quant. Biol. 40, 571-579. .AM D. M. K., LASATERE. M. & NAKA K . 4 . (1978) y-Aminobutyric acid: a neurotransmitter candidate for cone horizontal cells in the catfish retina. Proc. natn. Acad. Sci., U S A . 75, 631G6313. .ORENTZ K. (1976) A simple polyacrylamide gradient gel preparation for estimating molecular weight. Analyt. Biochem. 76, 214-220.
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F A R R A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J . hiol. Chem. 193, 265-275. MARCR. E., STELLW. K., BOK D. & LAMD. M. K. (1978) GABA-ergic pathways in the goldfish retina. J . cornp. Neurol. 182, 221-246. MCLAUCHLIN B. J.. WOOD J. G., SAITOK., BARBERR., E. & Wu J.-Y. (1974) Fine-strucVAUGHNJ. E., ROBERTS tural localization of glutamate decarboxylase in synaptic terminals of rodent cerebellum. Brain Res. 76, 377-391. MCLAUGHLIN B. J.. WOOD J. G., SAITOK., ROBERTSE. & Wu J.-Y. (1975) Fine-structural localization of glutamate decarboxylase in developing axonal process and presynaptic terminals of rodent cerebellum. Brain Rex 85, 355-371. MILLERG . L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. OBATA K. & TAKEDA K. (1969) Release of y-aminobutyric acid into fourth ventricle induced by stimulation of cat cerebellum. J . Nrurochem. 16, 1043-1047. ROBERTSE. & SIMONSEN D. G. (1963) Some properties of L-glutamic decarboxylase in mouse brain. Biochern. Pharmac. 12, 113-134. SAITOK. (1976) Immunochemical studies of G A D and GABA-T, in G A B A in Nervous System Function (ROBERTS E., CHASET . N . & TOWER D. B., eds.) pp. 103-1 11. Raven Press, New York. SAITOK., Wu J.-Y., MATSUDAT. & ROBERTSE. (1974~) Immunochemical comparisons of vertebrate glutamic acid decarboxylase. Brain Res. 65, 277-285. E. SAITOK., BARBER R., WU J.-Y., MATSUDAT., ROBERTS & VAUGHN J. E. (1974h) Immunohistochemical localization of glutamate decarboxylase in rat cerebellum. Proc. natn. Acad. Sci., U.S.A. 71, 269-213. TAKEUCHI A. & TAKEUCHI N. (1965) A study of inhibitory action of y-aminobutyric acid on neuromuscular transmission in crayfish. J . Physiol., Lond. 177, 225-238. WHITAKER J. R. (1963) Determination of molecular weights of proteins by gel filtration on Sephadex. Analyt. Chem. 35, 1950-1953. Wu J.-Y. (1976) Purification, characterization, and kinetic studies of GAD and GABA-T from mouse brain, in G A B A in Nervous System Function (ROBERTS E., CHASE D. B., eds.) pp. 7-55. Raven Press, New T. N. & TOWER York. WU J.-Y. & ROBERTSE. (1974) Properties of brain L-glutamate decarboxylase: inhibition studies. J . Neurochem. 23, 159-767. WU J.-Y., MATSUDAT. & ROBERTSE. (1973) Purification and characterization of glutamate decarboxylase from mouse brain. J . hiol. Chem. 248, 3029-3034. Wu J.-Y., WONG E., SATOK., ROBERTSE. & SCHOUSBO~ A. (1976) Properties of L-glutamate decarboxylase from brains of adult and newborn mice. J . Neurochem. 27. 653-659. WU J.-Y., CHUDEO., WEIN J., ROBERTS E.. SAITOK . & WONG E. (1978) Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15) J . Neurochem. 30. 849-851.
of Neurochrmtstry Vol. 33, pp. 169 to 179 Pergnmon Press Ltd 1979. Printed in Great Britain 0 International Society lor Neurochemirtry Ltd
Journal
PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN Y. Y. THOMAS Su's3, J.-Y. Wuz and DOMINICM. K. LAM' Cullen Eye Institute and
* Department
of Cell Biology, Baylor College of Medicine, Houston, TX 77030, U.S.A.
