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Purification and properties of rat brain succinic semialdehyde dehydrogenase
BIOCHIMIE, 1977, 59, 257-268.
Purification and properties of rat brain succinic semialdehyde dehydrogenase. C. CASH <>, L. CIESIELSKI, M. MAITRE a n d P. MANDEL. Centre de N e u r o c h i m i e du CNRS, Institut de Chimie Biologique, 11 rue H n m a n n , 67000 Strasbourg, France. (7-2-1977). Summary. - - Succinic semialdehyde dehydrogenase from rat brain has been purified to electrophoretic homogeneity. It has a molecular veeight of about 140,000 and is composed of Vwo apparently identical subunits. The reaction eatalized by the pure protein is entirely dependent on endogenous - - S H groups. The Km (limits) for NAD and sueeinic semialdehyde are 2 × 10-5 M and 1 X 10-4 M respectively at the optimum pH of 8.6. Inhibition studies showy that the reaction mechanism is a compulsory ordered one where NAD binds first followed by sueeinic semialdehyde. INTRODUCTION. 4-amino b u t y r i c acid (GABA) is d e g r a d e d in the b r a i n to s u c c i n i c acid by the successive actions of a t r a n s a m i n a s e (GABA-T) and a d e h y d r o g e n a s e , s u c c i n i c s e m i a l d e h y d e d e h y d r o g e n a s e (SSADH). T h e first of these e n z y m e s f r o m several sources has been p u r i f i e d to h o m o g e n i t y and e x t e n s i v e l y studied [1, 2, 3, 4]. H o w e v e r , a p a r t f r o m some wor;k on p a r t i a l l y purified SSADH [5, 6, 7, 8], m u c h less is knoxvn about this e n z y m e w h o s e a c t i v i t y is so great in b r a i n that the c e r e b r a l level of s u c c i n i c s e m i a l d e h y d e (SSA) is ahvays v e r y lo'w. It is thus i n t e r e s t i n g to study the fundam e n t a l c h a r a c t e r i s t i c s of this e n z y m e ~rhich controls the p r i n c i p a l r o u t e by ' w h i c h SSA is catabolized. In this p r e s e n t w o r k , we h a v e purified rat b r a i n SSADH to h o m o g e n i t y , and studied some of its s t r u c t u r a l and c a t a l y t i c p r o p e r t i e s . MATERIALS AND METHODS.
Reagents. S u c e i n i c s e m i a l d e h y d e was p r e p a r e d by h y d r o lysis of v - e t h o x y - b u t y r o l a c t o n e , a gift f r o m Dr. C. G. W e r m u t h , S t r a s b o u r g ; o t h e r reagents ~vere of a n a l y t i c a l grade. E n z y m e assays. F o r q u a l i t a t i v e assays of c o l u m n elution fractions, 50 ~1 aliquots (1 ,~I aliquots f r o m the affinity Abbreviations : GABA-T : 4 aminobutyrate transaminase (EC. 2.6.1. 19). GABA: 4 aminobutyrate. SSA : Suceinic semi aldehyde. SSADH : suceinic semi aldehyde dehydrogenase (EC 1.2.1.24). ADH : AleohoI dehydrogenase (EC 1.1.1.71). TCA : Triehloraeetie acid. SDS : sodium dodeeyl sulfate. To whom all eorrespondenee should be addressed.
column), w e r e p i p e t t e d into tubes c o n t a i n i n g cold buffer consisting of 100 mM Tris/HC1 p H 8.6, 50 mM KCI, 3 × 10 .4 M NAD, 0.1 mM EDTA a n d 20 mM m e r c a p t o e t h a n o l . T h e r e a c t i o n w a s started by r a p i d a d d i t i o n of SSA to a final c o n c e n t r a t i o n of 1 × 10 -4 M in a total v o l u m e of 1 ml, and the samples w e r e i n c u b a t e d at 37°C for five minutes. T h e r e a c t i o n w a s s t o p p e d by i m m e r s i n g the tubes in b o i l i n g w a t e r for five m i n u t e s and after cooling, the fluorescence was r e a d in a Zeiss f l u o r i m e t e r at e x c i t a t i o n 355 n m ; and emission 470 n m ( u n c o r r e c t e d values). F o r q u a n t i t a t i v e e n z y m e assays at each step of the p u r i f i c a t i o n p r o c e d u r e , the same i n c u b a t i o n m e d i u m as above w a s used w i t h the a d d i t i o n of T r i t o n X - 1 0 0 to a final c o n c e n t r a t i o n of 1 p e r cent. A d d i t i o n of T r i t o n i n c r e a s e d the a c t i v i t y y i e l d in p a r t i c u l a t e f r a c t i o n s but h a d no significant effect w h e n clear e n z y m e samples ~x'ere used. T h e i n c u b a t i o n m e d i u m w a s m a i n t a i n e d at 37°C in t h e r m o s t a t e d cuvettes in the f l u o r i m e t e r , and the r e a c t i o n w a s started by a d d i t i o n of 50 td of suitably d i l u t e d enzyme. T h e rate of i n c r e a s e of fluorescence was r e c o r d e d d i r e c t l y and the c h a n g e p e r m i n u t e w a s c o m p a r e d to the fluoresc e n c e u n d e r the same c o n d i t i o n s of a solution of NADH p r e v i o u s l y c a l i b r a t e d by its o p t i c a l density at 340 nm. F o r the p e r i o d of the m e a s u r e m e n t s the rate of i n c r e a s e of f l u o r e s c e n c e w a s constant.
Protein determinations. T h e m e t h o d of Lo~vry et al [9] w a s used for s a m p l e s o b t a i n e d b e f o r e D E A E cellulose c h r o m a t o g r a p h y . F o r the later stages, w h e r e the p r o t e i n c o n c e n t r a t i o n w a s v e r y low, the F l u o r e s c a m i n e
258
C. Cash, L. Ciesielski, M. Maitre and P. Mandel.
m e t h o d [10] w a s used. At the c o n c e n t r a t i o n s used, AMP and m e r c a p t o e t h a n o l did not interfere. Bovine s e r u m a l b u m i n 'was used as a s t a n d a r d .
Gel Electrophoresis. 7.5 p e r cent P o l y a c r y l a m i d e gels w e r e p r e p a r e d a c c o r d i n g to the t e c h n i q u e of J o i v i n et aI [ l l i . E l e c t r o p h o r e s i s w a s c a r r i e d out for about 12 h at 4°C and 3 m i l l i a m p s p e r gel. T h e b a n d s w e r e stained in Coomassie b r i l l a n t blue. ENZYME PURIFICATION.
Extraction. 40 W i s t a r rats w e r e killed by d e c a p i t a t i o n , the b r a i n s r a p i d l y r e m o v e d and s u s p e n d e d in a total v o l u m e of 240 ml of ice cold w a t e r c o n t a i n i n g 5 mM m e r c a p t o e t h a n o l a n d 0.1 mM EDTA. T h e suspension w a s h o m o g e n i z e d using a c o m m e r c i a l food b l e n d e r at m a x i m u m speed for a total of six minutes, a l l o w i n g i n t e r v a l s to p r e v e n t significant heating. T h e h o m o g e n a t e w a s c e n t r i f u g e d at 50,000 g for 30 m i n u t e s at 4°C, and the s¢lpernatants decanted. T h e pellets w e r e r e s u s p e n d e d in the above m e d i u m and the b l e n d i n g and c e n t r i fugation w a s r e p e a t e d . T h e s u p e r n a t a n t s w e r e pooled.
