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Pig brain 4-aminobutyrate 2-ketoglutarate transaminase. Purification, kinetics and physical properties
BIOCHIMIE, 1974, 56, 823-832.
Pig brain 4-aminobutyrate 2-ketoglutarate transaminase. Purification, kinetics and physical properties. Marcienne BLOCH-TARDY,Bernadette ROLLAND a n d P i e r r e GONNARD. D~partement de Biochimie, C.H.U. Henri-Mondor, 94010 Crdteil, France. (1-4-1974). Summary. - - Pig brain 4-am.inobutyrate 2-ketoglutarate transaminase, (E.C. 2.6.1.19) has been purified about 1200 to 1500 fold, using ammonium sulfate fractionation, heat precipitation and various chromatographic techniques. The enzyme was homogeneous in potyacrylamide gel electrophoresis and appeared as a dimer when electrophoresis was performed in presence of SDS or SDS and urea. A molecular weight of 105000 was calculated by gel filtration and confirmed by SDS polyacrvlamide gel electrophoresis. The optimum pH for the enzyme was 8.7 and the Km values for y-aminobutyrate and ¢c ketoglutarate were estimated to be 4.8 × 10-3 M and 1.3 X 10-3 M respectively. The pHt of the enzyme was 5.9 Two peaks of GABA,r were separated on DEAE Cellulose chromatography and separately purified. Differences in sedimentation constant, and enzyme kbaetic and the association of the second peak with aspartate amiuotransferase led us to distuss the possible existence of molecular complexes. INTRODUCTION. T h e r e is c o n s i d e r a b l e evidence for the involv e m e n t of v - a m i n o b u t y r i c acid (GABA) as an i n h i b i t o r y t r a n s m i t t e r at certains synapses i n the m a m m a l i a n c e n t r a l nervous system. It has become i n c r e a s i n g l y i m p o r t a n t to obtain better knowledge about the t~,o enzymes i n v o l v e d in the m e t a b o l i s m of GABA. 4 - a m i n o b u t y r a t e 2-ketoglntarate ~transaminase (E.C.2.6.1.19) (GABAT) has been p a r t i a l l y purified from rat a n d mouse b r a i n [1, 2]. This enzyme is chiefly located into m i t o c h o n drial m a t r i x (about 80 p. 100). The r e m a i n e d part is b o u n d to m i t o c h o n d r i a l m e m b r a n e s ( u n p u b l i s hed results). Recently, Schousboe, W u and Roberts [7] choosed this m e m b r a n o u s m a t e r i a l to purified GABAT from mouse b r a i n . The o b t a i n i n g of a highly purified soluble enzyme is necessary to study its localization in b r a i n a n d at p o s t s y n a p t i c level in the prospect to r e a c h a better u n d e r s t a n d i n g of the m e c h a n i s m of action of GABA i n the synapse. In this p a p e r we deal w i t h a p u r i f i c a t i o n procedure of a soluble GABA,r in pig b r a i n , a n d some p r o p e r t i e s of the enzyme. T w o peaks of enzyme activity were separated by DE 52 Cellulose c h r o m a t o g r a p h y a n d there The abbreviation used are : GABA, ~t-aminobutyric acid; GABAT, GABA transamina~se; PLP, phospho pyridoxal; GSH, reduced glutathione; AAT, aspartate aminotransf~rase, SDS, sodium dodecyl sulfate ; a KG, a ketoglutaric acid ; GA, glutamie acid.
were purified. The possibility for GABAT to form m o l e c u l a r complexes ~,ith other p r o t e i n s was discussed.
MATERIAL AND METHODS.
1. Enzyme assays. A method for d e t e r m i n a t i o n of GABA~, activity has been developed i n our l a b o r a t o r y [3]. [l-14C]a ketoglutarate a n d cold GABA were used as substrates to form [14C] glutamic acid. I n a second step, glutamate decarboxylase, commercially available from E. colt (Fhika) w a s added. The rate of 14CO2 evolved agrees stoechiometrically with the glutamate formed d u r i n g the transa m i n a t i o n , that is to say, ~¢qith GABA T activity. A less sensitive but r a p i d m e t h o d [2] has also been used as a s t a n d a r d assay method in p u r i fication p r o c e d u r e s d u r i n g w h i c h , m a n y fractions must be tested.
2. Polyacrylamide gel electrophoresis. P o l y a c r y l a m i d e gel electrophoresis was performed a c c o r d i n g to the original p r o c e d u r e of Davis [4]. The sample (200 to 300 ,~l) of enzyme solution was a p p l i e d on a 7 p. cent p o l y a c r y l a m i d e gel c o l u m n (1.3 × 8 cm) p H 8.9 w i t h a 3 p. cent c o n c e n t r a t i n g gel pH 6.7 (1.3 × 0.5 cm) using a m m o n i u m persulfate as p o l y m e r i z i n g agent. A Tris glycin buffer (0.6 g and 0.58 g glycin per 1) pH 8.3 was used as electrophoresis buffer, a n d the m i g r a t i o n was p e r f o r m e d at 4 ° for 3 h w i t h a c u r r e n t of 10 m A / c o l u m n .
824
M. Bloch-Tardy, B. R o l l a n d and P. Gonnard.
T h e samples c o n s i s t e d of 10 to 40 lug of e n z y m e c o n t a i n i n g one d r o p of B r o m o p h e n o l Blue as m a r k e r a n d t w o d r o p s of n e u t r a l glycerol. P r o t e i n b a n d s w e r e v i s u a l i z e d by Coomassie Blue R-250 s t a i n i n g for one n i g h t and d e s l a i n e d in acetic acid 10 p. cent. F o r e n z y m e assay, i m m e d i a t e l y after e l e c t r o p h o r e s i s , the gels w e r e cut in 5 m m slices, i n c u b a t e d i n d i v i d u a l l y in I ml of t r a n s a m i n a t i o n r e a c t i n g buffer at 4 ° u n d e r agitation for at least t w o hours, after that, the fractions w e r e s u b m i t t e d to f r e e z i n g and t h a w i n g and then, i n c u b a t e d at 37 ° for 60 rain.
3. Polyacrylamide - - SDS - - Urea electrophorests. Disc gel e l e c t r o p h o r e s i s in s o d i u m d o d e c y l sulfate w i t h or w i t h o u t urea 4 M w a s p e r f o r m e d as d e s c r i b e d by W e b e r and Osborn [5]. 100 td of e n z y m e solution i n c u b a t e d f o r 2 h o u r s at 37 ° or for 15 m i n at 600 in p r e s e n c e of 0.1 p. cent SDS a n d ~ m e r c a p t o e t h a n o l , w e r e a p p l i e d after addition of B r o m o p h e n o l Blue and n e u t r a l g l y c e r o l on a 5 p. cent p o l y a c r y l a m i d e ge! c o l u m n (0.5 × 1(~ cm) in a p h o s p h a t e buffer 0.1 M, p H 7.2. Protein b a n d s w e r e visualized by Coomassie Blue G-250 in TCA 5 p. cent for one night and destained w i t h 7 p. cent acetic acid as d e s c r i b e d by Diezel and al. [~6]. Gels w e r e r u n at 6 mA p e r tube at 25 ° for 3 to 4 h o u r s - - Relative m o b i l i t i e s w e r e calculated w i t h p r o t e i n s used as m a r k e r s . I n d i v i d u a l p r o t e i n c o n c e n t r a t i o n g e n e r a l l y ranged b e t w e e n : 200 and 400 i~g/ml.
4. Isoelectric focusing. T h e i s o e l e c t r i c f o c u s i n g c o l u m n (LKB 8100 Vol 110 ml) a n d p H 3-10 (40 p. cent solution) a m p h o line c a r r i e r a m p h o l y t e s w e r e o b t a i n e d f r o m LKB P r o d u c t e r AB (S-16125 BROMMA I Sweden). F i n a l c o n c e n t r a t i o n of c a r r i e r a m p h o l y t e s in the sucrose g r a d i e n t w a s 1 p. cent ( w / v ) . W h e n i s o e l e c t r i c f o c u s i n g w a s c o m p l e t e d , f r a c t i o n s of a p p r o x i m a t e l y 1 ud w e r e collected, p H and e n z y m e a c t i v i t y of e a c h sample w e r e d e t e r m i n e d . A p o w e r of 16.0'0 m W w a s a p p l i e d to the c o l u m n for 70 h o u r s all a l o n g the e l e c t r o f o c u s i n g migration. All o p e r a t i o n s w e r e p e r f o r m e d at 4 ° .
5. Determination of molecular weight by gel filtration. A 2.5 c m d i a m e t e r glass c o l u m n w a s p a c k e d at 4 ° to a bed h e i g h t of 95 c m w i t h S e p h a d e x G-100 e q u i l i b r a t e d w i t h p h o s p h a t e acetate buffer 0.1 M, p H 6.8. P r o t e i n s w e r e eluted at 4 ° w i t h the same buffer at a flow rate of about 11 m l / h o u r . T h e
BIOCHIMIE, 1974, 56, n ° 6-7.
f o l l o w i n g m a r k e r s w e r e used in v a r i o u s c o m b i n a tions to c a l i b r a t e the c o l u m n : c y t o c h r o m e c o v a l b u m i n - s e r u m a l b u m i n - alcohol d e s h y d r o g e nase - D e x t r a n blue,
6. Protein determination. P r o t e i n w a s d e t e r m i n e d r o u t i n e l y a c c o r d i n g to the m e t h o d of L o w r y and al. I8].
7. pH dependence of GABA,I,. T h e effect of pH on a c t i v i t y of a purified p r e p a r a t i o n of GABAT h a d been studied in a r a n g e of 7 to 9 w i t h Tris-HC1 buffer 0.2 M.
8. Effect of subslrate concentration on activity. Km values w e r e d e t e r m i n e d , u s i n g the i s o t o p i c t e c h n i c w i t h d i f f e r e n t substrate c o n c e n t r a t i o n s (GABA : I - 25 raM, a k e t o g l u t a r a t e : 0.2 - _9 mM).
9. Substrate specificity. T h e specificity of the e n z y m e for v a r i o u s a m i n o a c i d s w a s studied. A m i n o a c i d s in question w e r e substituted for GABA, usually at the same c o n c e n t r a t i o n (25 raM), or at t h r e e d i f f e r e n t conc e n t r a t i o n s (10 - 25 - 50, raM). 10. Sedimentation constant. A sucrose d e n s i t y g r a d i e n t f r o m 5 to 20 p. cent in saline solution ( p h o s p h a t e acetate 0.01 M, p H 6.8) w a s p r e p a r e d in tubes of SW 50 L s w i n g bucket rotor. T h e e n z y m e solution (0.1 ml c o n t a i n i n g about 20 j~g proteins) w e r e l a y e r e d on the top of the c o l u m n . T h , c e n t r i f u g a t i o n w a s p e r f o r m e d at 45.000 r.p.m, for 8 h at 3 ° in a S p i n c o L I p r e p a r a t i v e centrifuge. F o u r d r o p s f r a c t i o n s w e r e collected and tested for e n z y m e a c t i v i t y and for p r o t e i n c o n c e n t r a t i o n .