(Received 20 September 1978. Accepted 29 January 1979) Abstract-L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7-15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 2000 and 90,000 f 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 f 2000, 40,000 k 5000 and 90,000 k 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 pg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 6&70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1 :1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.
ALTHOUGH y-aminobutyric acid (GABA) has been functional architecture of GABA pathways in the censhown to be a major inhibitory neurotransmitter in tral nervous system. both invertebrate and vertebrate nervous systems For the past several years, we have been studying (KUFFLER & EDWARDS, 1958; TAKEUCHI & TAKEUCHI,the GABA pathways in retinas of teleosts and other lower vertebrates using biochemical, autoradio1965; OBATA & TAKEDA, 1969), very few GABA synapses have been precisely localized in the verte- graphic and electrophysiological techniques (LAM, brate central nervous system. The enzyme involved 1972, 1975a, b ; LAM et al., 1978; MARC et al., 1978). in the synthesis of GABA, glutamic acid decarboxy- The immunocytochemical localization of GAD in lase (GAD), has been shown to be concentrated only these retinas is clearly another important method for in GABAergic neurons. Localization of this enzyme the elucidation of GABA pathways. Unfortunately, at cellular and subcellular levels is, therefore, an im- the antibody against GAD from mouse brain does portant step toward identification of GABAergic not cross-react with the GAD from fish brains and neurons. GAD has been purified to homogeneity from retinas (SAITOet al., 1974a). Thus, as a first step to mouse brain and its properties extensively character- obtain an antibody against GAD from teleost brains ized (WU et al., 1973; Wu & ROBERTS, 1974; Wu, and retinas, we have purified G A D from brains of 1976; Wu et al., 1978). Furthermore, a specific anti- the channel catfish, Ictalurus punctatus. In this paper, body against GAD has been obtained and employed the procedures for such purification and for the profor immunochemical studies and immunocytochemiduction of a specific antibody against the purified cal localization of GAD-containing neurons (SAITOet enzyme are presented. al., 1974a, b ; MCLAUCHLIN et al., 1974, 1975; SAITO, 1976). These studies suggest that such an approach may ultimately lead to a detailed description of the MATERIALS AND METHODS To whom correspondence should be addressed. Abbreviations used: GAD, L-glutamic acid decarboxylase; GABA, y-aminobutyric acid; PP, pyridoxal phosphate; AET, 2-aminoethylisothiouroniumbromide hydrobromide. 169
Materials. Brains of channel catfish (Ictalurus puncfatus) were purchased from Adams Fish Farm, Angleton, TX. Pyridoxal phosphate (PP), 2-aminoethylisothiouronium bromide hydrobromide (AET), glutathione (reduced form, GSH), hyamine solution, and standard proteins for molecular weight determination were obtained from Sigma
170
Y. Y. THOMAS Su, J.-Y. Wu and DOMINIC M. K. LAM
Chemical Co., St. Louis, MO. Ultra pure ammonium sulfate was obtained from Schwarz-Mann, Orangeburg, NY: EDTA was from Mallinckrodt, St. Louis, MO; potassium phosphate salts were from Fisher Scientific, Pittsburgh, PA. LKB Ultro-gel was purchased from LKB, Rockville, MD; DEAE Sephacel was from Pharmacia Fine Chemicals, Piscatoway, NJ; calcium phosphate gel and materials for polyacrylamide gel electrophoresis were obtained from Bio-Rad Laboratories, Richmond, CA. Uniformly I4C-labeled glutamic acid was obtained from New England Nuclear, Boston, MA. Glass distilled water was used for the preparation of all solutions. Enzjime ussuy. GAD was assayed by a sensitive and simple radiometric method as previously described (ROBERTS & SIMONSEN.1963; Wu, 1976). Uniformly I4C-labeled glutamic acid was introduced into a disposable culture tube that had been kept in an ice water bath. The tube was sealed with a serum rubber stopper which held a central well containing 0.2ml hyamine solution. The reaction was started by injecting 0.2 ml of enzyme solution in standard buffer (0.05 M-potassium phosphate, pH 7.2, containing protectors 0.1 mM-PP, 1 mM-AET, 1 mM-EDTA and 1 mM-GSH) into the culture tube. The tube was then incubated in a water bath for 30min at 37°C. The reaction was terminated by injecting 0.2 ml of 0.5 N - H , S O ~into the reaction mixture. The reaction mixture was furtber incubated for another 60 min to ensure the complete release of CO, and its absorption into the hyamine base. The I4CO, absorbed in hyamine was then counted in a liquid scintillation counter. Enzyme activity was expressed as pmol I4CO2 formed per min per mg of protein at 37°C. Protein determination. The protein concentration was determined either by a modified Lowry method (LOWRY et al., 1951:MILLFR,1959) or by a more sensitive Coomassie Blue method (BRADFORD, 1976). Bovine serum albumin was used as standard protein.