Ammonium sulphate fractionation. T h e p o o l e d s u p e r n a t a n t s w e r e b r o u g h t to 45 p e r cent s a t u r a t i o n at ice t e m p e r a t u r e using solid (NH~). SO~, the pH b e i n g m a i n t a i n e d at 7.2 by a d d i t i o n of 2M (NH4)OH. T h e suspension w a s cent r i f u g e d at 30,000 g for 30 mn ; the pellets w e r e d i s c a r d e d and the s u p e r n a t a n t b r o u g h t to 70 p e r cent (NHa) 2 SO 4 s a t u r a t i o n as above. After centrifugation the r e s u l t a n t pellets w e r e dissolved in the m i n i m u m v o l u m e (about 5 inl) of 2 mM p h o s p h a t e buffer c o n t a i n i n g 5 mM m e r c a p t o e t h a n o l and 0.1 mM EDTA.
Column chromatography. All c h r o m a t o g r a p h y 'was p e r f o r m e d in a cold r o o m at about 4°C and all e q u i l i b r a t i o n and elution buffers c o n t a i n e d 5 mM m e r c a p t o e t h a n o l and 0.1 mM EDTA. Columns w e r e e x t e n s i v e l y equilib r a t e d w i t h starting buffer b e f o r e use.
Gel filtration. This w a s p e r f o r m e d on a c o l u m n of Sephader, G200 (1 m length, 2.6 cm d i a m e t e r ) . T h e e n z y m e w a s eluted 'with 2 mM p h o s p h a t e buffer p H 7.2 and c o l l e c t e d in 5 ml fractions.
Ion exchange chromatography. T h e active f r a c t i o n f r o m S e p h a d e x G200 chrom a t o g r a p h y w a s absorbed onto a c o l u m n of D E A E Cellulose ( W h a t m a n DE52 ; 15 cm length, 2.6 cm
BIOCHIMIE, 1977, 59, n ° 3.
d i a m e t e r ) . T h e colunln was e x t e n s i v e l y r i n c e d w i t h s t a r t i n g buffer before elution w i t h 500 ml of a l i n e a r g r a d i e n t of 0 to 45 mM KC1 c o n t a i n i n g 2 mM p h o s p h a t e buffer p H 7.2. T h e eluate w a s c o l l e c t e d in 5 ml fractions.
Affinity chromatography. T h e active f r a c t i o n f r o m D E A E cellulose chrom a t o g r a p h y w a s a b s o r b e d onto a c o l u m n of 5' AMP substituted S e p h a r o s e ( P h a r m a c i a Sepharose 4B, 5 cm length, 0.5 cm d i a m e t e r ) . T h e c o l u m n w a s w a s h e d ~vith 10 mM p h o s p h a t e buffer p H 7.2 and eluted in 0.5 ml f r a c t i o n s w i t h 1 mM AMP in the same buffer. Glycerol to a c o n c e n t r a tion of 10 p e r cent w a s a d d e d to the e n z y m e fraction before storage a t - - 3 0 ° C . P H Y S I C A L P R O P E R T I E S AND S T R U C T U R E ,
Molecular weight of native enzyme. A c o l u m n of S e p h a d e x G200 (2.6 × 100 cnl) w a s e q u i l i b r a t e d w i t h 10 mM p h o s p h a t e buffer p H 7.2 c o n t a i n i n g 100 mM KC1. SSADH plus the f o l l o w i n g m o l e c u l a r w e i g h t m a r k e r s w e r e a p p l i e d to the c o l u m n in a total v o l u m e of 1 m l : o v a l b u m i n , human v-globulin, h o r s e h a e m o g l o b i n , yeast a l c o h o l d e h y d r o g e n a s e and d e x t r a n blue. T h e p r o t e i n s w e r e eluted 'with e q u i l i b r a t i o n buffer in 5 ml fractions. T h e peaks w e r e d e t e r m i n e d by t h e i r a b s o r b a n c e at 280 n m ; e x c e p t ADH a n d SSADH w h i c h w e r e d e t e r u l i n e d e n z y m a t i c a l l y .
Molecular ,weight of subunits. This w a s d e t e r m i n e d on 10 p e r cent p o l y a c r y l a m i d e gels in 0.1 p e r cent SDS and 6 M u r e a as d e s c r i b e d by W e b c r and Osborn [..12]. 20 ~g e n z y m e w a s i n c u b a t e d at 37°C in e l e c t r o p h o r e s i s buffer c o n t a i n i n g 0.1 p e r cent SDS, 5 M u r e a and 20 mM m e r c a p t o e t h a n o l . T h e f o l l o w i n g molecular weight markers were u s e d : myoglobin (17,000) c h y x n o t r y p s i n o g e n A (25,000) p e p s i n (35,000) o v a l b u m i n (45,000) and h u m a n a l b u m i n m o n o m e r (69,000). B r o m o p h e n o l blue w a s used to m a r k the front. The m o b i l i t i e s w e r e c a l c u l a t e d a c c o r d i n g to the m e t h o d of W e b e r and Osborn
[12]. Subunit binding. About 20 r~g of e n z y m e w a s b r o u g h t to pH 5 with KH2PQ ; urea and mercaptoethanol were a d d e d to final c o n c e n t r a t i o n s of 6 M and 60 mM r e s p e c t i v e l y . T h e e n z y m e w a s i n c u b a t e d at 40°C for I h, then b r o u g h t to p H 8 w i t h 0.1 M NaOH. l o d o a c e t a m i d e 'was a d d e d to a c o n c e n t r a t i o n of 200 mM and the sample i n c u b a t e d for 30' at 40 °. T h e t r e a t e d e n z y m e w a s s u b m i t t e d to e l e c t r o p h o rests on an SDS gel as d e s c r i b e d u n d e r <( Mole-
R a t brain succinic s e m i a l d e h y d e dehydrogenase. c u l a r w e i g h t of subunits >>. At the same time, a n o t h e r e n z y m e sample w a s run w h i c h had been t r e a t e d w i t h urea and SDS, but not m e r c a p t o ethanol.