RESULTS. T h e successive steps in the p u r i f i c a t i o n of GABA~ f r o m about t h r e e p i g b r a i n s are d e s c r i b e d . Our e n z y m e p r e p a r a t i o n is 1200 to 1500 foId purified as c o m p a r e d to the o r i g i n a l a c e l b n e p o w d e r . Its specific a c t i v i t y ranges b e t w e e n 400 and 500 aM g l u t a m i c a c i d f o r m e d p e r h o u r p e r mg of protein. It can be stored at 4 ° for few days. T h e p u r i f i e d e n z y m e can be kept for a long p e r i o d at - - 20 .0 in the d a r k in p r e s e n c e of PLP, d i t h i o t r e i t o l , EDTA, as p r o t e c t o r s . E n z y m e solution is stable at pH 5.5 to pH 7. a k e t o g l u t a r i c acid as well as acetate ions w e r e f o u n d to p r o v i d e a good p r o t e c t i o n against h e a t as s h o w n in figure 1.
Pig brain $-aminobutyrale 2-keloglutarate transaminase.
825
I - - P u r i f i c a t i o n of p i g b r a i n G A B 4 r. All steps were performed at 4 ° and ration was protected from light.
the prepa-
After each step of purification, samples were taken for enzyme activity, protein concentration, and polyacrylamide electrophoresis.
D
Stev 1 : Extraction. Acetone powder obtained from brains of three pig was suspended with slow stirring in 600 ml of phosphate a c e t a t e b u f f e r p H 6.8, 0.01 M c o n t a i n i n g 10 -4 M P L P a n d G S H a n d 10 -a M E D T A and a ketoglutaric a c i d f o r 20 r a i n a n d t h e n ,
~ ! ',
I
~¢I"- I °
p h o s p h a t e a c e t a t e butler 0 . 0 1 - - p H 6 . 8
V EXTBACT
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p r e c i p i t a t i o n 56, - - p H 5 . 2 Y SUPERNATANT
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0 - - 55 p. c e n t
Y SUP~ERNA-TANT
SO,, ( N H , ) 2 - - 55 - - 75 p. c e n t Y PELLET
S e p h a d e x G - - 100 phosphate acetate 0.1 M --pH
Y
6.8
ELUATE
CM cellulose a c e t a t e buffer 0 . 0 l M -- p H 5 . 4 5 ELUATE
a c e t a t e buffer 0 . 1 M - - p H 5 . 7 D E A E cellulose Tris M a l e a t e buffer 0 . 0 1 M - - p H 7 . 2
i
-
¥
-
ELUATE
~
Tris - - M a l e a t e 0 . 0 9 M - - p H 7 . 2 to 5 . 2 g r a d i e n t
DEAE I DEAE calcium phosphate gel c h r o m a t o g r a p h y phosphate buffer 0.01 M - - p H 6 . 8 Gradient elution 0 . 0 5 to 0 . 3 M GABAT I
BIOCHIMIE, 1974, 56, n ° 6-7.
II
G A B A T II
p~l
L
FIG. 1. - - H e a t i n a c t i v a t i o n a t 56 ° f o r 30 m i n . V a r i a t i o n w i t h pH © i n p r e s e n c e of 10 m M a c e t a t e + P L P + GSH (10-4 M). • w i t h o u t a c e t a t e .
ACETONE POWDER
I
•
M. BIoch-Tardy, B. R o l l a n d and P. Gonnard.
826
homogenized in a large chilled potter homogen i z e r . A c e n t r i f u g a t i o n of 20 m i n at 17500 g i n a M S E S P 50 c e n t r i f u g e w a s p e r f o r m e d .
Step 2 : Heat precipitation. The clear supernatant was decanted and used f o r p r o t e i n p r e c i p i t a t i o n a t p H 5.3 a n d 56 ° f o r 10 m i n . T h e s u s p e n s i o n w a s c e n t r i f u g e d f o r 30 m i n a t 17500 g.
to g i v e 55 p. c e n t s a t u r a t i o n . A l o n g t h e t i m e of a d d i t i o n of t h e s o l u t i o n , p H w a s k e p t c o n s t a n t n e a r 6.8 b y a d d i t i o n of N H 4 0 H 2 N. A f t e r 30 m i n of e q u i l i b r a t i o n , t h e s u s p e n s i o n w a s c e n t r i f u g e d , a t 27500 g f o r 60 m i n . T h e p e l l e t w a s d i s c a r d e d a n d t h e s u p e r n a t a n t b r o u g h t to 75 p. c e n t s a t u r a t i o n w i t h t h e s a t u r a t e d s o l u t i o n of ammonium sulfate.
TABLE I.
Purification of pig brain GABA transaminase. Fraction
Total activity (')
Homogenate . . . . . . . . . . . . . . . . Extract .................... Heat treatment ............. (NH4) ~ SO 4 5 5 - - 7 5 p. cent.. Sephadex G - - 1 0 0 . . . . . . . . . . . CMGellulose . . . . . . . . . . . . . . D E A E Cellulose Peak I . . . . . . Peak II . . . . . 8. Hydroxylapatite Peak I . . . . . Peak II . . . . .
6900 6000 4500 2900 ~00 1740 900 750 600 70
1. 2. 3. 4. 5. 6. 7.
Total protein (g) 69 39 0.79 0.14 0.044 0.010 0.003 0,005 0.0012 0.0007
Specific activity (")
Yield p. cent
0.3 1.7 '5.7 20 52 174 300 150 500 100
I00 90 65 40 33 25 13 I0 9 1
(*) Activity m e a s u r e d w i t h the isotopic m e t h o d in I~M g l u t a m i c acid f o r m e d per hour. (**) Specific activity in .~M g l u t a m i e acid formed per h o u r per mg of protein.
The suspension was centrifuged in the same conditions than above. The last pellet was dissolv e d i n a 0.01 M p h o s p h a t e a c e t a t e b u f f e r c o n t a i ning PLP, GSH and EDTA.
Step ~ : G-IO0 Gel filtration. 5 ts*'!/,.
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35
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50
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60
G'~BAT ,
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I
70
FRACTION
NUMBER
FIG. 2. - - Gel filtration on Sephadex G-100. The prorein was applied a n d eluted w i t h 0.1 M sodium phosp h a t e acetate buffer pH 6.8. P r o t e i n c o n c e n t r a t i o n e - o - o is given in U.D.O. at 280 n m a n d e n z y m e activity e - - e in I~M g l u t a m i c acid f o r m e d per h o u r per ml of solution 6 ml was collected in each fractions.
Step 3 : Ammonium sulfate precipitations. The supernatant was collected and a saturated s o l u t i o n of a m m o n i u m s u l f a t e (in p h o s p h a t e a c e t a t e b u f f e r 0.01 M, p H 6.8) w a s a d d e d s l o w l y
BIOCHIMIE, 1974, 56, n ° 6-7.
A c o l u m n (5 × 100 c m P h a r m a c i a ) w a s p a c k e d with Sephadex G-100 a n d e q u i l i b r a t e d with a c e t a t e p h o s p h a t e b u f f e r 0.1 M, p H 6.8 b e f o r e t h e a p p l i c a t i o n of t h e d i s s o l v e d p e l l e t f r o m t h e 75 p. cent precipitation. The protein was eluted with t h e s a m e b u f f e r , as u s e d f o r t h e e q u i l i b r a t i o n at 20 ml~/h,o u r ,and a t y p i c a l p r o f i l e i s s h o w n i n f i g u r e 2. R e c o v e r y is a b o u t 90 p. c e n t .
Step 5 : CM Chromatography. A c o l u m n (1.5 × 30 c m ) w a s p a c k e d w i t h CM c e l l u l o s e (CM 52 W h a t m a n ) p r e v i o u s l y e q u i l i b r a t e d w i t h 0.01 M a c e t a t e b u f f e r , p H 5.45. After the application, the column was washed with the same buffer and the enzyme was eluted w i t h a l i n e a r c o n c e n t r a t i o n g r a d i e n t of s o d i u m a c e t a t e b u f f e r of p H 5.7 f r o m 0.01 M to 0.2 M.
Pig brain 4 - a m i n o b u t y r a l e T h e e n z y m e s t a r t e d to a p p e a r a t 0.08 M a n d t h e p e a k f r a c t i o n c o r r e s p o n d e d to 0.1 M b u f f e r c o n centration.
2-ketoglutarate t r a n s a m i n a s e . Tris-Maleate buffer 10-3 M E D T A .
0.01
827
M, p H
6.8
containing
Enzyme preparation was applied on the colunm. The column had been washed with the same buffer and elution accomplished a linear pH grad i e n t of 7.2 to 5.2, i n T r i s - M a l e a t e 0.09 M. T w o g o o d s e p a r a t e d GABA~ p e a k s w e r e e l u t e d . T h e first p e a k a p p e a r e d at p H 6.9 (GABA T I) a n d t h e s e c o n d o n e (GABA T II) at p H 6.7 (fig. 4).
i
Step 7 : Calcium Phosphate graphy.
A c o l u m n (1 X 13 c m ) w a s p a c k e d w i t h calcium phosphate (Biorad HT) and equilibrated w i t h 0.01 M s o d i u m p h o s p h a t e b u f f e r , p l I 6.8 .-k P I , P + GSH, 104 M. E n z y m e p r e p a r a t i o n p r e viously dialyzed one night against the equilibrated buffer, was applied on the column.
FRACTION N U M B E R
Fro. 3. - - CM cellulose c h r o m a t o g r a p h y of the gel filtration active fraction. The protein was applied in 0.01 M acetate buffer pH 5.4, w a s h e d w i t h the same huffer and eluted w i t h a g r a d i e n t of sodium actetatc buffer pH 5.7 from 0.01 M to 0.2 M. o--o GABAT activity in ~M glutamic acid f o r m e d / ml. O - - © P r o t e i n c o n c e n t r a t i o n (O.D. at 280 nm). T h e r e c o v e r y ~vas a b o u t 80 p. c e n t a n d t h e p u r i f i c a t i o n of a b o u t 3 t o 4 f o l d . T h e GABA~, active fractions were pooled and dialyzed against a T r i s - M a l e a t e b u f f e r 0.01 M a n d p H 7.2 (fig. 3).
A c o l u m n (1.5 X 30) w a s p a c k e d w i t h D E A E c e l l u l o s e (DE 52 W h a t m a n ) a n d e q u i l i b r a t e d w i t h
.%
/\,',',, / \
/
/
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0.1t M
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5
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/ %...
Step 6 : DEAE Chromatography.
i
Gel Chromato-
1"0
ZO
so
• io
s'o FRACTION
i0 NUMBER
Fro. 5. - - C h r o m a t o g r a p h y on calcium p h o s p h a t e gel, The p r o t e i n was applied i n 0.01 M sodium phosp h a t e pH 6.8. E l u t i o n was p e r f o r m e d w i t h a l i n e a r g r a d i e n t of sodium p h o s p h a t e f r o m 0.05 M to 0.3 M. • a b s o r b a n c e at 280 nm. O GABA~ II activity in jxM GLU. f o r m e d per h o u r per ml. • AAT activity.
ta
200-
'-
! 10o-
........ o.7.o .....
The column was then washed with an equal v o l u m e of 0.01 M b u f f e r a n d tile e n z y m e e l u t e d u s i n g a l i n e a r c o n c e n t r a t i o n g r a d i e n t of 0.05 M p h o s p h a t e b u f f e r p H 6.8. Figure 5 GABA~ II.
gives
the
profile
of
elution
for
FRACTION NUMBER
F](;. 4. - - DEAE cellulose c h r o m a t o g r a p h y of the CM peak fraction, The p r o t e i n 'was applied in 0.01 M TrisMaleate buffer pH 6.8 and eluted w i t h a g r a d i e n t of pH f r o m 7.2 to 5.2. o--o GABA~, activity in IlxM g l u t a m i e acid formed per ml. © - - - © p r o t e i n c o n c e n t r a t i o n (O.D. 280 nm). x---$ AAT activity.
BIOCHIMIE, 1974, 56, n ° 6-7.