Prepurution of crude extract. Catfish brains were first weighed and then homogenized in about 10 times volume of ice-cold, double distilled water containing 1 mM-AET, 0.1 mM-PP, 1 mM-EDTA and 1 mM-GSH, pH 7.2. About 2 ml of this homogenate were saved and the rest was centrifuged at 100,000g for 45 min. Concentrated potassium phosphate buffer and protector solution, pH 7.2, were added to the supernatant (crude extract) to give each component a final concentration the same as that of the standard buffer. About 2ml of this solution were saved and the rest served as the starting material for further purification. All the operations were carried out at 4°C unless otherwise mentioned. Ammonium sulfate fractionation. Solid ammonium sulfate was added gradually to the well-stirred crude extract solution to give approx 40% saturation (243g/l.). The pH of the solution was maintained at 7.2 by gradual addition of 0.1 N-NH,OH during the addition of ammonium sulfate. After the addition of ammonium sulfate was completed, the solution was stirred for an additional 20 min and then centrifuged at 1 3 ,2 0 0 ~for 30 min. The pellet was saved and more ammonium sulfate was added to the supernatant to give 75% of saturation (245 g/l.). The solution was centrifuged as before and the pellets thus obtained were dissolved in a minimal volume of standard buffer. The solutions were dialyzed against a large volume of standard buffer to remove ammonium sulfate. All enzyme preparations were stored at -20°C unless otherwise mentioned. First geljltration chromatography. Ultro-gel AcA 34 was equilibrated with standard buffer and packed into a column of 2.5 x 100cm. About 15 ml of enzyme solution obtained from the 4&75% ammonium sulfate fractionation were applied to the column. The column was eluted with standard buffer at a flow rate of 26 ml,h and approx 12 ml per fraction (Fig. I ) were collected. Two peaks with GAD
FRACTION NUMBER
FIG. 1. Gel filtration of GAD from ammonium sulfate fractionation. Fractions of approx 12 ml were was in mg/ml, and enzyme activity (@-a was ) in collected. Protein concentration (A-A) pmol/min.
FRACTION NUMBER FIG. 2. Calcium phosphate gel chromatography of GAD from gel filtration. The column WQS first equilibrated and washed with 0.01 M-phosphate buffer (fractions 1-7) and then washed with 0.05 ~ . p h o s phate buffer (fractions 8-15). Fractions of 12 ml were collected during this washing period. A linear gradient from 0.05 M to 0.3 M-phosphate buffer was applied (fractions 16-76). When the gradient reached 0.3 M, the column was further washed with 0.3 M-phosphate buffer. Fractions of 6 ml were collected. Protein concentration (A-A) was in mg/ml and enzyme activity (&--o) was in pmol/min.