Isoelectric focussing. This was p e r f o r m e d on c y l i n d r i c a l gels (5 m m by 7.3 cm) using the system devised by Alien E13]. The final c o n c e n t r a t i o n of a c r y l a m i d e in the gel was 6 p e r cent w i t h 2.5 p e r cent A m p h o l i n e pH 3 to 10. The e l e c t r o d e solutions 'were 0.1 M HC1 for the l o w e r bath (anode) and 0.15 M e t h a n o l a m i n e for the u p p e r bath (cathode) E l e c t r o p h o r e s i s was c a r r i e d out for about 8 hours at 4°C w i t h a c o n s t a n t c u r r e n t of 1 mA per gel, the voltage starting at 200 volts and r e a c h i n g 500 volts at the end of the run. Then, the gel was fixed for 2 h o u r s w i t h 2 changes of 12 p e r cent TCA, and afterw a r d s stained for 4 hours in a solution c o n t a i n i n g acetic acid, ethanol and 0.2 p e r cent aqueous Coomassie b r i l l i a n t blue in the p r o p o r t i o n s 1 : 2.5 : 2.5 respectively. The gel 'was destained w i t h several changes of acetic acid, ethanol, w a t e r , 1 : 2 . 5 : 6 . 5 by volume. An u n s t a i n e d gel focussed u n d e r i d e n t i c a l c o n d i t i o n s but ~without e n z y m e was cut into p o r t i o n s of about 2 mm. E a c h p o r tion w a s e x t r a c t e d o v e r n i g h t at 4°C w i t h one m l of w a t e r . The p H of each e x t r a c t Yeas m e a s u r e d to d e t e r m i n e the true p H gradient.
N-terminal analysis. The d a n s y l a t i o n m e t h o d of Zanetta was used E14] starting w'ith 100 :~g of p r o t e i n p r e c i p i t a t e d w i t h 10 p e r cent TCA and w a s h e d w i t h 1 ml M HC1 and t h e n t~vice w i t h 1 ml acetone. The chrom a t o g r a p h y 'was p e r f o r m e d both on silica gel and p o l y a m i d e sheets. The spots c o r r e s p o n d i n g to (x-dansyl-leucine and to e-dansyl-lysine w e r e eluted f r o m the silica gel and the fluorescence m e a s u r e d as d e s c r i b e d by Zanetta el at.
Amino acid composition. About 100 ~tg of e n z y m e 'was p r e c i p i t a t e d w i t h 10 p e r cent TCA acid and after c e n t r i f u g a t i o n , the p r o t e i n was h y d r o l y s e d in a sealed tube in 500 ~xl 6 M HC1 for 18 h o u r s at l l 0 ° C . T h e a m i n o - a c i d composition was determined with a Technicon a m i n o acid analyser. T r y p t o p h a n ~ . h i c h is d e s t r o y e d by this m e t h o d 'was not d e t e r m i n e d . KINETIC STUDIES.
Determination of Michaelis constants. The w e r e p e r f o r m e d at (a) 25°C and (b) 37°C. The i n c u b a t i o n m e d i u m consisted of 100 mM Tris HC1 c o n t a i n i n g 50 mM KC1, 20 mM m e r c a p t o ethanol and 0.1 mM EDTA. Various substrate con-
BIOCHIMIE, 1977, 59, n" 3.
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c e n t r a t i o n s w e r e a d d e d at c o n s l a n t volume. The r e a c t i o n w a s started by a d d i t i o n of p u r e e n z y m e and the rate of i n c r e a s e of fluorescence m e a s u r e d directly. The slopes vcere a l w a y s o b t a i n e d f r o m the l i n e a r part of the r e c o r d i n g and w e r e thus initial rates.
Reverse reaction. The rate of the r e v e r s e r e a c t i o n ~vas m e a s u r e d by d e t e r m i n i n g the a m o u n t of NAD f o r m e d by the strong alkali m e t h o d of L o w r y et al [15] at various time intervals up to 7 h o u r s at 37°C in the usual i n c u b a t i o n m e d i u m but c o n t a i n i n g the reverse substrate p a i r i.e. s u c c i n i c acid and NADH at a c o n c e n t r a t i o n each of 1.75 × 10-~ M. This rate was c o m p a r e d to the f o r w a r d r e a c t i o n rate u n d e r i d e n t i c a l c o n d i t i o n s and e n z y m e c o n c e n tration but w i t h the f o r w a r d r e a c t i o n substrate p a i r at 1.75 X 10 -4 M. Controls w e r e run w i t h o u t enzyme.
Reaction equilibrium. The usual i n c u b a t i o n m e d i u m w a s used, but the substrates NAD and SSA w e r e e a c h at 1.75 × 10 4 M. The r e a c t i o n was started by a d d i t i o n of purified e n z y m e to the m e d i u m m a i n t a i n e d at 37°C in f l u o r i m e t e r cuvettes. R e a d i n g w e r e taken until no f u r t h e r i n c r e a s e in fluorescence could be detected. The f l u o r i m e t e r w a s then r e c a l i b r a t e d and the NADH p r e s e n t d e t e r m i n e d .
Determination of pH optimum. F o r p H ' s b e l o w 9, 100 mM s o d i u m p y r o p h o s phate buffer w a s u s e d ; above p H 9, g l y e i n e / NaOH buffers. The p H m e d i a all c o n t a i n e d NAD 3 × 10 -~M, SSA 1 × 10 -4 M, KC150 mM, m e r c a p t o e t h a n o l 20 mM and E D T A 0.1 raM. The rales of f l u o r e s c e n c e i n c r e a s e 'were r e c o r d e d d i r e c t l y (initial rates).
Substrate specificity. The initial r e a c t i o n rates w i t h various aldeh y d e s at 1 × 10 -~ M c o n c e n t r a t i o n s and NAD at 3 X 10 .4 M veere c o m p a r e d to a c o n t r o l w i t h SSA at 1 × 10 -~ M. S i m i l a r l y the r e a c t i o n rate using 3 × 10 -4 M NADP yeas c o m p a r e d to that o b t a i n e d using 3 × 10 -4 M NAD.
Inhibition studies. 1) Aldehydes. R e a c t i o n rates w e r e m e a s u r e d in the p r e s e n c e of v a r i o u s a l d e h y d e s and a range of para-substitared b e n z a l d e h y d e s at 1 X 10 -3 M, veith 3 × 10 -4 M NAD and 1 × 10 -~ M SSA. The m e c h a n i s m of i n h i b i t i o n by p - h y d r o x y b e n z a l d e h y d e was det e r m i n e d by m e a s u r i n g the initial rates at several i n h i b i t o r c o n c e n t r a t i o n s w i t h v a r i o u s fixed con-
C. Cash, L . C i e s i e l s k i , M. M a i t r e a n d P . M a n d e i .
260
c e n t r a t i o n s of o n e s u b s t r a t e a n d t h e n v a r i o u s fixed c o n c e n t r a t i o n s of the o t h e r substrate. 2) Substrate inhibition by SSA. I n h i b i t i o n b y h i g h c o n c e n t r a t i o n s o f SSA w a s m e a s u r e d u s i n g v a r i a b l e c o n c e n t r a t i o n s o f NAD. 3) AMP. T h e m e c h a n i s m o f i n h i b i t i o n b y AMP w a s stud i e d u s i n g s e v e r a l c o n c e n t r a t i o n s o f AMP, f i r s t u s i n g v a r i o u s f i x e d c o n c e n t r a t i o n s of NAD, t h e n v a r i o u s f i x e d c o n c e n t r a t i o n s o f SSA.
rious c o n c e n t r a t i o n s of m e r c a p t o e t h a n o l b e t w e e n 0 a n d 10 raM. T h e r e a c t i o n ~,as s t a r t e d b y a d d i tion of e n z y m e .