I I - - Phgsico-chemical caracterization of GABA T I. 1 - - P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of p u r i f i e d GABA T I, a f t e r t h e 7 th s t e p of t h e p u r i f i c a tion revealed a single protein band, carrying the GABA r activity. DO
M. Bloch-Tardy, B. Rolland and P. Gonnard.
828
2 -- Molecular weight determination.
M=
a) S e d i m e n t a t i o n c o e f f i c i e n t . The sedimentation coefficient was evaluated by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n c o m p a r a t i v e l y to three marker proteins : --
yeast alcohol dehydrogenase ( $ 2 0 , w = 7.4 S) ;
--pig heart soluble aspartate r a s e [9] ($20, w = 5.5S) ; --
T h e m o l e c u l a r w e i g h t is e s t i m a t e d to 105 000 ___ 7 000.
[10] anainotransfe-
c y t o c h r o m e c ([1!1] (S 20; w = 1.9 S).
Figure 6 shows the GABA T I a n d GABA~ II.
S RT - - X - - D (I-~ ~)
z
relative
mobilities
of
71s
: LOG. DIFFUSION
81
L
COEFFJC|ENT
FIG. 7. - - Molecular weight e s t i m a t i o n by gel filtration. The column 2.5 cm i.d. was packed w i t h Sephadex G-100 to a heigh of 85 cm. The elution volumes in milliliters (___ 1 ml) of the markers and of GABAx are indicated.
O
u~
3. D i s s o c i a t i o n of GABA T b y SDS a n d u r e a .
\
I
5
110 FRACTION
115
NUMBER
E l e c t r o p h o r e s i s o f GABA T I i n SDS p o l y a c r y l a m i d e gels w a s i n v e s t i g a t e d u n d e r t h e f o l l o w i n g conditions :
FIG. 6. - - S e d i m e n t a t i o n of GABAT and m a r k e r s in sucrose gradients. 200 ~1 of each p r e p a r a t i o n (60 to 80 [~g proteins) were sedimented in identical sucrose gradients (5 to 20 p. cent in p h o s p h a t e buffer 0.01 M, pH 6.8 -4- PLP q- GSH 10-5 M) for 8 hours at 45 000 r.p.m, in a Beckman SW 50 L Rotor. Q-) GABAT I. (~ GABAT II. .a
o
4.4 1
T h e s e d i m e n t a t i o n c o e f f i c i e n t w a s of 5.5 S f o r t h e p u r i f i e d GABA T I. b) D i f f u s i o n c o e f f i c i e n t . The diffusion coefficient was determined using a G-100 S e p h a d e x c o l u m n . T h e d e t e r m i n a t i o n o f t h e v o i d v o l u m e of t h e c o l u m n (Vo) a n d of t h e e l u t i o n v o l u m e (V) f o r p r o t e i n m a r k e r s w e r e d e t e r m i n e d i n a 1'0.0 m M s o d i u m a c e t a t e b u f f e r p H 6.8 c o n t a i n i n g 10 -5 M G S t t a n d P L P . T h e d i f f u s i o n c o e f f i c i e n t D 20 w w a s e s t i m a t e d f o r GABA T I to b e 4.45 X 107 cm~ sec-1 (fig. 7).
c) Molecular weight determination. I f w e c o n s i d e r t h a t t h e v a l u e o f v i s 0.73 as f o r g l o b u l a r p r o t e i n s , a n d t h a t ~ ---- 1, t h e m o l e c u l a r w e i g h t o f GABA.rI w i l l b e o b t a i n e d f r o m t h e equation
BIOCHIMIE, 1974, 56, n ° 6-7.
i
..J
43
42
4.1
RELATIVE
MOBILITY
Fro. 8. - - D e t e r m i n a t i o n of the subunit molecular weight of GABAT in 7 p. cent polyacrylamide gels with dodecyl sulfate. About 100 I~xg of each protein were applied to gels a f t e r dissociation for 2 h at 37 ° in sodium p h o s p h a t e pH 7 containing 0.1 p. cent SDS and 0.1 p. cent I~ mercaptoethanol. Standards are : ~3 Bovine serum a l b u m i n dimer. Ovalbumin dimer. (-3) Bovine serum a l b u m i n monomer. (~ Ovalbumin monomer. (~) Cytochrome C.
Pig brain 4-aminobutyrate 2.ketoglutarate transaminase. a) i n 0.1 p. c e n t ( w / v ) SDS a n d 0.1 ( w / v ) I~ m e r c a p t o e t h a n o l w i t h 2 h i n c u b a t i o n f o r t h e e n z y m e s o l u t i o n at 37 ° .
pH
829
E s t i m a t i o n of m o l e c u l a r w e i g h t for this b a n d , o n t h e b a s i s of t h e r e l a t i v e m o b i l i t i e s of s t a n d a r d p r o t e i n s gave a v a l u e s of 51 000 ± 2 200. T h e s e r e s u l t s s u g g e s t e d t h a t GABA T I w a s a d i m e r a n d
..t"
t* vf
u /
.,/,.,..,j4""
/" /"
t,
>
S*"
!
PURIFICATION
t ! Z0
40
$0
,~
80
TUBE
NUMBER
FI.?,. 9. - - Isoelectrofoeusing of partially purified GABAT (after gel filtration) pH range of a m p h o l i n e s was 3 to 10 e - - e GabaT activity in uM glutamie acid formed per hour, per ml. e - e - e pH in each fraction.
STEPS
Fro. 10a. - - Relative degrees of pnrifieation during f o u r steps. (~) Aspartate transamination. ~) ~ Alanine transamination. GABA transamination.
~) in 1 p. c e n t ( w / v ) SDS a n d 1 p. c e n t ( w / v ) mercaptoethanol, and 4 M urea with incubation at 45 ° f o r 1 h. V) i n 0.1 p. c e n t ( w / v ) SDS a n d ~ m e r c a p t o e t h a n o l , w i t h i n c u b a t i o n at 60 ° f o r 15 rain. 5) i n 8 M u r e a + 0.1 p. c e n t ( w / v ) 13 m e r c a p t o e t h a n o l w i t h l h i n c u b a t i o n at 45 °. All gels s h o w e d a s i n g l e b a n d of p r o t e i n .
TABLE 1I. SubstraL
Relative activity p. cent
o
~r . . . . .
1
~--;~
3
PURIFICATION
4
5
STEP
Fro. 10b. - - Specific activity in :aM of glutamic acid f o r m e d per hour, per mg proteins of the three activities during the four first steps of the purification. (9 GABA t r a n s a m i n a t i o n . ~) I~ alanine t r a n s a m i n a t i o n . (~) Aspartate t r a n s a m i n a t i o n . that the two s u b u n i t s w e r e i d e n t i c a l in m o l e c u l a r w e i g h t a n d i n c h a r g e (fig. 8). 4. I s o e l e c t r i c f o c u s i n g .
Gaba . . . . . . . . . . . . . . . . . . . . . . . . . Glutamie acid . . . . . . . . . . . . . . . . . . A!anine . . . . . . . . . . . . . . . . . . . . . Tryptophane . . . . . . . . . . . . . . . . . . Leucine . . . . . . . . . . . . . . . . . . . . . . . Phenylalanine . . . . . . . . . . . . . . . . . M~thionine . . . . . . . . . . . . . . . . . . . . Glyeine . . . . . . . . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . . . . . . . . Arginine . . . . . . . . . . . . . . . . . . . . . . Threonine . . . . . . . . . . . . . . . . . . . . . Proline . . . . . . . . . . . . . . . . . . . . . . . . Taurine . . . . . . . . . . . . . . . . . . . . . . . Hypotaurine . . . . . . . . . . . . . . . . . . Para Chloro Phenyl Gaba . . . . . . .
BIOCHIMIE, 1974, 56, n ° 6-7.
100 136 126 21 10 3 0
T h e d e t e r m i n a t i o n of t h e i s o e l e c t r i c p o i n t w a s o b t a i n e d w i t h p a r t i a l l y p u r i f i e d e n z y m e ( a f t e r gel f i l t r a t i o n ) b e f o r e t h e s e p a r a t i o n o n DE-52 c o l u m n , b e c a u s e of t h e f r a g i l i t y of t h e t w o GABA.r f r a c t i o n s a n d t h e p o w e r f u l l d e n a t u r i n g a c t i o n of s y n t h e t i c c a r r i e r a m p h o l y t e s ; p H i w a s e v a l u a t e d to 5.8 - 5.9 u s i n g a m p h o l y t e s f r o m p H 3 to 10 (fig. 9).
~a ~a
III - -
Fnnclionnal properties of GABAT 1.
aa
a) S u b s t r a t e s p e c i f i c i t y . am
T a b l e II s h o w s t h a t c e r t a i n L - a - a m i n o a c i d s c a n s e r v e as s u b s t r a t e s of GABA z.
M. B l o c h - T a r d y , B. R o l l a n d and P. Gonnard.
830
A s p a r t i c a c i d is a s u b s t r a t e u n t i l t h e D E A E s t e p . A f t e r s e p a r a t i o n i n t o p e a k I a n d p e a k II, .1. V
To prove whether a protein fraction possesses a single enzyme with two catalytic functions, rather than two separate enzymes with two d i s t i n c t f u n c t i o n s , i t i s c u s t o m a r y to f o l l o w t h e r a t i o of t h e t w o c a t a l y t i c f u n c t i o n s t h r o u g h o u t a s e r i e of p r o t e i n f r a c t i o n a t i o n p r o c e d u r e s . W e s t u d i e d t h e s e a c t i v i t i e s o v e r t h e f o u r first s t e p s of t h e p u r i f i c a t i o n v i s - h - v i s GABA a n d a l a n i n e a n d f o u n d t h e m e x a c t l y p a r a l l e l (fig. 10a et b).
/
I 1
I 2
I 3
I 4
~
• 1
T h i s m e a n s t h a t ~ a l a n i n e is t r a n s a m i n a t e d to t h e s a m e e x t e n t as GABA. ~ a l a n i n e is a g o o d s u b s t r a t e f o r GABA T w h e r e a s a s p a r t i c a c i d s e a m s to b e t h e s u b s t r a t e of a s e p a r a t e e n z y m e o r of a n o t h e r m o l e c u l a r f o r m of GABA T.
(o K G )
Fro. l l a . - - Lineweaver-Burk, plot of GABAT I. a k e t o g l u t a r a t e c o n c e n t r a t i o n s varied f r o m 0..9. to 2 mM a n d for GABA f r o m 1 to 25 mM. A p p a r e n t reciprocal K,n values 'were o b t a i n e d for one s u h s t r a t e at infinite c o n c e n t r a t i o n of the other.
/ xx
o.,f
x
0.3
/
0.2
i
r
1
I
3
i
/
x
/
.
I
5
1 ~KG
r
0.,
FIG. l l b . - - Lineweaver-Burk, plot of GABAT II.
r
7
7.,
'
~
815
~
I
,,,,
.
FIG. 12. - - Specific activity of GABAT as a f u n c t i o n of pH in the i n c u b a t i o n m e d i u m . The isotopic assay was employed a n d Tris-HC1 buffer (0.2 M - p H 7 to 9) was used to o b t a i n the desired pH.
b) D e t e r m i n a t i o n
of K,n v a l u e s .
The apparent Michaelis constant value for 2 - o x o g l u t a r a t e w a s 1.3 m M a n d Kw. v a l u e f o r GABA w a s e v a l u a t e d t o 4.8 m M (fig. l l a , l l c ) . c) p H p r o f i l e .
2
/ 1 GABA
Fro. lle. - - Lineweaver-Burk, plot of GABAT I.
o n l y t h e s e c o n d o n e is f o u n d to e x h i b i t a s p a r t a t e aminotransferase activity.