activity were obtained. The first peak appeared in the void volume, suggesting a high molecular species. Five batches of second peak fractions were pooled. Ammonium sulfate fractionation was carried out as before and the precipitate formed with the 4&75% of saturation was dissolved in and dialyzed against the standard buffer. Chromatography of calcium phosphate gel. The enzyme solution from the preceding step was dialyzed against 10 mM-potassium phosphate buffer, pH 7.2, containing 0.1 mM-PP, 1 mM-AET and 1 mM-GSH. The dialyzed enzyme solution was applied to a calcium phosphate gel column (2.5 x 15cm) which had been equilibrated with 1 mM-potassium phosphate buffer, pH 7.2, containing 0.1 mM-PP, 1 mM-AET and 1 mM-GSH. After the application of the sample, the column was washed with 1 column volume of 10 mM-potassium phosphate, followed by 1 column volume of 50 mM-potassium phosphate buffer. A linear gradient made from 140ml of 50mM and 140ml of 300 mM-potassium phosphate buffer, pH 7.2, was then applied to the column (Fig. 2). Three peaks with GAD activity were obtained. Since excess calcium phosphate was used, the enzyme activity started to appear at about 0.22 M and the peak fraction appeared at 0 . 2 5 ~ Fractions . with enzyme activity higher than 3.4 pmol/min were pooled and fractionated with ammonium sulfate as before for further purification. Second gel filtration chromatography. The enzyme solution from the preceding step was applied to and eluted from the same Ultro-gel column as described in Fig. 1. The elution profile is shown in Fig. 3. Only one peak with GAD activity was obtained. Fractions with enzyme activity higher than 7 pmol/min were pooled and concentrated with (NH,),SO, as described before. Polyacrylarnide gel electrophoresis and enzyme assay of gel sfices. Five per cent polyacrylamide slab gels, 0.127 x 15 x 15cm or 0.127 x 15 x I O c m , were pre-
pared. The electrophoresis running buffer contained 0.025 M-Tris, 0.192 M-glycine, 1 mM-AET, 1 mM-EDTA and 0.1 mM-PP, pH 8.3. Before application of the sample, the gel was prerun for at least 30min at a current of 1.0 mA/cm of gel. The running buffer used for the prerun contained 0.5 mM-mercaptoethanol in order to remove ammonium persulfate and other ions. Samples consisting of 50-250 pg of protein were prepared in 10% glycerol containing bromophenol blue to mark the front. Electrophoresis was carried out at 4°C at a constant current of 1.5 mA/cm of gel for about 5 h. After electrophoresis, the gel for protein staining was first fixed in 50% trichloracetic acid overnight (usually 40 min-2 h is enough) and then stained with 1% Coomassie Brilliant Blue in 7% acetic acid for 2 h. Destaining was carried out by diffusion in 7% acetic acid. The unstained gel for the enzyme assay was cut into 1 cm slices. Each slice was chopped into small pieces and incubated overnight in 0.2 ml standard buffer, pH 6.2. Enzyme activities were measured as described before. For preparative polyacrylamide gel electrophoresis, the procedures were exactly the same as those of analytical gel electrophoresis with the exception that a thicker gel, 0.38 cm, was used. The enzyme solution obtained from the second gel filtration step was applied to a preparative polyacrylamide gel. Electrophoresis was carried out at a constant current of 50 mA. After electrophoresis the gel slices containing GAD were chopped into small pieces and then incubated overnight in standard buffer at -20°C. The enzyme was extracted several times from the gel by homogenizing the gel in the standard buffer. The gel was removed by a brief centrifugation. Gradient polyacrylamide gel electrophoresis. A 3.7-1 5% polyacrylamide gel was prepared according to the method of LORENTZ(1976). The procedures were the same as those described for the regular polyacrylamide gel electrophoresis except that the running buffer consisted of
Y. Y. THOMAS Su, J.-Y. Wu and DOMINIC M. K. LAM
172
//
s '\ I \ I\
I I
\ \
I
\
I
I I J
0
\
\ \
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0
20
28
36
FRACTlON NUMBER FIG.3. Gel filtration of GAD from calcium phosphate gel. Fractions of 12 ml were collected. Protein concentration (A-A) was in mg/ml, and enzyme activity (@--O) was in pmol/min.