Iodoacetamide inactivation. I n c u b a t i o n s w e r e c a r r i e d o u t at 37°C w i t h SSA at 1 × 10 -4 M a n d NAD at 3 × 10 -4, 1.5 × 10 -4 a n d 0.5 × 10 -4 M. T h e m e d i u m w a s 100 m M T r i s HCI b u f f e r c o n t a i n i n g 50 m M KC1, 0.1 m M E D T A a n d 5 m M m e r c a p t o e t h a n o l . T h e p H w a s 7.5 as a t t h i s p H , t h e c a r b o x y m e t h y l a t i o n r e a c t i o n is s p e cific to c y s t e i n r e s i d u e s . T h e r e a c t i o n w a s s t a r t e d
TABLE I,
Purification of s u c c i n i c s e m i a l d e h y d e dehgdrogenase f r o m rat brain. Fraction
Volume (ml)
Units/ml
Homogenate . . . . . . . . . . . . . . . . . . Pooled supernatants . . . . . . . . . . J Sephadex G200 eluate . . . . . . . . . DEAE cellulose eluate . . . . . . . . . A M P affinity column eluate . . . . I
240 306 94 116 2.2
0.48
0.3 0.2 0.08 3.07
! [ I I
mg/protein/ml
Units/rag protein
Yield
Purification
30 15 2.5 0.1 0.11
0.016 0. 021 0.08 0.87 27
100 80 16 8 6
1 1.3 5 55 1710
1 unit = 1 ~mol NADH f o r m e d per minute.
Mercaptoethanol reactivation. Mercaptoethanol-free enzyme was prepared by r e a b s o r p t i o n of diluted e n z y m e on an affinity c o l u m n a n d e l u t i o n w i t h 1 m M AMP i n b u f f e r containing no mercaptoethanol. Reactivation curves w e r e o b t a i n e d by fluorescence m e a s u r e m e n t i n t h e u s u a l m e d i u m at 37°C b u t c o n t a i n i n g va-
M I
5
2
8
4
I0'_ - - r - - - v - IOO
150
FIG. 2. - - Molecular pweight determination of native enzyme by Sephadex G200 filtration : l. ~/ Globulin ; 2. Alcohol dehydrogenase ; 3. H a e m o g l o b i n ; 4. Ovalb u m i n ; 5. SSADH.
FIo. 1. - - SDS-Polyacrylamide gel electrophoresis of purified enzyme. BIOCHIMIE, 1977, 59, n ° 3.
b y a d d i t i o n of e n z y m e to t h e m e d i u m c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of i o d o a c e t a m i d e . T h e inactivation period was measured by direct record i n g of the f l u o r e s c e n c e change.
Bat brain succinic semialdehyde dehydrogenase. RESULTS,
Puri[ieation. T h e e n z y m e w a s p u r i f i e d 1,76(} f o l d c ~ p a r e d t o t h e i n i t i a l h o m o g e n a l e ' w i t h a y i e l d of a b o u t 6 p e r c e n t . T h e f i n a l e l u a l e f r o m t h e a f f i n i t y col u m n (2.2 ml) c o n t a i n e d 250 ~g p r o t e i n a n d h a d a s p e c i f i c a c t i v i t y o f 27 u n i t s p e r m i l l i g r a m , (ta-
~o~
261
ble I), 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 u s i n g non-denaturing conditions gave a single band cont a i n i n g all t h e e n z y m e a c t i v i t y , SDS gel e l e c t r o p h o r e s i s a l s o r e v e a l e d a s i n g l e b a n d (see fig. i ) .
31olecular weights and subunits.
of the native enzyme
S e p h a d e x G 200 gel f i l t r a t i o n u s i n g m o l e c u l a r w e i g h t m a r k e r s g a v e a m o l e c u l a r w e i g h t of a b o u t 140,000 (see fig. 2). F r o m SDS gel e l e c t r o p h o r e s i s , a m o l e c u l a r w e i g h t of a b o u t 68,000 w a s c a l c u -
5 0
04
M-W
1@ ~b~ x ~-"~
~1
i
t
1
I
I
2
3
~rfm
Fro. 3. - - Molecular ~meiyht delerrainalion af enzgme s n b u n i t s by SDS 9el electrophoresis. 1. Myoglobin ; 2, C h y m o t r y p s i n o g e n A ; 3, P e p s i n ; 4, Ovalbumin ; 5, Serum Albumin. SSADH subunits have the same m o b i l i t y as s e r u m albumin. TABLF. lI.
A m i n o a c i d c o m p o s i t i o n o[ r a t b r a i n succinic semialdehyde dehydrogenase.
,> *
i
I
[;'to. 4, - - Lin~weaver-BurJ~ rates at ~5~C as a f u n c t i o n of variable concentrations of the 8 × 10-s M ; 2. SSA 4 X 10-o and 4. SSA 1,5 X 10-5 M.
plot of initial reaclion NAD concentration, and second substrale. 1. SSA M ; 3. SSA 2.5 × 10-5 M
l u t e d (fig. 3). P r e t r e a t m e n t o f t h e e n z y m e w i t h o r without mercaptoethanol and iodoacetamide did n o t c h a n g e t h e r.f. o n SDS gels.
Isoelectric point. Amino acid
Glutamate . . . . . . . . . . . . . . Aspartate . . . . . . . . . . . . . . . Threonine . . . . . . . . . . . . . . . Serine . . . . . . . . . . . . . . . . . . Glyeine . . . . . . . . . . . . . . . . . Cysteine . . . . . . . . . . . . . . . . Methionine . . . . . . . . . . . . . . Valine . . . . . . . . . . . . . . . . . . Praline . . . . . . . . . . . . . . . . . Arginine . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . Lysine . . . . . . . . . . . . . . . . . . Phenylalanine . . . . . . . . . . . Tyrosine . . . . . . . . . . . . . . . . Leueine . . . . . . . . . . . . . . . . . lsoleueine . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . . Tryptaphan
Residues per Mol
100 80 5~ 78 120 16 14 88 38 90 14 70 46 30 86 40 116
. . . . . . . . . . . . .
Total . . . . . . . . . . . . . . . . . . .
BIOCHIMIE, 1977, 59, n ° 3.
1078
T h e i s o e l e c t r i c p o i n t is a b o u t 7,2.
104 KNAD
40 30
10 i
///
t
2
L
4
i
d
! 8
FIG, ft. - - Replot f r o m figure 1~ o f intercepts on :e axis (~s' NAD) against reciprocal SSA eoneenlralions.
C. Cash, L. C i e s i e l s k i , M. M a i t r e a n d P. M a n d e l .
262
N - T e r m i n a l analysis.
Sabstrate s p e c i f i c i t y .
A single N-Terminal amino-acid corresponding to l e u c i n e w a s f o u n d , t h u s s u g g e s t i n g t w o i d e n tical subunits.