BIOCHIMIE, 1974, 56, n ° 6-7.
S p e c i f i c a c t i v i t y of GABAx i n T r i s - H C 1 0.2 b u f f e r as a f u n c t i o n .of p H is s h o w n i n f i g u r e 12 -- The pH optimum ( p H o) f o r t h e p u r i f i e d GABA T I 'was f o u n d to b e b e t w e e n 8.6 a n d 8.7,
Aspartate aminotransferase GABA transaminase association.
IV --
GABA T e x i s t s i n t w o d i f f e r e n t m o l e c u l a r f o r m s dissociated on a DEAE cellulose column. The first
Pig brain 4-aminobutgrate 2-ketoglutarate transaminase. m o l e c u l a r f o r m (GABA~ I) is the purified enzyme. The second f o r m (GABA~II), is associated w i t h AAT activity. A study of this s e c o n d associated f o r m w a s u n t e r d a k e n to v e r i f y w h e t h e r it is an a u t h e n t i c association or an artefact. a) D e t e r m i n a t i o n of K m values. T h e a p p a r e n t Micbealis c o n s t a n t value for 2-oxoglutaratc w a s f o u n d to be 0.43 mM for GABA,r II that is to say m u c h l o w e r than that of GABATI. Km value for GABA w a s found to be 4.8 mM for the t w o m o l e c u l a r f o r m s (fig. 11 b). b) S e d i m e n t a t i o n coefficient.
831
$-alanine w a s an effective a m i n o group d o n n o r to t h e e n z y m e as w e k n o w that r e l a t i v e t r a n s a m i n a t i o n s of GABA and ~-alanine d u r i n g some steps of the p u r i f i c a t i o n seem to be strongly parallel. T h e basic p r o p e r t i e s of this e n z y m e w e r e differ e n t f r o m those o b t a i n e d f r o m p a r t i a l l y p u r i f i e d p r e p a r a t i o n s [l, 2, 73 Kr,~ values for ~ a m i n o b u l y r i c a c i d and (x k e t o g l u t a r i c acid w e r e h i g h e r than those r e p o r t e d by A. S e h o u s b o e et al. [7] in mouse brain. K m for GABA w a s near the value r e p o r t e d by I. S. Sytinsky [2] in rat brain, but K m for (~ k e t o g l u t a r i c acid w a s l o w e r than in p a r t i a l l y purified e n z y m e of rat brain. T h e optim u m pH is h i g h e r than that r e p o r t e d by W a k s m a n and Roberts [1] and Schousboe el al. [71.
T h e s e d i m e n t a t i o n coefficient for GABA,r II was f o u n d to be to 6.3 S i n d i c a t i n g a h i g h e r m o l e c u l a r w e i g h t than GABA,r I (fig. 6).
We have also found that GAB:~ 1, is able to exist in two different forms, d i s s o c i a t e d by DEAE cellulose c h r o m a t o g r a p h y .
c) If this association exists it could be possible to t r a n s f o r m the second form into the first one.
T h e first p e a k of GABAT a c t i v i t y consists of purified GABA~, the second p e a k (GABAr II) was f o u n d to be associated w i t h aspartate a m i n o t r a n s ferase activity. This peak r e p r e s e n t e d about 35 to 40 p. cent of the total GABA,r a c t i v i t y and s e e m e d to be m o r e stable than the first one (GABAT I).
H y d r o x y l a p a t i t e c h r o m a t o g r a p h y could separate AAT a c t i v i t y f r o m GABAr but w i t h ahnost total loss of the GABAT activity.
DISCUSSION. All the steps of p u r i f i c a t i o n d e s c r i b e d h e r e p r o v e d to be r e p r o d u c t i b l e . GABAT has p r e v i o u s l y been 150 fold purified f r o m mouse b r a i n ~1~, about 400 fold f r o m rat b r a i n [2] and 1 200 fold f r o m a m e m b r a n o u s f r a c t i o n of mouse b r a i n mitoc h o n d r i a [7]. In the p r e s e n t w o r k , w e purified the soluble f r a c t i o n of the e n z y m e f r o m p i g brain. This e n z y m e has been o b t a i n e d in a h i g h l y purified state, about 1 500 fold as c o m p a r e d to the specific a c t i v i t y of an i n i t i a l e x t r a c t of acetone p o w d e r . D u r i n g p u r i f i c a t i o n on DEAE cellulose w i t h Tris-Maleate, at pH 7.2 to 5.2, pig b r a i n GABAT was s e p a r a t e d into two peaks. T h e first peak consisted after the 7 th step of the most h i g h l y purified GABAT ever obtained f r o m m a m m a l i a n tissue. This f r a c t i o n w a s found to be h o m o g e n e o u s , as only one p r o t e i n b a n d ~vas o b t a i n e d after polya c r y l a m i d e e l e c t r o p h o r e s i s . T h i s band c o i n c i d e d w i t h the GABAT a c t i v i t y obtained after slicing of h a l f part of the gel. We have d e m o n s t r a t e d that this purified GABAT exists in a d i m e r i c form. The two subunits e x h i b i t i d e n t i c a l m o l e c u l a r w e i g h t and charge. T h e molecular w e i g h t of GABAT is about 105 000 ___ 5 000 and the r e l a t i v e m i g r a t i o n in the p r e s e n c e of SDS or urea s h o w e d a single band of about 51 000 _+__ 2 200.
BIOCHIMIE, 1974, 56, n ° 6-7.
After h y d r o x y l a p a t i t e c h r o m a t o g r a p h y , GABA T II w a s d i s s o c i a t e d f r o m AAT a c t i v i t y but w i t h a loss most of its activity. F o r GABAT I I the Kn: value of GABA is found to be i d e n t i c a l to that of GABATI, but K m for a k e t o g l u t a r i c acid w a s m u c h lower. Such a differ e n c e m a y be of i m p o r t a n c e for the a c t i v i t y of the e n z y m e in vioo, because as w e know, the conc e n t r a t i o n of a k e t o g l u t a r i c acid in b r a i n is s o m e w h a t l o w e r than the K,, value for this substrate [12]. The s e d i m e n t a t i o n constant for GABAT II has been found to be 6.3, w h e r e a s that of the first peak was found to be 5.5. T h e different elution p a t t e r n s o b t a i n e d after DE-52 c h r o m a t o g r a p h y different K m values and a h i g h e r s e d i m e n t a t i o n constant of this second GABAT peak led us to c o n s i d e r the e x i s t e n c e of a m o l e c u l a r c o m p l e x b e t w e e n AAT and GA13AT. Since AAT is a m i d w a y e n z y m e b e t w e e n the c i t r i c acid c y c l e and the v a m i n o b u t y r i c acid shunt, it m a y be assumed that in the brain, such a c o m p l e x m a y p l a y a role in the c o n t r o l of conc e n t r a t i o n of i n t r a c e l l u l a r pool of glutamic acid and GABA. T w o h y p o t h e s i s m a y be posed, one c o n s i d e r i n g an association b e t w e e n GABAa, and AAT and the s e c o n d one an h y b r i d i z a t i o n b e t w e e n subunits of these t w o enzymes. To p r e c i s e these possibilities, f u r t h e r i n v e s t i g a t i o n s are u n d e r study in o u r laboratory.
M. B l o c h - T a r d y , B. R o l l a n d a n d P. G o n n a r d .
832
Acknolewledgmenls. T h i s s t u d y w a s s u p p o r t e d b y a g r a n t f r o m D.R.M.E. ( C o n v e n t i o n 72 34 814). T h e a u t h o r s a r e i n d e b t e d to P r o f . M. E. G o l d b e r g (Institut Pasteur) for helpful discussions concerning t h e s u b j e c t a n d t h a n k Mrs. C. F a g e s f o r h e r t e c h n i c a l assistance.
La transaminase 4 aminobutyrique 2 cStoglutarique (E.C. 2.6.1.19) a 6t5 purifi6e 1 200 h 1 500 f o i s h p a r t i r de c e r v e a u de pore. La p u r i f i c a t i o n c o m p o r t c les ~ t a p e s s u i v a n t e s : e x t r a c t i o n , ~ l i m i n a t i o n p a r la c h a l e u r de p r o t ~ i n e s i n a c t i v e s , p r e c i p i t a t i o n s p a r le s u l f a t e d ' a m m o n i u m , f i l t r a t i o n s u r S e p h a d e x G-100, c h r o m a t o g r a p h i c s u r CM c e l l u l o s e , c h r o m a t o g r a p h i c s u r D E A E c e l l u l o s e , c h r o m a t o g r a p h i c s u r gel de p h o s p h a t e de c a l c i u m . L ' e n z y m e e s t h o m o g ~ n e c n e l e c t r o p h o r ~ s e s u r gel de p o l y a c r y l a m i d e et a p p a r a l t s o u s f o r m e d ' u n d i m ~ r e d o n t l a s o u s - u n i t 6 a u n p o i d s m o l ~ c u l a i r e de 51 000 ___ 2 200, q u a n d l'61ectrophor~se e s t effectu~e e n prSs e n c e de SDS ou SDS et ur6e. Le p o i d s m o l ~ c u l a i r e de l ' c n z y m e a ~t~ d ~ t e r m i n 6 p a r f i l t r a t i o n s u r S e p h a d e x G-100 et p e u t 5tre e s t i m 6 h 105 000 -4- 5 O00 (confirm~ p a r 61ectrophor~se s u r gel de p o l y a c r y l a m i d e SDS). Le p H o p t i m u m d ' a c t i o n de l ' c n z y m e e s t de 8.7, s o n p H i s o S l e c t r i q u e d e 5.9. L e s Km d ~ t e r m i n 6 s p a r r a p p o r t h l ' a c i d e a c ~ t o g l u t a r i q u e et h l ' a c i d e 7 a m i n o b u t y r i q u e s o n t r e s p e c t i v e m e n t de 1.3 et 4.8 raM.
BIOCHIMIE, 1974, 56, n" 6-7.
L a c h r o m a t o g r a p h i c s u r D E A E c e l l u l o s e m e t e n 6vid e n c e d e u x pies d ' a c t i v i t 6 q u i o u t 6t6 purifl~s s 6 p a r 6 m e n t . Le 1 °r pie : GABAT I r e p r 6 s e n t e l ' e n z y m e s e u l e , le 2" pie : GABAT II r e p r 6 s e n t e u n e a s s o c i a t i o n de la GABAT a v e e l ' a s p a r t a t e a m i n o t r a n s f 6 r a s e . L e s c o n s t a n t e s de s 6 d i m e n t a t i o n , les c i n 6 t i q u e s d ' a e t i v i t 6 l~ar r a p p o r t h l ' a c i d e a c 6 t o g l u t a r i q u e m o n t r e n t d e s diff6rences sensibles. L ' a s s o c i a t i o n 6 t r o i t e de la GABAT II avec l ' a s p a r t a t e aminotransf~rase nous a amen~ h envisager l'existence d'un complexe mol6culaire entre les deux enzymes. REFERENCES.