0.046 M-Tris, 0.192 M-glycine and protectors. The electrophoresis was carried out at 4°C at a constant voltage of 7 V/cm of gel for 15 h. Sodium dodecyl suqate (SDS) polyacrylamide gel electrophoresis. 12% SDS-polyacrylamide gel electrophoresis was carried out on 1.5 mm thick slab gels at 1.5 mA/cm, as described by LAEMMLI (1970). The samples of GAD or marker proteins were incubated at 100°C for 3min in 0.06 M-Tris buffer, pH 6.7, containing 0.104 SDS and 0.1% 2-mercaptoethanol. Electrophoresis was carried out at 4°C at 1.5 mA/cm for 3 h in a buffer containing 0.046 M-Tris, 0.192~-glycineand 0.1% SDS. Staining and destaining of the gel was as described above. Estimation of molecular weight. The molecular weight of GAD was estimated by gel filtration according to the method of ANDREWS(1964) and WHITAKER (1963) and by gradient polyacrylamide gel electrophoresis as described by LORENTZ (1976). The molecular weight of subunits was estimated by SDS-polyacrylamide gel electrophoresis as reported by LAEMMLI (1970). Ferritin, aldolase, ovalbumin, pepsin and bovine serum albumin were included as marker proteins. In gel electrophoresis, the relative mobility of each protein band was calculated in the following manner: Relative mobility
=
distance of protein migration length of gel after staining length of gel before staining distance of bromophenol blue migration
Production of G A D antiserum. The electrophoretically homogeneous enzyme was employed as an antigen. Antiserum was produced in a rabbit by biweekly injections into the infrascapular muscles of 20pg of the enzyme in complete Freund's adjuvant. A total of 80 pg of the purified GAD was used. The rabbit was bled through the ear vein
I week after the fourth injection. IgG from the GAD antiserum (anti-GAD IgG) and serum from the unimmunized rabbits (normal IgG) were prepared by DEAE-Sephacel column chromatography as described earlier (FAHEY, 1967). The anti-GAD IgG and normal IgG were stored at -20°C in small batches and thawed before use. Inhibition studies. The studies of the inhibition of enzyme activity by GAD antiserum were carried out with previously described procedures (SAITOet al., 19740). Inhibition of the enzyme by antiserum at different time intervals was measured as follows: 12 pg of GAD in 100 pl of the standard buffer were incubated at 0°C with 240 ml of antiserum (19.2 pg protein). The enzymatic activities of portions of the mixtures were measured after 18, 24, 48, 72, 96, 120, 144 and 168 h incubation. Inhibition of the enzyme by increasing amounts of antiserum was measured as follows: 100 pl of GAD (12 pg of protein) were incubated at 0°C for 4 days with 20 p1 (1.64 pg) to 240 pl (19.2 pg) of the antiserum. The standard buffer was added to make a final volume of 5002. In control experiments the same amount of unimmunized rabbit serum was used in place of the antiserum. In addition to the incubation mixture, the supernatant fluid and the precipitate obtained from the mixture by centrifugation at lOOOg for 30min were also used for the enzyme assay. The precipitates formed between the enzyme and the antiserum were washed 3 times with the standard buffer and then suspended in the same buffer before the assay.
RESULTS
The successive steps in the purification of G A D from 600 catfish brains are summarized in Table 1. The enzyme was partially inactivated during electrophoresis. The extent of enzyme inactivation was deter-
GEL SLICE NUMBER
2
0
I
I
I
4 I
I
I
10
8
6 1
1
I
1
I
I
FIG.4. 7.57;, polyacrylamide gel electrophoresis of G A D from gel filtration and G A D from preparative gel electrophoresis. The protein pattern of G A D from gel filtration is at the bottom and that extracted from preparative gel electrophoresis is at the center. About 5 0 ~ gof each sample were applied to the gel. Migration was from left to right. The enzyme activity measured in slices of a parallel gel was in terms of c.p.m./gel slice ( I cm).
173
DISTANCE MIGRATED
0
-
(cm)
2
4
6
8
I
I
I
I
FIG. 5. 3.7-15”/, gradient polyacrylamide gel electrophoresis of G A D from gel filtration and G A D from preparative gel electrophoresis. The protein pattern from gel filtration is at the top and that extracted from gel electrophoresis at the bottom. About 40pg of each sample were applied to the gel. Migration was from left to right. The enzyme activity measured in slices of B parallel gel was in terms of c.p.m./gel slice ( I cm). 174
MW
90000----
40000- - - -
22000- - - -
FIG.