Other aldehydes. T h e a l d e h y d e s tested c a n n o t be c o n s i d e r e d as significant s u b s t r a t e s for SSADH in v i e w of t h e i r
A m i n o acid composition. T h e q u a n t i t i e s a r e r e p r e s e n t e d o n t a b l e II. T a k i n g a c c o u n t t h e 100 I~g o f p r o t e i n h y d r o l y s e d a n d t h e m o l e c u l a r w e i g h t f o u n d o f a b o u t 136,000, w e c a l c u l a t e d 539 a m i n o a c i d r e s i d u e s p e r c h a i n . T h e m a i n 3 a m i n o a c i d p r e s e n t a r e Glu, A s p , Ala a n d Gly. T h e y r e p r e s e n t a b o u t 30 p e r c e n t o f t h e t o t a l a m i n o a c i d s of t h e m o l e c u l e .
1
3
1
I
,/
I
I
I
I
I
6
7
8
9
10
h
pH
2
I 5
I 10 .M.SSA
I 15
•
I , 20
FIG. 7. - - Subslrate inhibition by SSA Plot of reciprocal velocity against SSA concentration. t. NAD 3 X 10-4 M ; 2. NAD 1 X 10-4 M.
FIG. 6. - - Relative activity of SSADH
as a function of pH.
f e e b l e r a t e of o x i d a t i o n i n c o m p a r i s o n w i t h SSA o x i d a t i o n (see t a b l e III). KINETIC
STUDIES.
TABLE IV.
K m determinations. By m e a s u r i n g i n i t i a l r a t e s at v a r i o u s f i x e d c o n c e n t r a t i o n s of one s u b s t r a t e w h i l s t v a r y i n g the c o n c e n t r a t i o n s o f t h e o t h e r , K m ' s ( l i m i t ) ~vere o b t a i n e d f r o m s e c o n d a r y plots of the initial Linew e a v e r - B u r k p l o t s . T h e s e a r e a b o u t 2 X 10-~ lVl f o r NAD a n d 1 X 10 -4 M f o r SSA (see fig. 4, fig. 5).
I n h i b i t i o n by aldehydes. Aldehyde (t 0-:aM)
Per cent residual activity
Valeraldehyde . . . . . . . . . . . Benzaldehyde . . . . . . . . . . . Chloral hydrate . . . . . . . . . . p-hydroxyhenzaldehyde . . .
84 70 60 0
pH optimum. I n h i b i t i o n by a series of para-sabstituted benzaldehydes (10-.3 M).
(see fig. 6). T h e r e is a b r o a d p H o p t i m u m w i t h a m a x i m u m at a b o u t p H 8.6.
R group
Per cent residual activity
TABLE III.
Relative rates of oxidation of some other aldehydes.
OH
(Rate Related to SSA = 100 per cent).
Br
F
.................... ......................
Cl ...................... .....................
I ......................
Aldehyde (t0-4M)
Per cent activity
Valeraldehyde . . . . . . . . . . . Chloral hydrate . . . . . . . . . . Acetaldehyde . . . . . . . . . . . . Benzaldehyde . . . . . . . . . . . p-hydroxybenzaldehyde...
2.6
BIOCH1M1E, 1977, 59 n ° 3.
1.0 0.5 0.5 0
NO:~ . . . . . . . . . . . . . . . . . . . . NH 2 . . . . . . . . . . . . . . . . . . . . C~N ................... CH:~ . . . . . . . . . . . . . . . . . . . .
! I
CH(CH~)~ . . . . . . . . . . . . . . . O-CH:~ . . . . . . . . . . . . . . . . . . O-C2H.,, . . . . . . . . . . . . . . . . . .
! , ,
C._,H:, . . . . . . . . . . . . . . . . . . . .
0 81 62 62 48 80 100 82 44
49 27 58 58
Rat brain succinic semialdehyde dehydrogenase.
263
NADP. W i t h N A D P a s s u b s t r a t e a b o u t 13 p e r c e n t o f the activity was observed compared to a control using equimolar NAD as substrate.
8 slope
6
,°t
/
3O-
_t Vo
2O0
2
4 6 8 4 H B X 106 M
10
Fro. 9. - - Secondary plot of slopes versus
inhibitor concentration from [igure 8.
I0-
o
1
20
X 104M
Flo. 8. - - Double reciprocal plot of inhibition o[ SSADH by #HB at 3 concentrations of SSA. NAD c o n c e n t r a t i o n is 3 X 10-4 M. 1. No i n h i b i t o r ; 2. 4 HB 2 X 10-6 M ; 3. 4HB 5 × 10-6 M a n d 4. 4HB 1 X 10-5 M. TABLE V.
Reactivation of suecinic s e m i a l d e h y d e dehydrogenase by ~-mercaptoethanol. ~-mercaptoethanol concentration 3 1.5 4.5 3 1.5 0
X X X X ×
10-eM 10-UM 10-3M 10-aM 10-aM
Per cent ol m a x i m u m rate achieved 100 100 78 72 32 0
Reactivation period
30" 30" 1'30" 2'30" *~
7' 0
Inhibition. 1) Substrate inhibition by SSA. SSA b e c o m e s i n h i b i t o r y at c o n c e n t r a t i o n s a b o v e 10 -3 M (fig. 7). At i n h i b i t o r S S A c o n c e n t r a t i o n s ,
BIOCHIMIE, 1977, 59, n ° 3.
i 2
I 4
i 6
I 6
10
Fta. 10. - - Double reciprocal plot o[ SSADH inhibition
by ~HB at 3 concentrations o[ NAD. SSA c o n c e n t r a t i o n is 1 × 10-4 M. 1. No i n h i b i t o r ; 2. 4HB 2 X 10-6 M ; 3. 4HB 5 × 10-G M ; 4. 4HB 1 × 10-5 M. 20
C. Cash, L . C i e s i e l s k i , ill. M a i t r e a n d P . M a n d e l .
264
t h e r e is, as has been p r e v i o u s l y d e m o n s t r a t e d by K a m m e r a t and Veldstra [5], a l i n e a r i t y b e t w e e n the SSA c o n c e n t r a t i o n a n d the r e c i p r o c a l value of the i n i t i a l r e a c t i o n v e l o c i t y (see fig. 7). F o r d i f f e r e n t c o n c e n t r a t i o n s of NAD, a plot of 1/(SSA) ( i n h i b i t o r y c o n c e n t r a t i o n s ) versus 1 / V 0 gives p a r a l l e l lines s h o w i n g that the i n h i b i t i o n is u n e o m p e t i t i v e w i t h r e s p e c t to NAD, suggesting a r e a c t i o n b e t w e e n one SSA m o l e c u l e a n d a substrate e n z y m e c o m p l e x .
3) I n h i b i t i o n by 5' AMP. As can be seen f r o m a competitive inhibitor a K i of about 7,5 10 -5 inhibitor with respect
figs 12, 13, 14, 15, AMP is w i t h r e s p e c t to NAD w i t h M and a non c o m p e t i t i v e to SSA.