1. WAKSMANN, A..& ROBERTS, E. (1965) Biochemistry, 4, 2132-2138. 2. SYTINSKV, I. A. & VASILIJEV, (1970) Enzymologia, 32, 1-11. 3. GONNAED, P., W1CKER, A., KOUYOUMDJIAN, J. C. & BLOCH-TARI)Y, M. (1973) Biochimie, 55, 509-510. 4. DAVISS, B. J. (1964) A n n . N.Y. Acad. Sci., 121, 404410. 5. WEBER, K. & OSBOR~, M. (1969) J. Biol. Chem., 244, 4406-4413. 6. DIEZEL, W., KOPPERSEHI~GER, G. ~, HOFFMANN, E. (1972) Anal. Biochem., 48, 617-620. 7. SCHOUSBOE, A., W u , J.-Y. & ROBERTS, E. (1973) Biochemistry, 12, 2868-2873. 8. LOWRY, O. H., ROSEBROUGH, N. Y., FARE, A. & RANDALL, R. J. (1951) J. Biol. Chem., 193, 265-275. 9. JENKINS, W. T., YPHANTIS, D. A. • SIZER, 1. W . (1959) J. Biol. Chem., 234, 51-57. 10. HAKES, J. E. & VELICK, J. R. (1954) d. Biol. Chem., 207, 225-230. 11. MARGOLIASH, E. & LUSTGARTEN, J. (1962) J. Biol. Chem., 237, 3397-3405. 12. CHEN~, S. C. (1971) Handb. Neurochem., 5, 283-306.
Pig brain 4-aminobutyrate 2-ketoglutarate transaminase. Purification, kinetics and physical properties. Marcienne BLOCH-TARDY,Bernadette ROLLAND a n d P i e r r e GONNARD. D~partement de Biochimie, C.H.U. Henri-Mondor, 94010 Crdteil, France. (1-4-1974). Summary. - - Pig brain 4-am.inobutyrate 2-ketoglutarate transaminase, (E.C. 2.6.1.19) has been purified about 1200 to 1500 fold, using ammonium sulfate fractionation, heat precipitation and various chromatographic techniques. The enzyme was homogeneous in potyacrylamide gel electrophoresis and appeared as a dimer when electrophoresis was performed in presence of SDS or SDS and urea. A molecular weight of 105000 was calculated by gel filtration and confirmed by SDS polyacrvlamide gel electrophoresis. The optimum pH for the enzyme was 8.7 and the Km values for y-aminobutyrate and ¢c ketoglutarate were estimated to be 4.8 × 10-3 M and 1.3 X 10-3 M respectively. The pHt of the enzyme was 5.9 Two peaks of GABA,r were separated on DEAE Cellulose chromatography and separately purified. Differences in sedimentation constant, and enzyme kbaetic and the association of the second peak with aspartate amiuotransferase led us to distuss the possible existence of molecular complexes. INTRODUCTION. T h e r e is c o n s i d e r a b l e evidence for the involv e m e n t of v - a m i n o b u t y r i c acid (GABA) as an i n h i b i t o r y t r a n s m i t t e r at certains synapses i n the m a m m a l i a n c e n t r a l nervous system. It has become i n c r e a s i n g l y i m p o r t a n t to obtain better knowledge about the t~,o enzymes i n v o l v e d in the m e t a b o l i s m of GABA. 4 - a m i n o b u t y r a t e 2-ketoglntarate ~transaminase (E.C.2.6.1.19) (GABAT) has been p a r t i a l l y purified from rat a n d mouse b r a i n [1, 2]. This enzyme is chiefly located into m i t o c h o n drial m a t r i x (about 80 p. 100). The r e m a i n e d part is b o u n d to m i t o c h o n d r i a l m e m b r a n e s ( u n p u b l i s hed results). Recently, Schousboe, W u and Roberts [7] choosed this m e m b r a n o u s m a t e r i a l to purified GABAT from mouse b r a i n . The o b t a i n i n g of a highly purified soluble enzyme is necessary to study its localization in b r a i n a n d at p o s t s y n a p t i c level in the prospect to r e a c h a better u n d e r s t a n d i n g of the m e c h a n i s m of action of GABA i n the synapse. In this p a p e r we deal w i t h a p u r i f i c a t i o n procedure of a soluble GABA,r in pig b r a i n , a n d some p r o p e r t i e s of the enzyme. T w o peaks of enzyme activity were separated by DE 52 Cellulose c h r o m a t o g r a p h y a n d there The abbreviation used are : GABA, ~t-aminobutyric acid; GABAT, GABA transamina~se; PLP, phospho pyridoxal; GSH, reduced glutathione; AAT, aspartate aminotransf~rase, SDS, sodium dodecyl sulfate ; a KG, a ketoglutaric acid ; GA, glutamie acid.
were purified. The possibility for GABAT to form m o l e c u l a r complexes ~,ith other p r o t e i n s was discussed.
MATERIAL AND METHODS.
1. Enzyme assays. A method for d e t e r m i n a t i o n of GABA~, activity has been developed i n our l a b o r a t o r y [3]. [l-14C]a ketoglutarate a n d cold GABA were used as substrates to form [14C] glutamic acid. I n a second step, glutamate decarboxylase, commercially available from E. colt (Fhika) w a s added. The rate of 14CO2 evolved agrees stoechiometrically with the glutamate formed d u r i n g the transa m i n a t i o n , that is to say, ~¢qith GABA T activity. A less sensitive but r a p i d m e t h o d [2] has also been used as a s t a n d a r d assay method in p u r i fication p r o c e d u r e s d u r i n g w h i c h , m a n y fractions must be tested.
2. Polyacrylamide gel electrophoresis. P o l y a c r y l a m i d e gel electrophoresis was performed a c c o r d i n g to the original p r o c e d u r e of Davis [4]. The sample (200 to 300 ,~l) of enzyme solution was a p p l i e d on a 7 p. cent p o l y a c r y l a m i d e gel c o l u m n (1.3 × 8 cm) p H 8.9 w i t h a 3 p. cent c o n c e n t r a t i n g gel pH 6.7 (1.3 × 0.5 cm) using a m m o n i u m persulfate as p o l y m e r i z i n g agent. A Tris glycin buffer (0.6 g and 0.58 g glycin per 1) pH 8.3 was used as electrophoresis buffer, a n d the m i g r a t i o n was p e r f o r m e d at 4 ° for 3 h w i t h a c u r r e n t of 10 m A / c o l u m n .
824
M. Bloch-Tardy, B. R o l l a n d and P. Gonnard.
T h e samples c o n s i s t e d of 10 to 40 lug of e n z y m e c o n t a i n i n g one d r o p of B r o m o p h e n o l Blue as m a r k e r a n d t w o d r o p s of n e u t r a l glycerol. P r o t e i n b a n d s w e r e v i s u a l i z e d by Coomassie Blue R-250 s t a i n i n g for one n i g h t and d e s l a i n e d in acetic acid 10 p. cent. F o r e n z y m e assay, i m m e d i a t e l y after e l e c t r o p h o r e s i s , the gels w e r e cut in 5 m m slices, i n c u b a t e d i n d i v i d u a l l y in I ml of t r a n s a m i n a t i o n r e a c t i n g buffer at 4 ° u n d e r agitation for at least t w o hours, after that, the fractions w e r e s u b m i t t e d to f r e e z i n g and t h a w i n g and then, i n c u b a t e d at 37 ° for 60 rain.
3. Polyacrylamide - - SDS - - Urea electrophorests. Disc gel e l e c t r o p h o r e s i s in s o d i u m d o d e c y l sulfate w i t h or w i t h o u t urea 4 M w a s p e r f o r m e d as d e s c r i b e d by W e b e r and Osborn [5]. 100 td of e n z y m e solution i n c u b a t e d f o r 2 h o u r s at 37 ° or for 15 m i n at 600 in p r e s e n c e of 0.1 p. cent SDS a n d ~ m e r c a p t o e t h a n o l , w e r e a p p l i e d after addition of B r o m o p h e n o l Blue and n e u t r a l g l y c e r o l on a 5 p. cent p o l y a c r y l a m i d e ge! c o l u m n (0.5 × 1(~ cm) in a p h o s p h a t e buffer 0.1 M, p H 7.2. Protein b a n d s w e r e visualized by Coomassie Blue G-250 in TCA 5 p. cent for one night and destained w i t h 7 p. cent acetic acid as d e s c r i b e d by Diezel and al. [~6]. Gels w e r e r u n at 6 mA p e r tube at 25 ° for 3 to 4 h o u r s - - Relative m o b i l i t i e s w e r e calculated w i t h p r o t e i n s used as m a r k e r s . I n d i v i d u a l p r o t e i n c o n c e n t r a t i o n g e n e r a l l y ranged b e t w e e n : 200 and 400 i~g/ml.
4. Isoelectric focusing. T h e i s o e l e c t r i c f o c u s i n g c o l u m n (LKB 8100 Vol 110 ml) a n d p H 3-10 (40 p. cent solution) a m p h o line c a r r i e r a m p h o l y t e s w e r e o b t a i n e d f r o m LKB P r o d u c t e r AB (S-16125 BROMMA I Sweden). F i n a l c o n c e n t r a t i o n of c a r r i e r a m p h o l y t e s in the sucrose g r a d i e n t w a s 1 p. cent ( w / v ) . W h e n i s o e l e c t r i c f o c u s i n g w a s c o m p l e t e d , f r a c t i o n s of a p p r o x i m a t e l y 1 ud w e r e collected, p H and e n z y m e a c t i v i t y of e a c h sample w e r e d e t e r m i n e d . A p o w e r of 16.0'0 m W w a s a p p l i e d to the c o l u m n for 70 h o u r s all a l o n g the e l e c t r o f o c u s i n g migration. All o p e r a t i o n s w e r e p e r f o r m e d at 4 ° .
5. Determination of molecular weight by gel filtration. A 2.5 c m d i a m e t e r glass c o l u m n w a s p a c k e d at 4 ° to a bed h e i g h t of 95 c m w i t h S e p h a d e x G-100 e q u i l i b r a t e d w i t h p h o s p h a t e acetate buffer 0.1 M, p H 6.8. P r o t e i n s w e r e eluted at 4 ° w i t h the same buffer at a flow rate of about 11 m l / h o u r . T h e
BIOCHIMIE, 1974, 56, n ° 6-7.
f o l l o w i n g m a r k e r s w e r e used in v a r i o u s c o m b i n a tions to c a l i b r a t e the c o l u m n : c y t o c h r o m e c o v a l b u m i n - s e r u m a l b u m i n - alcohol d e s h y d r o g e nase - D e x t r a n blue,
6. Protein determination. P r o t e i n w a s d e t e r m i n e d r o u t i n e l y a c c o r d i n g to the m e t h o d of L o w r y and al. I8].
7. pH dependence of GABA,I,. T h e effect of pH on a c t i v i t y of a purified p r e p a r a t i o n of GABAT h a d been studied in a r a n g e of 7 to 9 w i t h Tris-HC1 buffer 0.2 M.
8. Effect of subslrate concentration on activity. Km values w e r e d e t e r m i n e d , u s i n g the i s o t o p i c t e c h n i c w i t h d i f f e r e n t substrate c o n c e n t r a t i o n s (GABA : I - 25 raM, a k e t o g l u t a r a t e : 0.2 - _9 mM).
9. Substrate specificity. T h e specificity of the e n z y m e for v a r i o u s a m i n o a c i d s w a s studied. A m i n o a c i d s in question w e r e substituted for GABA, usually at the same c o n c e n t r a t i o n (25 raM), or at t h r e e d i f f e r e n t conc e n t r a t i o n s (10 - 25 - 50, raM). 10. Sedimentation constant. A sucrose d e n s i t y g r a d i e n t f r o m 5 to 20 p. cent in saline solution ( p h o s p h a t e acetate 0.01 M, p H 6.8) w a s p r e p a r e d in tubes of SW 50 L s w i n g bucket rotor. T h e e n z y m e solution (0.1 ml c o n t a i n i n g about 20 j~g proteins) w e r e l a y e r e d on the top of the c o l u m n . T h , c e n t r i f u g a t i o n w a s p e r f o r m e d at 45.000 r.p.m, for 8 h at 3 ° in a S p i n c o L I p r e p a r a t i v e centrifuge. F o u r d r o p s f r a c t i o n s w e r e collected and tested for e n z y m e a c t i v i t y and for p r o t e i n c o n c e n t r a t i o n .