6. SDS-polyacrylarnide gel electrophoresis of purified GAD. About 15 jig of purified GAD were
electrophoresed on a 12.5% SDS-polyacrylarnide gel. See text for details.
175
FIG.7. Ouchterlony double diffusion test of anti-GAD IgG from rabbit. Enzyme crude extract was placed in the center well. and wells a and f contained undiluted anti-GAD I& and unimmunized rabbit IgG. respectively. Wells b, c. d and e contained anti-GAD IgG at 2. 4. 8 and 16-fold dilutions. respectively.
176
L-Glutamic acid decarboxylase from catfish brain
177
TABLE1
Homogenate Supernatant 40-75%* Ultro-gel (4&75%)* Calcium phosphate (40-750/:,)* Ultro-gel (0-75%)* Gel extraction
Total volume (ml)
Total protein (mg)
600 550 60 22
6750 3052.5 976.2 264
12 6 8
Specific enzyme activity (pmol/mg/min) 103
23.6 10.8 1.12
Total enzyme activity (pmol/min)
4 5.8 29.6 64.6
27.0 17.7 17.6 17.0
454 826 2415t 4830$
9.1 8.9 2.7 5.4
* Fractionation with ammonium sulfate at per cent of saturation as indicated. t Specific activity actually obtained from gel extract without any correction. $ Specific activity calculated with the correction for the inactivation of the enzyme during electrophoresis.
mined to be about 50% by comparing the enzyme activity of equal amounts of GAD before and after electrophoresis. If the specific activity of the enzyme extracted from the gel was corrected for the inactivation, it represents about a 1200-fold purification over the original homogenate. The purified enzyme protected with AET, pyridoxal phosphate and reduced glutathione is stable at - 20°C in the dark for several months. Thawing and freezing cause a slight decrease in activity.
as demonstrated in imrnunodiffusion experiments in which a sharp, single precipitin band was obtained with GAD antiserum and crude enzyme preparations (Fig. 7). Furthermore, the precipitin band also contained GAD activity, while a comparable piece of gel did not show any enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex.
Criteria of purity
by
(a) Polyacrylamide gel electrophoresis of 50 pg of the purified enzyme revealed a single protein band with the location of the enzyme activity corresponding to the location of the protein band (Fig. 4). (b) On 3.7 to 15% gradient polyacrylamide gel electrophoresis, the purified enzyme preparation also migrated as a sharp, single protein band which contained all the enzyme activity (Fig. 5).
Inhibition test
The activity of the enzyme was partially inhibited incubating with the GAD antisera
loo
I
4t IW
Molecular weights of the native enzymes
The molecular weight of GAD was estimated by gel filtration to be 84,000 2000 and by gradient polyacrylamide gel electrophoresis as 90,000 4000, assuming that GAD was similar to the marker proteins-ferritin, aldolase, ovalbumin, pepsin and bovine serum albumin-as a globular protein.
+
Molecular weight of the subunits
GAD was treated with SDS and 2-mercaptoethanol prior to electrophoresis on the SDS-polyacrylamide gel system. Three major protein bands were obtained which correspond to molecular weights of 22,000 F 2000, 40,000 5000 and 90,000 5 6000 respectively (Fig. 6). Ouchterlony double immunodiflusion tests with G A D antiserum
Antibodies against the purified GAD appeared to be specific only to GAD, even in crude preparations,
TIME ( D A Y S ) FIG.8. Inhibition of GAD by the antiserum as a function of incubation time at 0°C. Twelve micrograms of GAD were incubated with 19.2 pg of antiserum. GAD activities were assayed after the incubation period. The percentage of inhibition was obtained by comparing the activity of the sample and that of control (GAD incubated with unimmunized rabbit serum).