4
40
2{3
1
Vo
30 3 I
15 intercept 2E
10 10
0
I 2
I 4
I 6
I 8
I 10
N A D X 10-4M
1
I 0
2
I
I
4 6 4HB ~ 106M
I 8
10
Fro. 11. - - Secondary plot of intercepts versus
FIG. 12. - - Double reciprocal plot of SSADH inhibition by AMP at 3 concentrations of NAD. SSA is 1 X 10-4 M. 1. No i n h i b i t o r ; 2. AMP 0.1 m M ; 3. AMP 0.2 mM and 4. AMP 0.5 raM.
inhibitor concentration from figure 10.
It should be noted that s u e e i n i c acid, the product of SSA o x i d a t i o n , was not i n h i b i t o r y even at 100 mM. 2) I n h i b i t i o n by aldehydes. p - h y d r o x y b e n z a l d e h y d e (4 HB) is the most p o w e r f u l of the i n h i b i t o r y a l d e h y d e s studied. No e n z y m e a c t i v i t y could be d e t e c t e d ~vith 4 HB at 10 ~a M and SSA and NAD at 1 × 10 -4 and 3 × 10-~ M r e s p e c t i v e l y . A series of p-substitued benz a l d e h y d e s w e r e in g e n e r a l m o r e i n h i b i t o r y as the substituent group b e c a m e less e l e c t r o n e g a t i v e (see table 4). It can be seen (Figs 8, 9, 10, 11) thai 4 HB is a c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t to SSA ",vith a K i of about 3 × 10 -6 M and is an u n c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t to NAD.
BIOCHIMIE, 1977, 59, n ° 3.
ADP and ATP but not a d e n i n e or a d e n o s i n e i n h i b i t s SSADH to a s i m i l a r extent to AMP.
Mercaptoethanol reactivation. A p u r e p r e p a r a t i o n of SSADH is c o m p l e t e l y i n a c t i v e in the absence of m e r c a p t o e t h a n o l . By a d d i n g v a r i o u s l o w c o n c e n t r a t i o n s of m e r c a p t o ethanol to the i n c u b a t i o n m e d i u m c o n t a i n i n g the enzyme, a d i m i n i s h i n g p e r i o d of r e a c t i v a t i o n ~vith i n c r e a s i n g m e r c a p t o e t h a n o l c o n c e n t r a t i o n s ~vas o b s e r v e d (table V). About 10 -2 M m e r c a p t o e t h a n o l w a s n e c e s s a r y for full e n z y m e activity. An analysis of the r e a c t i v a t i o n c u r v e s (see a p p e n d i x ) i n d i c a t e s that the r e a c t i v a t i o n m e c h a n i s m follows pseudo-first o r d e r kinetics.
Iodoacelamide inactivation. With 10 -2 M i o d o a c e t a m i d e , no e n z y m e a c t i v i t y was detected, u n d e r the c o n d i t i o n s e m p l o y e d .
265
Rat brain succinic semialdehyde dehydrogenase. W i t h l o w e r c o n c e n t r a t i o n s , a d e c r e a s e in r e a c t i o n rate could be o b s e r v e d , l e a d i n g a l w a y s to total
i n a c t i v a t i o n . A n a l y s i s of t h e i n c u b a t i o n c u r v e (see a p p e n d i x ) s h o w s t h a t i n a c t i v a t i o n is p s e u d o first o r d e r . It c a n b e s e e n f r o m t a b l e VI t h a t t h e i n a c t i v a t i o n p e r i o d i n c r e a s e s w i t h NAD c o n c e n tration. TABLE ¥ I .
Inactivation of succinic semialdehyde dehydrogenase by iodoacetamide.
slope
3 3
X 1D-4M X 10--~M
1.5
X
Inactivation period
lodoacetamide concentration
NAD concentration
4 2 2 2
10 -~ M
0.75 X 10 -4 M
1'30"
mM mM mM mM
13'45" 3'10" 10"
10
I
,/
I 0.1
I 0.2
I 03
I 04
I 0.5
MM. A M P
FI~. 13. - - Secondary plot of slopes uersus inhibitor
concentration from figure 12.
t a w
1 O
10
I 1
I 2
I 3
I 4
I 5
~M. AMP
FI(~. 15. - - Secondary plot of intercepts versus
inhibitor concentration from figure 1-~,
Reverse reaction.
,,-f//
I
t
I
1
I
0
1
2
3
4
~AX
10"4 M
Under identical reaction conditions save for the s u b s t i t u t i o n of t h e f o r w a r d s u b s t r a t e b y e q u i m o l a r c o n c e n t r a t i o n s of t h e r e v e r s e s u b s t r a t e p a i r , t h e r e v e r s e r e a c t i o n p r o c e e d e d at 1/1230 o f t h e r a t e of the f o r w a r d r e a c t i o n .
FI(L 14. - - Double reciprocal plot of SSADH inhibition
by AMP at 3 concentrations of SSA.
NAD is 3 X 10-4 M. 1. No i n h i b i t o r ; 2. AMP 1 mM ; 3. AMP 2 mM and 4. AMP 5 mM.
BIOCHIMIE, 1977, 59, n ° 3.
Equilibrium. At e q u i l i b r i u m , 92 p e r c e n t o f t h e NAD w a s r e d u c e d to N A D H . T h u s t h e e q u i l i b r i u m c o n s t a n t