RESULTS. T h e successive steps in the p u r i f i c a t i o n of GABA~ f r o m about t h r e e p i g b r a i n s are d e s c r i b e d . Our e n z y m e p r e p a r a t i o n is 1200 to 1500 foId purified as c o m p a r e d to the o r i g i n a l a c e l b n e p o w d e r . Its specific a c t i v i t y ranges b e t w e e n 400 and 500 aM g l u t a m i c a c i d f o r m e d p e r h o u r p e r mg of protein. It can be stored at 4 ° for few days. T h e p u r i f i e d e n z y m e can be kept for a long p e r i o d at - - 20 .0 in the d a r k in p r e s e n c e of PLP, d i t h i o t r e i t o l , EDTA, as p r o t e c t o r s . E n z y m e solution is stable at pH 5.5 to pH 7. a k e t o g l u t a r i c acid as well as acetate ions w e r e f o u n d to p r o v i d e a good p r o t e c t i o n against h e a t as s h o w n in figure 1.
Pig brain $-aminobutyrale 2-keloglutarate transaminase.
825
I - - P u r i f i c a t i o n of p i g b r a i n G A B 4 r. All steps were performed at 4 ° and ration was protected from light.
the prepa-
After each step of purification, samples were taken for enzyme activity, protein concentration, and polyacrylamide electrophoresis.
D
Stev 1 : Extraction. Acetone powder obtained from brains of three pig was suspended with slow stirring in 600 ml of phosphate a c e t a t e b u f f e r p H 6.8, 0.01 M c o n t a i n i n g 10 -4 M P L P a n d G S H a n d 10 -a M E D T A and a ketoglutaric a c i d f o r 20 r a i n a n d t h e n ,
~ ! ',
I
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p h o s p h a t e a c e t a t e butler 0 . 0 1 - - p H 6 . 8
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p r e c i p i t a t i o n 56, - - p H 5 . 2 Y SUPERNATANT
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0 - - 55 p. c e n t
Y SUP~ERNA-TANT
SO,, ( N H , ) 2 - - 55 - - 75 p. c e n t Y PELLET
S e p h a d e x G - - 100 phosphate acetate 0.1 M --pH
Y
6.8
ELUATE
CM cellulose a c e t a t e buffer 0 . 0 l M -- p H 5 . 4 5 ELUATE
a c e t a t e buffer 0 . 1 M - - p H 5 . 7 D E A E cellulose Tris M a l e a t e buffer 0 . 0 1 M - - p H 7 . 2
i
-
¥
-
ELUATE
~
Tris - - M a l e a t e 0 . 0 9 M - - p H 7 . 2 to 5 . 2 g r a d i e n t
DEAE I DEAE calcium phosphate gel c h r o m a t o g r a p h y phosphate buffer 0.01 M - - p H 6 . 8 Gradient elution 0 . 0 5 to 0 . 3 M GABAT I
BIOCHIMIE, 1974, 56, n ° 6-7.
II
G A B A T II
p~l
L
FIG. 1. - - H e a t i n a c t i v a t i o n a t 56 ° f o r 30 m i n . V a r i a t i o n w i t h pH © i n p r e s e n c e of 10 m M a c e t a t e + P L P + GSH (10-4 M). • w i t h o u t a c e t a t e .
ACETONE POWDER
I
•
M. BIoch-Tardy, B. R o l l a n d and P. Gonnard.
826
homogenized in a large chilled potter homogen i z e r . A c e n t r i f u g a t i o n of 20 m i n at 17500 g i n a M S E S P 50 c e n t r i f u g e w a s p e r f o r m e d .
Step 2 : Heat precipitation. The clear supernatant was decanted and used f o r p r o t e i n p r e c i p i t a t i o n a t p H 5.3 a n d 56 ° f o r 10 m i n . T h e s u s p e n s i o n w a s c e n t r i f u g e d f o r 30 m i n a t 17500 g.
to g i v e 55 p. c e n t s a t u r a t i o n . A l o n g t h e t i m e of a d d i t i o n of t h e s o l u t i o n , p H w a s k e p t c o n s t a n t n e a r 6.8 b y a d d i t i o n of N H 4 0 H 2 N. A f t e r 30 m i n of e q u i l i b r a t i o n , t h e s u s p e n s i o n w a s c e n t r i f u g e d , a t 27500 g f o r 60 m i n . T h e p e l l e t w a s d i s c a r d e d a n d t h e s u p e r n a t a n t b r o u g h t to 75 p. c e n t s a t u r a t i o n w i t h t h e s a t u r a t e d s o l u t i o n of ammonium sulfate.
TABLE I.
Purification of pig brain GABA transaminase. Fraction
Total activity (')
Homogenate . . . . . . . . . . . . . . . . Extract .................... Heat treatment ............. (NH4) ~ SO 4 5 5 - - 7 5 p. cent.. Sephadex G - - 1 0 0 . . . . . . . . . . . CMGellulose . . . . . . . . . . . . . . D E A E Cellulose Peak I . . . . . . Peak II . . . . . 8. Hydroxylapatite Peak I . . . . . Peak II . . . . .
6900 6000 4500 2900 ~00 1740 900 750 600 70
1. 2. 3. 4. 5. 6. 7.
Total protein (g) 69 39 0.79 0.14 0.044 0.010 0.003 0,005 0.0012 0.0007
Specific activity (")
Yield p. cent
0.3 1.7 '5.7 20 52 174 300 150 500 100
I00 90 65 40 33 25 13 I0 9 1
(*) Activity m e a s u r e d w i t h the isotopic m e t h o d in I~M g l u t a m i c acid f o r m e d per hour. (**) Specific activity in .~M g l u t a m i e acid formed per h o u r per mg of protein.
The suspension was centrifuged in the same conditions than above. The last pellet was dissolv e d i n a 0.01 M p h o s p h a t e a c e t a t e b u f f e r c o n t a i ning PLP, GSH and EDTA.
Step ~ : G-IO0 Gel filtration. 5 ts*'!/,.
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.
.
.
.
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FRACTION
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FIG. 2. - - Gel filtration on Sephadex G-100. The prorein was applied a n d eluted w i t h 0.1 M sodium phosp h a t e acetate buffer pH 6.8. P r o t e i n c o n c e n t r a t i o n e - o - o is given in U.D.O. at 280 n m a n d e n z y m e activity e - - e in I~M g l u t a m i c acid f o r m e d per h o u r per ml of solution 6 ml was collected in each fractions.
Step 3 : Ammonium sulfate precipitations. The supernatant was collected and a saturated s o l u t i o n of a m m o n i u m s u l f a t e (in p h o s p h a t e a c e t a t e b u f f e r 0.01 M, p H 6.8) w a s a d d e d s l o w l y
BIOCHIMIE, 1974, 56, n ° 6-7.
A c o l u m n (5 × 100 c m P h a r m a c i a ) w a s p a c k e d with Sephadex G-100 a n d e q u i l i b r a t e d with a c e t a t e p h o s p h a t e b u f f e r 0.1 M, p H 6.8 b e f o r e t h e a p p l i c a t i o n of t h e d i s s o l v e d p e l l e t f r o m t h e 75 p. cent precipitation. The protein was eluted with t h e s a m e b u f f e r , as u s e d f o r t h e e q u i l i b r a t i o n at 20 ml~/h,o u r ,and a t y p i c a l p r o f i l e i s s h o w n i n f i g u r e 2. R e c o v e r y is a b o u t 90 p. c e n t .
Step 5 : CM Chromatography. A c o l u m n (1.5 × 30 c m ) w a s p a c k e d w i t h CM c e l l u l o s e (CM 52 W h a t m a n ) p r e v i o u s l y e q u i l i b r a t e d w i t h 0.01 M a c e t a t e b u f f e r , p H 5.45. After the application, the column was washed with the same buffer and the enzyme was eluted w i t h a l i n e a r c o n c e n t r a t i o n g r a d i e n t of s o d i u m a c e t a t e b u f f e r of p H 5.7 f r o m 0.01 M to 0.2 M.
Pig brain 4 - a m i n o b u t y r a l e T h e e n z y m e s t a r t e d to a p p e a r a t 0.08 M a n d t h e p e a k f r a c t i o n c o r r e s p o n d e d to 0.1 M b u f f e r c o n centration.
2-ketoglutarate t r a n s a m i n a s e . Tris-Maleate buffer 10-3 M E D T A .
0.01
827
M, p H
6.8
containing
Enzyme preparation was applied on the colunm. The column had been washed with the same buffer and elution accomplished a linear pH grad i e n t of 7.2 to 5.2, i n T r i s - M a l e a t e 0.09 M. T w o g o o d s e p a r a t e d GABA~ p e a k s w e r e e l u t e d . T h e first p e a k a p p e a r e d at p H 6.9 (GABA T I) a n d t h e s e c o n d o n e (GABA T II) at p H 6.7 (fig. 4).
i
Step 7 : Calcium Phosphate graphy.
A c o l u m n (1 X 13 c m ) w a s p a c k e d w i t h calcium phosphate (Biorad HT) and equilibrated w i t h 0.01 M s o d i u m p h o s p h a t e b u f f e r , p l I 6.8 .-k P I , P + GSH, 104 M. E n z y m e p r e p a r a t i o n p r e viously dialyzed one night against the equilibrated buffer, was applied on the column.
FRACTION N U M B E R
Fro. 3. - - CM cellulose c h r o m a t o g r a p h y of the gel filtration active fraction. The protein was applied in 0.01 M acetate buffer pH 5.4, w a s h e d w i t h the same huffer and eluted w i t h a g r a d i e n t of sodium actetatc buffer pH 5.7 from 0.01 M to 0.2 M. o--o GABAT activity in ~M glutamic acid f o r m e d / ml. O - - © P r o t e i n c o n c e n t r a t i o n (O.D. at 280 nm). T h e r e c o v e r y ~vas a b o u t 80 p. c e n t a n d t h e p u r i f i c a t i o n of a b o u t 3 t o 4 f o l d . T h e GABA~, active fractions were pooled and dialyzed against a T r i s - M a l e a t e b u f f e r 0.01 M a n d p H 7.2 (fig. 3).
A c o l u m n (1.5 X 30) w a s p a c k e d w i t h D E A E c e l l u l o s e (DE 52 W h a t m a n ) a n d e q u i l i b r a t e d w i t h
.%
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Step 6 : DEAE Chromatography.
i
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s'o FRACTION
i0 NUMBER
Fro. 5. - - C h r o m a t o g r a p h y on calcium p h o s p h a t e gel, The p r o t e i n was applied i n 0.01 M sodium phosp h a t e pH 6.8. E l u t i o n was p e r f o r m e d w i t h a l i n e a r g r a d i e n t of sodium p h o s p h a t e f r o m 0.05 M to 0.3 M. • a b s o r b a n c e at 280 nm. O GABA~ II activity in jxM GLU. f o r m e d per h o u r per ml. • AAT activity.
ta
200-
'-
! 10o-
........ o.7.o .....