178
Y. Y. THOMAS Su, J.-Y. Wu and DOMINIC M. K. LAM
homogenizing medium; secondly, the use of preparative gel electrophoresis as the last step of purification. In hypotonic medium, most of the soluble proteins were liberated into the supernatant. About 70% of GAD activity was obtained in the supernatant compared to about 25% obtained from the crude mitochondrial fraction in the mouse brain preparation using isotonic medium. A similar result with mouse brain has also been reported from this laboratory using hypotonic medium (Wu et al., 1976). Hence, the use of hypotonic solution provided a general and efficient method for extracting enzyme from brain. Preparative polyacrylamide gel electrophoresis provides a simple and easy method for protein purification. Two drawbacks are the partial enzyme inactivation during electrophoresis and low recovery of enzyme from the gel. However, this method is particularly useful when a small quantity of homogeneous 0 100 200 3 3 protein is needed, for instance, as an antigen for antiANTISERUM (11) body production. The enzyme preparation obtained FIG.9. Inhibition of GAD by increasing amounts of anti- by a combination of various column chromatoserum after 4 days incubation at 0°C. Twelve micrograms graphies and preparative gel electrophoresis appears to of GAD were incubated with 1.64-19.2pg of antiserum; be homogeneous. This is based on the following A-A, reaction mixture; 0---0, supernatant from observations: first, in polyacrylamide gel electroreaction mixture; M, precipitate obtained from the phoresis, which separates proteins according to their reaction mixture by brief centrifugation. charge and size, the purified enzyme preparation migrates as a single protein band which contains all GADj19.2pg GAD antiserum) at 0°C for varying the enzyme activity; secondly, in gradient polyacrylperiods of time (Fig. 8). The maximum inhibition, amide gel electrophoresis, which separates proteins 64%, was obtained after 4 days incubation. Inhibition according to their size only, the purified enzyme soluwas expressed as percentage of the activity of the tion also migrates as a single protein band which is enzyme which was incubated with the same amount coincident with the enzyme activity ; thirdly, the antiof serum from unimmunized rabbits. The enzyme ac- body against the purified enzyme preparation forms tivity was stable at 0°C during the course of incuba- a single and sharp precipitin band when it is tested tion. Serum from the unimmunized rabbits inhibited aganst a crude enzyme preparation in immunodiffuthe enzyme activity slightly (1&20%). The degree of sion tests. Furthermore, the precipitin band obtained inhibition of enzyme activity also varied as a function in immunodiffusion tests or the precipitate obtained of the amount of GAD antisera which were incubated in the enzyme inhibition study also contains GAD with a constant amount of GAD protein at 0°C for activity, suggesting that the complex is indeed a 4 days (Fig. 9). The maximum inhibition, 62%, which GAD-anti-GAD complex. It also suggests that the was similar to the results obtained in Fig. 8, was antibody is monospecific to GAD and the antigenobtained at a ratio of GAD protein/GAD antiserum purified GAD preparation-used for antibody proprotein of about 1 :1.6. When the enzyme activity was duction is homogeneous. The molecular weight of measured after the incubation mixtures had been cen- GAD estimated from gradient polyacrylamide gel trifuged, it was found that the activity in the superna- electrophoresis is somewhat higher than the one tant fluid decreased with the increase of GAD anti- obtained from gel filtration, possibly due to a partial sera and the opposite was true for the activity in the denaturation of protein molecules during electroprecipitate. In the presence of 19.2pg of GAD anti- phoresis as reflected in the partial inactivation of the serum, all the activity in the mixture was recovered enzyme activity. SDS-polyacrylamide gel electroin the precipitate. phoresis revealed three major proteins with molecular weights of 22,000 f 2000, 40,000 5000 and 90,000 6000. Since the native enzyme has a molecular weight around 80,0G+90,000, these three proteins DISCUSSION may represent a monomer, dimer, and tetramer if the The methods described in this paper for the purifi- smallest subunit has a molecular weight around cation of GAD from catfish brain are somewhat dif- 20,000. It is interesting that the mouse brain enzyme ferent from those previously employed for the purifi- also showed multiple protein bands on SDS-polyacrylcation of GAD from mouse brain (Wu et al., 1973; amide gel electrophoresis representing monomer, Wu, 1976). Two major modifications were made: first, dimer and higher degrees of aggregation. However, the use of hypotonic instead of isotonic solutions as more vigorous physical and chemicaI studies are
L-Glutamic acid decarboxylase from catfish brain
needed before the precise subunit structure of GAD can be established. Another interesting similarity between the mouse brain enzyme and catfish brain enzyme is that the enzyme preparations from the hypotonic medium show two enzyme activity peaks o n gel filtration column corresponding to molecular weights of higher than 200,000 and 84,000, respectively (Wu et al., 1976). The nature of the high molecular weight GAD, whether it is merely an aggregated form of the low mo1:cular weight GAD, a complex of the low molecular weight GAD and other macromolecules, or a new type of GAD, is now under invest iga t i on. Acknowledgements-This investigation was supported in part by the Retina Research Foundation (Houston), NIH grants EY 02423 and NS 13224 and a grant from Huntington's Chorea Foundation in memory of Mrs. RUTH BtRMAN.