C. Cash, L. Ciesielski, M. Maitre and P. Mandel.
266 K ----
[NAD]
[Succinate]
= 132. and the h G ° [NAD] [SSA] for the f o r w a r d r e a c t i o n is - - 3,000 c a l / m o l . DISCUSSION. Using affinity c h r o m a t o g r a p h y as a final step, we o b t a i n e d SSADH w h i c h was h o m o g e n o u s a c c o r d i n g to the c r i t e r i a of both n o n - d e n a t u r i n g and SDS 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 a n d by N - t e r m i n a l analysis. An a p p a r e n t p u r i f i c a t i o n f a c t o r of 1,700 fold w i t h r e s p e c t to the i n i t i a l h o m o g e n a t e w a s obtained, the specific a c t i v i t y w a s 27 units p e r m i l l i g r a m of p r o t e i n . No isoenzymes w e r e detected. To study the k i n e t i c p a r a m e t e r s a n d m e c h a nism, w e ~vere obliged to w o r k w i t h i n a s o m e w h a t l i m i t e d r a n g e of substrate c o n c e n t r a t i o n s ; the u p p e r limits b e i n g fixed by the r e l a t i v e l y l o w Km's and the p h e n o m e n o n of substrate i n h i b i tion ; the l o w e r limits b e i n g fixed by the need to h a v e an a c c u r a t l y m e a s u r a b l e i n i t i a l r e a c t i o n rate for s e v e r a l minutes. If the e n z y m e c o n c e n t r a t i o n w e r e r e d u c e d to a l l o w l o n g e r i n c u b a t i o n p e r i o d s , its stability b e c a m e a l i m i t i n g factor. L i n e w e a v e r - B u r k plots of i n i t i a l rates w h e n one substrate 'was v a r i e d at s e v e r a l fixed conc e n t r a t i o n s of the o t h e r gave lines w i t h slopes w h i c h are not significantly different. This is unusual for d e h y d r o g e n a s e , but has been observed with g l y c e r a l d e h y d e - p h o s p h a t e dehydrogenase f r o m Pisum sativum [16]. F r o m the m e c h a nistic p o i n t of v i e w , this can be e x p l a i n e d in one of the f o l l o w i n g w a y s : 1) T h e i u e c h a n i s m is ping-pong, i.e., a p r o d u c t is released b e f o r e the a d d i t i o n of the s e c o n d substrate. 2) T h e m e c h a n i s m is sequential, but i n v o l v e s a t h i r d s a t u r a t i n g substrate [17] a d d i n g in the s e c o n d position. 3) T h e e n z y m e substrate d i s s o c i a t i o n c o n s t a n t is m u c h s m a l l e r than the Kin. In this case, the c h a n g e in slope is v e r y small in c o m p a r i s o n to the c h a n g e in i n t e r c e p t for a l i m i t e d range of substrate c o n c e n t r a t i o n s [18]. Thus, in o r d e r to f u r t h e r d e l i n e a t e the m e c h a n i s m , w e h a v e studied d e a d - e n d i n h i b i t i o n patterns. AMP, an analogue of NAD, w a s f o u n d to be a c o m p e t i t i v e i n h i b i t o r vis h vis NAD, but a non c o m p e t i t i v e i n h i b i t o r vis h vis SSA. H o w e v e r , p-hydroxybenzaldehyde, a competitive inhibitor w i t h r e s p e c t to SSA, gave u n c o m p e t i t i v e i n h i b i tion w h e n NAD w a s the v a r i a b l e substrate. A
BIOCHIMIE, 1977, 59, n ° 3.
s e c o n d a r y plot of the i n t e r c e p t versus i n h i b i t o r c o n c e n t r a t i o n s is linear. This is i n d i c a t i v e of an o r d e r e d bi-bi or ter-bi m e c h a n i s m w h e r e NAD must b i n d first to the enzyme, f o l l o w e d by SSA, before release of products. That is, p - h y d r o x y b e n z a l d e h y d e b i n d s only w i t h the enzyme-NAD c o m p l e x and h e n c e competes w i t h the s e c o n d substrate SSA, but is u n c o m p e t i t i v e s vis ~ vis NAD as it has no affinity for the free enzyme. T h e possible r e a c t i o n m e c h a n i s m is s u m m a r i z e d in the s c h e m e : E-AMP
hMl~
E
E-NADH
NAD'
.
,
E-NAO
" E-NAD-H20-SSA
5ucclr~te
E NAD-H20 SSA - SSA -
-
T h e m a i n i n d i c a t i o n s for an o r d e r e d ter-bi m e c h a n i s m w i t h w a t e r as a substrate are that : 1) W a t e r is n e c e s s a r y for the s t o e c h i o m e t r y of the c h e m i c a l r e a c t i o n R-C-H q- NAD + H20 ~-~ R-C-OH + NADH ~- H ~ I[ 0 O 2) A ter-bi o r d e r e d m e c h a n i s m w i t h w a t e r as the second substrate w o u l d e x p l a i n the p a r a l l e l lines of the L i n e w e a v e r - B u r k plot. O r d e r e d ter-bi m e c h a n i s m w i t h the s e c o n d substrate at s a t u r a t i n g level can be d i s t i n g u i s e d f r o m o r d e r e d bi-bi m e c h a n i s m by p r o d u c t i n h i b i t i o n . I n h i b i t i o n w i t h NADH w i t h r e s p e c t to SSA w o u l d be u n c o m p e t i t i v e in the ease of a ter-bi m e c h a nism, non c o m p e t i t i v e in the ease of a bi-bi mechanism. E x p e r i m e n t s h a v e been c o n d u c t e d but are i n c o n c l u s i v e o'wing to the love i n h i b i t i o n constants. P r o d u c t s are ~xeak i n h i b i t o r s w h e n the f o r w a r d r e a c t i o n is s t r o n g l y favoured. To distinguish b e t w e e n these t'wo o r d e r e d m e c h a n i s m o t h e r i n v e s t i g a t i o n s should be c o n d u c t e d .
APPENDIX.
D e t e r m i n a t i o n of a c t i v a t i o n and i n a c t i v a t i o n p e r i o d s f r o m the f l u o r e s c e n c e r e c o r d i n g s u s i n g 1st o r d e r r e a c t i o n kinetics.
Rat brain succinic semialdehyde dehydrogenase. 1) I n a c t i v a t i o n b y I o d o a c e t a m i d e c e n t p r o d u c t (fig. 16).
--
Fluores-
2) R e a c t i v a t i o n b y m e r c a p t o e t h a n o l . Eo Initial enzyme inactive E A c t i v e e n z y m e at t i m e t T Reactivation period
E o A c t i v e e n z y m e at z e r o t i m e E A c t i v e e n z y m e at t i m e t
t E = E o (1-e - - Log2) T dF v --- K E (2) dt
v Reaction rate T Inactivation period F Fluorescence (Arbitrary Units) F o F l u o r e s c e n c e at z e r o t i m e F~
267
t x all o t h e r s y m b o l s ~ as a b o v e (8)
F l u o r e s c e n c e at i n f i n i t e t i m e / /
F
/
%" ,'
//
///
///
/ / /
//
/
//
~///
///
>t
',. )//
,t FIG.
16.
/
/
/
/
/
/
(1) E : (2)
v --
Eo.e dF
t
---
T
Log2 Fro, 17.
KE
--
dt
F r o m (8) a n d (2) F :
F r o m 1) a n d 2) KT
KT
+ Fo
F~ -- F . . . . . e Log2
(4)
t
__ ~t Log2
T
= a constant --
T - - - - Log2 __ 1)) F = K E o (t + - e T Log2
(5)
t Log2 T
(6)
F~--F are the values experimentaly determined from the fluorescence recordings; Log (F~--F) is t r e a t e d b y t h e least m e a n s q u a r e m e t h o d . If e x p e r i m e n t a l l y a s t r a i g h l i n e is o b t a i n e d ~ i t h a g o o d c o e f f i c i e n t of c o r r e l a t i o n t h e i n a c t i v a t i o n p e r i o d c a n be e x p r e s s e d b y t h e relation T
Log2 slope
BIOCHIMIE, 1977, 59, n ° 3.