The column was then washed with an equal v o l u m e of 0.01 M b u f f e r a n d tile e n z y m e e l u t e d u s i n g a l i n e a r c o n c e n t r a t i o n g r a d i e n t of 0.05 M p h o s p h a t e b u f f e r p H 6.8. Figure 5 GABA~ II.
gives
the
profile
of
elution
for
FRACTION NUMBER
F](;. 4. - - DEAE cellulose c h r o m a t o g r a p h y of the CM peak fraction, The p r o t e i n 'was applied in 0.01 M TrisMaleate buffer pH 6.8 and eluted w i t h a g r a d i e n t of pH f r o m 7.2 to 5.2. o--o GABA~, activity in IlxM g l u t a m i e acid formed per ml. © - - - © p r o t e i n c o n c e n t r a t i o n (O.D. 280 nm). x---$ AAT activity.
BIOCHIMIE, 1974, 56, n ° 6-7.
I I - - Phgsico-chemical caracterization of GABA T I. 1 - - P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of p u r i f i e d GABA T I, a f t e r t h e 7 th s t e p of t h e p u r i f i c a tion revealed a single protein band, carrying the GABA r activity. DO
M. Bloch-Tardy, B. Rolland and P. Gonnard.
828
2 -- Molecular weight determination.
M=
a) S e d i m e n t a t i o n c o e f f i c i e n t . The sedimentation coefficient was evaluated by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n c o m p a r a t i v e l y to three marker proteins : --
yeast alcohol dehydrogenase ( $ 2 0 , w = 7.4 S) ;
--pig heart soluble aspartate r a s e [9] ($20, w = 5.5S) ; --
T h e m o l e c u l a r w e i g h t is e s t i m a t e d to 105 000 ___ 7 000.
[10] anainotransfe-
c y t o c h r o m e c ([1!1] (S 20; w = 1.9 S).
Figure 6 shows the GABA T I a n d GABA~ II.
S RT - - X - - D (I-~ ~)
z
relative
mobilities
of
71s
: LOG. DIFFUSION
81
L
COEFFJC|ENT
FIG. 7. - - Molecular weight e s t i m a t i o n by gel filtration. The column 2.5 cm i.d. was packed w i t h Sephadex G-100 to a heigh of 85 cm. The elution volumes in milliliters (___ 1 ml) of the markers and of GABAx are indicated.
O
u~
3. D i s s o c i a t i o n of GABA T b y SDS a n d u r e a .
\
I
5
110 FRACTION
115
NUMBER
E l e c t r o p h o r e s i s o f GABA T I i n SDS p o l y a c r y l a m i d e gels w a s i n v e s t i g a t e d u n d e r t h e f o l l o w i n g conditions :
FIG. 6. - - S e d i m e n t a t i o n of GABAT and m a r k e r s in sucrose gradients. 200 ~1 of each p r e p a r a t i o n (60 to 80 [~g proteins) were sedimented in identical sucrose gradients (5 to 20 p. cent in p h o s p h a t e buffer 0.01 M, pH 6.8 -4- PLP q- GSH 10-5 M) for 8 hours at 45 000 r.p.m, in a Beckman SW 50 L Rotor. Q-) GABAT I. (~ GABAT II. .a
o
4.4 1
T h e s e d i m e n t a t i o n c o e f f i c i e n t w a s of 5.5 S f o r t h e p u r i f i e d GABA T I. b) D i f f u s i o n c o e f f i c i e n t . The diffusion coefficient was determined using a G-100 S e p h a d e x c o l u m n . T h e d e t e r m i n a t i o n o f t h e v o i d v o l u m e of t h e c o l u m n (Vo) a n d of t h e e l u t i o n v o l u m e (V) f o r p r o t e i n m a r k e r s w e r e d e t e r m i n e d i n a 1'0.0 m M s o d i u m a c e t a t e b u f f e r p H 6.8 c o n t a i n i n g 10 -5 M G S t t a n d P L P . T h e d i f f u s i o n c o e f f i c i e n t D 20 w w a s e s t i m a t e d f o r GABA T I to b e 4.45 X 107 cm~ sec-1 (fig. 7).
c) Molecular weight determination. I f w e c o n s i d e r t h a t t h e v a l u e o f v i s 0.73 as f o r g l o b u l a r p r o t e i n s , a n d t h a t ~ ---- 1, t h e m o l e c u l a r w e i g h t o f GABA.rI w i l l b e o b t a i n e d f r o m t h e equation
BIOCHIMIE, 1974, 56, n ° 6-7.
i
..J
43
42
4.1
RELATIVE
MOBILITY
Fro. 8. - - D e t e r m i n a t i o n of the subunit molecular weight of GABAT in 7 p. cent polyacrylamide gels with dodecyl sulfate. About 100 I~xg of each protein were applied to gels a f t e r dissociation for 2 h at 37 ° in sodium p h o s p h a t e pH 7 containing 0.1 p. cent SDS and 0.1 p. cent I~ mercaptoethanol. Standards are : ~3 Bovine serum a l b u m i n dimer. Ovalbumin dimer. (-3) Bovine serum a l b u m i n monomer. (~ Ovalbumin monomer. (~) Cytochrome C.
Pig brain 4-aminobutyrate 2.ketoglutarate transaminase. a) i n 0.1 p. c e n t ( w / v ) SDS a n d 0.1 ( w / v ) I~ m e r c a p t o e t h a n o l w i t h 2 h i n c u b a t i o n f o r t h e e n z y m e s o l u t i o n at 37 ° .
pH
829
E s t i m a t i o n of m o l e c u l a r w e i g h t for this b a n d , o n t h e b a s i s of t h e r e l a t i v e m o b i l i t i e s of s t a n d a r d p r o t e i n s gave a v a l u e s of 51 000 ± 2 200. T h e s e r e s u l t s s u g g e s t e d t h a t GABA T I w a s a d i m e r a n d
..t"
t* vf
u /
.,/,.,..,j4""
/" /"
t,
>
S*"
!
PURIFICATION
t ! Z0
40
$0
,~
80
TUBE
NUMBER
FI.?,. 9. - - Isoelectrofoeusing of partially purified GABAT (after gel filtration) pH range of a m p h o l i n e s was 3 to 10 e - - e GabaT activity in uM glutamie acid formed per hour, per ml. e - e - e pH in each fraction.
STEPS
Fro. 10a. - - Relative degrees of pnrifieation during f o u r steps. (~) Aspartate transamination. ~) ~ Alanine transamination. GABA transamination.
~) in 1 p. c e n t ( w / v ) SDS a n d 1 p. c e n t ( w / v ) mercaptoethanol, and 4 M urea with incubation at 45 ° f o r 1 h. V) i n 0.1 p. c e n t ( w / v ) SDS a n d ~ m e r c a p t o e t h a n o l , w i t h i n c u b a t i o n at 60 ° f o r 15 rain. 5) i n 8 M u r e a + 0.1 p. c e n t ( w / v ) 13 m e r c a p t o e t h a n o l w i t h l h i n c u b a t i o n at 45 °. All gels s h o w e d a s i n g l e b a n d of p r o t e i n .
TABLE 1I. SubstraL
Relative activity p. cent
o
~r . . . . .
1
~--;~
3
PURIFICATION
4
5
STEP
Fro. 10b. - - Specific activity in :aM of glutamic acid f o r m e d per hour, per mg proteins of the three activities during the four first steps of the purification. (9 GABA t r a n s a m i n a t i o n . ~) I~ alanine t r a n s a m i n a t i o n . (~) Aspartate t r a n s a m i n a t i o n . that the two s u b u n i t s w e r e i d e n t i c a l in m o l e c u l a r w e i g h t a n d i n c h a r g e (fig. 8). 4. I s o e l e c t r i c f o c u s i n g .
Gaba . . . . . . . . . . . . . . . . . . . . . . . . . Glutamie acid . . . . . . . . . . . . . . . . . . A!anine . . . . . . . . . . . . . . . . . . . . . Tryptophane . . . . . . . . . . . . . . . . . . Leucine . . . . . . . . . . . . . . . . . . . . . . . Phenylalanine . . . . . . . . . . . . . . . . . M~thionine . . . . . . . . . . . . . . . . . . . . Glyeine . . . . . . . . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . . . . . . . . Arginine . . . . . . . . . . . . . . . . . . . . . . Threonine . . . . . . . . . . . . . . . . . . . . . Proline . . . . . . . . . . . . . . . . . . . . . . . . Taurine . . . . . . . . . . . . . . . . . . . . . . . Hypotaurine . . . . . . . . . . . . . . . . . . Para Chloro Phenyl Gaba . . . . . . .
BIOCHIMIE, 1974, 56, n ° 6-7.
100 136 126 21 10 3 0
T h e d e t e r m i n a t i o n of t h e i s o e l e c t r i c p o i n t w a s o b t a i n e d w i t h p a r t i a l l y p u r i f i e d e n z y m e ( a f t e r gel f i l t r a t i o n ) b e f o r e t h e s e p a r a t i o n o n DE-52 c o l u m n , b e c a u s e of t h e f r a g i l i t y of t h e t w o GABA.r f r a c t i o n s a n d t h e p o w e r f u l l d e n a t u r i n g a c t i o n of s y n t h e t i c c a r r i e r a m p h o l y t e s ; p H i w a s e v a l u a t e d to 5.8 - 5.9 u s i n g a m p h o l y t e s f r o m p H 3 to 10 (fig. 9).
~a ~a
III - -
Fnnclionnal properties of GABAT 1.
aa
a) S u b s t r a t e s p e c i f i c i t y . am
T a b l e II s h o w s t h a t c e r t a i n L - a - a m i n o a c i d s c a n s e r v e as s u b s t r a t e s of GABA z.
M. B l o c h - T a r d y , B. R o l l a n d and P. Gonnard.
830
A s p a r t i c a c i d is a s u b s t r a t e u n t i l t h e D E A E s t e p . A f t e r s e p a r a t i o n i n t o p e a k I a n d p e a k II, .1. V
To prove whether a protein fraction possesses a single enzyme with two catalytic functions, rather than two separate enzymes with two d i s t i n c t f u n c t i o n s , i t i s c u s t o m a r y to f o l l o w t h e r a t i o of t h e t w o c a t a l y t i c f u n c t i o n s t h r o u g h o u t a s e r i e of p r o t e i n f r a c t i o n a t i o n p r o c e d u r e s . W e s t u d i e d t h e s e a c t i v i t i e s o v e r t h e f o u r first s t e p s of t h e p u r i f i c a t i o n v i s - h - v i s GABA a n d a l a n i n e a n d f o u n d t h e m e x a c t l y p a r a l l e l (fig. 10a et b).
/
I 1
I 2
I 3
I 4
~
• 1
T h i s m e a n s t h a t ~ a l a n i n e is t r a n s a m i n a t e d to t h e s a m e e x t e n t as GABA. ~ a l a n i n e is a g o o d s u b s t r a t e f o r GABA T w h e r e a s a s p a r t i c a c i d s e a m s to b e t h e s u b s t r a t e of a s e p a r a t e e n z y m e o r of a n o t h e r m o l e c u l a r f o r m of GABA T.
(o K G )
Fro. l l a . - - Lineweaver-Burk, plot of GABAT I. a k e t o g l u t a r a t e c o n c e n t r a t i o n s varied f r o m 0..9. to 2 mM a n d for GABA f r o m 1 to 25 mM. A p p a r e n t reciprocal K,n values 'were o b t a i n e d for one s u h s t r a t e at infinite c o n c e n t r a t i o n of the other.
/ xx
o.,f
x
0.3
/
0.2
i
r
1
I
3
i
/
x
/
.
I
5
1 ~KG
r
0.,
FIG. l l b . - - Lineweaver-Burk, plot of GABAT II.
r
7
7.,
'
~
815
~
I
,,,,
.