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F A R R A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J . hiol. Chem. 193, 265-275. MARCR. E., STELLW. K., BOK D. & LAMD. M. K. (1978) GABA-ergic pathways in the goldfish retina. J . cornp. Neurol. 182, 221-246. MCLAUCHLIN B. J.. WOOD J. G., SAITOK., BARBERR., E. & Wu J.-Y. (1974) Fine-strucVAUGHNJ. E., ROBERTS tural localization of glutamate decarboxylase in synaptic terminals of rodent cerebellum. Brain Res. 76, 377-391. MCLAUGHLIN B. J.. WOOD J. G., SAITOK., ROBERTSE. & Wu J.-Y. (1975) Fine-structural localization of glutamate decarboxylase in developing axonal process and presynaptic terminals of rodent cerebellum. Brain Rex 85, 355-371. MILLERG . L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. OBATA K. & TAKEDA K. (1969) Release of y-aminobutyric acid into fourth ventricle induced by stimulation of cat cerebellum. J . Nrurochem. 16, 1043-1047. ROBERTSE. & SIMONSEN D. G. (1963) Some properties of L-glutamic decarboxylase in mouse brain. Biochern. Pharmac. 12, 113-134. SAITOK. (1976) Immunochemical studies of G A D and GABA-T, in G A B A in Nervous System Function (ROBERTS E., CHASET . N . & TOWER D. B., eds.) pp. 103-1 11. Raven Press, New York. SAITOK., Wu J.-Y., MATSUDAT. & ROBERTSE. (1974~) Immunochemical comparisons of vertebrate glutamic acid decarboxylase. Brain Res. 65, 277-285. E. SAITOK., BARBER R., WU J.-Y., MATSUDAT., ROBERTS & VAUGHN J. E. (1974h) Immunohistochemical localization of glutamate decarboxylase in rat cerebellum. Proc. natn. Acad. Sci., U.S.A. 71, 269-213. TAKEUCHI A. & TAKEUCHI N. (1965) A study of inhibitory action of y-aminobutyric acid on neuromuscular transmission in crayfish. J . Physiol., Lond. 177, 225-238. WHITAKER J. R. (1963) Determination of molecular weights of proteins by gel filtration on Sephadex. Analyt. Chem. 35, 1950-1953. Wu J.-Y. (1976) Purification, characterization, and kinetic studies of GAD and GABA-T from mouse brain, in G A B A in Nervous System Function (ROBERTS E., CHASE D. B., eds.) pp. 7-55. Raven Press, New T. N. & TOWER York. WU J.-Y. & ROBERTSE. (1974) Properties of brain L-glutamate decarboxylase: inhibition studies. J . Neurochem. 23, 159-767. WU J.-Y., MATSUDAT. & ROBERTSE. (1973) Purification and characterization of glutamate decarboxylase from mouse brain. J . hiol. Chem. 248, 3029-3034. Wu J.-Y., WONG E., SATOK., ROBERTSE. & SCHOUSBO~ A. (1976) Properties of L-glutamate decarboxylase from brains of adult and newborn mice. J . Neurochem. 27. 653-659. WU J.-Y., CHUDEO., WEIN J., ROBERTS E.. SAITOK . & WONG E. (1978) Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15) J . Neurochem. 30. 849-851.