FIG. 12. - - Specific activity of GABAT as a f u n c t i o n of pH in the i n c u b a t i o n m e d i u m . The isotopic assay was employed a n d Tris-HC1 buffer (0.2 M - p H 7 to 9) was used to o b t a i n the desired pH.
b) D e t e r m i n a t i o n
of K,n v a l u e s .
The apparent Michaelis constant value for 2 - o x o g l u t a r a t e w a s 1.3 m M a n d Kw. v a l u e f o r GABA w a s e v a l u a t e d t o 4.8 m M (fig. l l a , l l c ) . c) p H p r o f i l e .
2
/ 1 GABA
Fro. lle. - - Lineweaver-Burk, plot of GABAT I.
o n l y t h e s e c o n d o n e is f o u n d to e x h i b i t a s p a r t a t e aminotransferase activity.
BIOCHIMIE, 1974, 56, n ° 6-7.
S p e c i f i c a c t i v i t y of GABAx i n T r i s - H C 1 0.2 b u f f e r as a f u n c t i o n .of p H is s h o w n i n f i g u r e 12 -- The pH optimum ( p H o) f o r t h e p u r i f i e d GABA T I 'was f o u n d to b e b e t w e e n 8.6 a n d 8.7,
Aspartate aminotransferase GABA transaminase association.
IV --
GABA T e x i s t s i n t w o d i f f e r e n t m o l e c u l a r f o r m s dissociated on a DEAE cellulose column. The first
Pig brain 4-aminobutgrate 2-ketoglutarate transaminase. m o l e c u l a r f o r m (GABA~ I) is the purified enzyme. The second f o r m (GABA~II), is associated w i t h AAT activity. A study of this s e c o n d associated f o r m w a s u n t e r d a k e n to v e r i f y w h e t h e r it is an a u t h e n t i c association or an artefact. a) D e t e r m i n a t i o n of K m values. T h e a p p a r e n t Micbealis c o n s t a n t value for 2-oxoglutaratc w a s f o u n d to be 0.43 mM for GABA,r II that is to say m u c h l o w e r than that of GABATI. Km value for GABA w a s found to be 4.8 mM for the t w o m o l e c u l a r f o r m s (fig. 11 b). b) S e d i m e n t a t i o n coefficient.
831
$-alanine w a s an effective a m i n o group d o n n o r to t h e e n z y m e as w e k n o w that r e l a t i v e t r a n s a m i n a t i o n s of GABA and ~-alanine d u r i n g some steps of the p u r i f i c a t i o n seem to be strongly parallel. T h e basic p r o p e r t i e s of this e n z y m e w e r e differ e n t f r o m those o b t a i n e d f r o m p a r t i a l l y p u r i f i e d p r e p a r a t i o n s [l, 2, 73 Kr,~ values for ~ a m i n o b u l y r i c a c i d and (x k e t o g l u t a r i c acid w e r e h i g h e r than those r e p o r t e d by A. S e h o u s b o e et al. [7] in mouse brain. K m for GABA w a s near the value r e p o r t e d by I. S. Sytinsky [2] in rat brain, but K m for (~ k e t o g l u t a r i c acid w a s l o w e r than in p a r t i a l l y purified e n z y m e of rat brain. T h e optim u m pH is h i g h e r than that r e p o r t e d by W a k s m a n and Roberts [1] and Schousboe el al. [71.
T h e s e d i m e n t a t i o n coefficient for GABA,r II was f o u n d to be to 6.3 S i n d i c a t i n g a h i g h e r m o l e c u l a r w e i g h t than GABA,r I (fig. 6).
We have also found that GAB:~ 1, is able to exist in two different forms, d i s s o c i a t e d by DEAE cellulose c h r o m a t o g r a p h y .
c) If this association exists it could be possible to t r a n s f o r m the second form into the first one.
T h e first p e a k of GABAT a c t i v i t y consists of purified GABA~, the second p e a k (GABAr II) was f o u n d to be associated w i t h aspartate a m i n o t r a n s ferase activity. This peak r e p r e s e n t e d about 35 to 40 p. cent of the total GABA,r a c t i v i t y and s e e m e d to be m o r e stable than the first one (GABAT I).
H y d r o x y l a p a t i t e c h r o m a t o g r a p h y could separate AAT a c t i v i t y f r o m GABAr but w i t h ahnost total loss of the GABAT activity.
DISCUSSION. All the steps of p u r i f i c a t i o n d e s c r i b e d h e r e p r o v e d to be r e p r o d u c t i b l e . GABAT has p r e v i o u s l y been 150 fold purified f r o m mouse b r a i n ~1~, about 400 fold f r o m rat b r a i n [2] and 1 200 fold f r o m a m e m b r a n o u s f r a c t i o n of mouse b r a i n mitoc h o n d r i a [7]. In the p r e s e n t w o r k , w e purified the soluble f r a c t i o n of the e n z y m e f r o m p i g brain. This e n z y m e has been o b t a i n e d in a h i g h l y purified state, about 1 500 fold as c o m p a r e d to the specific a c t i v i t y of an i n i t i a l e x t r a c t of acetone p o w d e r . D u r i n g p u r i f i c a t i o n on DEAE cellulose w i t h Tris-Maleate, at pH 7.2 to 5.2, pig b r a i n GABAT was s e p a r a t e d into two peaks. T h e first peak consisted after the 7 th step of the most h i g h l y purified GABAT ever obtained f r o m m a m m a l i a n tissue. This f r a c t i o n w a s found to be h o m o g e n e o u s , as only one p r o t e i n b a n d ~vas o b t a i n e d after polya c r y l a m i d e e l e c t r o p h o r e s i s . T h i s band c o i n c i d e d w i t h the GABAT a c t i v i t y obtained after slicing of h a l f part of the gel. We have d e m o n s t r a t e d that this purified GABAT exists in a d i m e r i c form. The two subunits e x h i b i t i d e n t i c a l m o l e c u l a r w e i g h t and charge. T h e molecular w e i g h t of GABAT is about 105 000 ___ 5 000 and the r e l a t i v e m i g r a t i o n in the p r e s e n c e of SDS or urea s h o w e d a single band of about 51 000 _+__ 2 200.
BIOCHIMIE, 1974, 56, n ° 6-7.
After h y d r o x y l a p a t i t e c h r o m a t o g r a p h y , GABA T II w a s d i s s o c i a t e d f r o m AAT a c t i v i t y but w i t h a loss most of its activity. F o r GABAT I I the Kn: value of GABA is found to be i d e n t i c a l to that of GABATI, but K m for a k e t o g l u t a r i c acid w a s m u c h lower. Such a differ e n c e m a y be of i m p o r t a n c e for the a c t i v i t y of the e n z y m e in vioo, because as w e know, the conc e n t r a t i o n of a k e t o g l u t a r i c acid in b r a i n is s o m e w h a t l o w e r than the K,, value for this substrate [12]. The s e d i m e n t a t i o n constant for GABAT II has been found to be 6.3, w h e r e a s that of the first peak was found to be 5.5. T h e different elution p a t t e r n s o b t a i n e d after DE-52 c h r o m a t o g r a p h y different K m values and a h i g h e r s e d i m e n t a t i o n constant of this second GABAT peak led us to c o n s i d e r the e x i s t e n c e of a m o l e c u l a r c o m p l e x b e t w e e n AAT and GA13AT. Since AAT is a m i d w a y e n z y m e b e t w e e n the c i t r i c acid c y c l e and the v a m i n o b u t y r i c acid shunt, it m a y be assumed that in the brain, such a c o m p l e x m a y p l a y a role in the c o n t r o l of conc e n t r a t i o n of i n t r a c e l l u l a r pool of glutamic acid and GABA. T w o h y p o t h e s i s m a y be posed, one c o n s i d e r i n g an association b e t w e e n GABAa, and AAT and the s e c o n d one an h y b r i d i z a t i o n b e t w e e n subunits of these t w o enzymes. To p r e c i s e these possibilities, f u r t h e r i n v e s t i g a t i o n s are u n d e r study in o u r laboratory.
M. B l o c h - T a r d y , B. R o l l a n d a n d P. G o n n a r d .
832
Acknolewledgmenls. T h i s s t u d y w a s s u p p o r t e d b y a g r a n t f r o m D.R.M.E. ( C o n v e n t i o n 72 34 814). T h e a u t h o r s a r e i n d e b t e d to P r o f . M. E. G o l d b e r g (Institut Pasteur) for helpful discussions concerning t h e s u b j e c t a n d t h a n k Mrs. C. F a g e s f o r h e r t e c h n i c a l assistance.
La transaminase 4 aminobutyrique 2 cStoglutarique (E.C. 2.6.1.19) a 6t5 purifi6e 1 200 h 1 500 f o i s h p a r t i r de c e r v e a u de pore. La p u r i f i c a t i o n c o m p o r t c les ~ t a p e s s u i v a n t e s : e x t r a c t i o n , ~ l i m i n a t i o n p a r la c h a l e u r de p r o t ~ i n e s i n a c t i v e s , p r e c i p i t a t i o n s p a r le s u l f a t e d ' a m m o n i u m , f i l t r a t i o n s u r S e p h a d e x G-100, c h r o m a t o g r a p h i c s u r CM c e l l u l o s e , c h r o m a t o g r a p h i c s u r D E A E c e l l u l o s e , c h r o m a t o g r a p h i c s u r gel de p h o s p h a t e de c a l c i u m . L ' e n z y m e e s t h o m o g ~ n e c n e l e c t r o p h o r ~ s e s u r gel de p o l y a c r y l a m i d e et a p p a r a l t s o u s f o r m e d ' u n d i m ~ r e d o n t l a s o u s - u n i t 6 a u n p o i d s m o l ~ c u l a i r e de 51 000 ___ 2 200, q u a n d l'61ectrophor~se e s t effectu~e e n prSs e n c e de SDS ou SDS et ur6e. Le p o i d s m o l ~ c u l a i r e de l ' c n z y m e a ~t~ d ~ t e r m i n 6 p a r f i l t r a t i o n s u r S e p h a d e x G-100 et p e u t 5tre e s t i m 6 h 105 000 -4- 5 O00 (confirm~ p a r 61ectrophor~se s u r gel de p o l y a c r y l a m i d e SDS). Le p H o p t i m u m d ' a c t i o n de l ' c n z y m e e s t de 8.7, s o n p H i s o S l e c t r i q u e d e 5.9. L e s Km d ~ t e r m i n 6 s p a r r a p p o r t h l ' a c i d e a c ~ t o g l u t a r i q u e et h l ' a c i d e 7 a m i n o b u t y r i q u e s o n t r e s p e c t i v e m e n t de 1.3 et 4.8 raM.
BIOCHIMIE, 1974, 56, n" 6-7.
L a c h r o m a t o g r a p h i c s u r D E A E c e l l u l o s e m e t e n 6vid e n c e d e u x pies d ' a c t i v i t 6 q u i o u t 6t6 purifl~s s 6 p a r 6 m e n t . Le 1 °r pie : GABAT I r e p r 6 s e n t e l ' e n z y m e s e u l e , le 2" pie : GABAT II r e p r 6 s e n t e u n e a s s o c i a t i o n de la GABAT a v e e l ' a s p a r t a t e a m i n o t r a n s f 6 r a s e . L e s c o n s t a n t e s de s 6 d i m e n t a t i o n , les c i n 6 t i q u e s d ' a e t i v i t 6 l~ar r a p p o r t h l ' a c i d e a c 6 t o g l u t a r i q u e m o n t r e n t d e s diff6rences sensibles. L ' a s s o c i a t i o n 6 t r o i t e de la GABAT II avec l ' a s p a r t a t e aminotransf~rase nous a amen~ h envisager l'existence d'un complexe mol6culaire entre les deux enzymes. REFERENCES.
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