constant
T
T h e c o n s t a n t is d e t e r m i n e d f r o m F o = T constant = Log2
Log2 KT
t Log2) +
e
(3)
Log2
Log (F~--F)
Log2
F = t (1-e - - -T L o g 2 ) + F o
Foo --
T
K E o (t +
(7)
(9) O
(9)
U s i n g a s u p p l e m e n t a r y f u n c t i o n F u w h i c h is the F l u o r e s c e n c e w h i c h ~vould be o b s e r v e d in all t h e e n z y m e s w e r e a c t i v e at z e r o t i m e F u = K E o t , we can write : t
X = Fu-F : Xoo :
KEo (1--e
KEoXoo--X
L o g (Xoo - - X) : - - - -
T
t
---
t
(10)
Log2
(11)
Log2 + a c o n s t a n t
(12)
= KEoe T
Log2) T
C. Cash, L. C i e s i e l s k i , M. M a i t r e a n d P. M a n d e l .
268
( X ~ - - X) is a n e x p e r i m e n t a l v a l u e d e t e r m i n e d f r o m t h e f l u o r e s c e n c e r e c o r d i n g . L o g ( X ~ - - X) is t r e a t e d as a b o v e . T h e r e a c t i v a t i o n p e r i o d of t h e e n z y m e is g i v e n b e t h e r e l a t i o n : Log2
T
slope R~su~i~. La semi aldt~hyde succinique d6shydrog6nase de cerveau de rat a 6t6 purifi6e j n s q u ' h homog6n6it6 selon les crit6res d'Slectrophor6se. Son poids mol6culaire est d ' e n v i r o n 140.000 Daltons et elle est formde de deux sous-unitSs a p p a r e m m e n t identiques. La r~action catalys~e par la prot~ine pure est enti~r e m e n t d6pendante de la pr6sence de groupes sulfhydriles endogbnes. Les Km limites pour ]e NAD et le semi ald6hyde succinique sont r e s p e c t i v e m e n t de 2 × 10-5 M e t 1 X 10-4 M a u pH o p t i m u m de 8,6. Des 6tudes cin6tiques et d ' i n h i b i t i o n m o n t r e n t que le m6canisme de la r6action est u n m6canisme ordonn6, h ordre obligatoire d ' a d d i t i o n des substrats : le NAD dolt se fixer le premier, le semi ald6hyde succinique le dernier. REFERENCES. 1. Schousboe, A., Wfl, J. Y. & Roberts, E. (1973) Biochem., 17, 2868-2873. 2. Maitre, M., Ciesielski, L., Cash, C. a Mande], P. (1975) Eur. J. Biochem., 57, 15.7-169.
BIOCHIMIE, 1977, 59, n ° 3.
3. Cash, C., Maitre, M., Ciesielski, L. ,~ Mandel, P. 0974) FEBS Letters, 47, 199-203. 4. Bloch-Tardy, M., Rolland, B. ~ Gonnard, P. (1974) Biochimie, 5.6, 823-837. 5. K a m m e r a a t , C. ,~ Veldstra, H. (1967) Biochem. Biophys. Acla, 15'1, 1-10. 6. Embree, L. J. ~ Albers, R. W. (1963) Biochem. Pharmacol., 13, 1209-1217. 7. Albers, R. W. ~ Koval, G. J. (1961) Biochem. Biophys. Acta, 57, 29-35. 8. Cash, C., Ciesielsld, L., Maitre, M. ~ Mandel, P. (1975) C. R. Soc. Biol. (Paris), 169, 884-887. 9. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. ,~ Randel, R. J. (1951) J. Biol. Chem., 11~3, 265-275. 10. Udenfried, S., Stein, S., Bohlen, P., Dairman, W'., Leimgruber, W. ,~ Weigele, M. (1972) Science, 178, 871-872. 1I. Joivin~ T., Chrombaeh, A..~ Naughton, M. A. (1964) Anal. Biochem., 9, 351-369. 12. Weber, K..a Osborn, M. (1969) J. Biol., 244, 44064412. 13. Allen in <( Disc Electrophoresis >>. Ind. Ed. Mauer (1971) p. 134, W a l t e r de Gruyter, Berlin. 14. Zanetta, J. P., Vincendon, G., Mandel, P. ~ Gombos, G. (19'70) J. Chromatog., 5, 441-548. 15. Lowry, O. ~ P a s s o n n e a u , J. in (( A Flexible System of Enzymic Analysis >> (1972) Academic Press, p. 10-11. 16. Duggelby, R. ~ Dennis, D. (1973) J. Biol. Chem., 249, 167-174. 17. Cleland, W. (1967) Annual Review of Biochemistry, ~6, 77-117. 18. Dalziel, K. (< The enzymes >>. Third Edition Academic Press, Vol. II, Ed. Boyer, p. 1-60.
Purification and properties of rat brain succinic semialdehyde dehydrogenase. C. CASH <>, L. CIESIELSKI, M. MAITRE a n d P. MANDEL. Centre de N e u r o c h i m i e du CNRS, Institut de Chimie Biologique, 11 rue H n m a n n , 67000 Strasbourg, France. (7-2-1977). Summary. - - Succinic semialdehyde dehydrogenase from rat brain has been purified to electrophoretic homogeneity. It has a molecular veeight of about 140,000 and is composed of Vwo apparently identical subunits. The reaction eatalized by the pure protein is entirely dependent on endogenous - - S H groups. The Km (limits) for NAD and sueeinic semialdehyde are 2 × 10-5 M and 1 X 10-4 M respectively at the optimum pH of 8.6. Inhibition studies showy that the reaction mechanism is a compulsory ordered one where NAD binds first followed by sueeinic semialdehyde. INTRODUCTION. 4-amino b u t y r i c acid (GABA) is d e g r a d e d in the b r a i n to s u c c i n i c acid by the successive actions of a t r a n s a m i n a s e (GABA-T) and a d e h y d r o g e n a s e , s u c c i n i c s e m i a l d e h y d e d e h y d r o g e n a s e (SSADH). T h e first of these e n z y m e s f r o m several sources has been p u r i f i e d to h o m o g e n i t y and e x t e n s i v e l y studied [1, 2, 3, 4]. H o w e v e r , a p a r t f r o m some wor;k on p a r t i a l l y purified SSADH [5, 6, 7, 8], m u c h less is knoxvn about this e n z y m e w h o s e a c t i v i t y is so great in b r a i n that the c e r e b r a l level of s u c c i n i c s e m i a l d e h y d e (SSA) is ahvays v e r y lo'w. It is thus i n t e r e s t i n g to study the fundam e n t a l c h a r a c t e r i s t i c s of this e n z y m e ~rhich controls the p r i n c i p a l r o u t e by ' w h i c h SSA is catabolized. In this p r e s e n t w o r k , we h a v e purified rat b r a i n SSADH to h o m o g e n i t y , and studied some of its s t r u c t u r a l and c a t a l y t i c p r o p e r t i e s . MATERIALS AND METHODS.
Reagents. S u c e i n i c s e m i a l d e h y d e was p r e p a r e d by h y d r o lysis of v - e t h o x y - b u t y r o l a c t o n e , a gift f r o m Dr. C. G. W e r m u t h , S t r a s b o u r g ; o t h e r reagents ~vere of a n a l y t i c a l grade. E n z y m e assays. F o r q u a l i t a t i v e assays of c o l u m n elution fractions, 50 ~1 aliquots (1 ,~I aliquots f r o m the affinity Abbreviations : GABA-T : 4 aminobutyrate transaminase (EC. 2.6.1. 19). GABA: 4 aminobutyrate. SSA : Suceinic semi aldehyde. SSADH : suceinic semi aldehyde dehydrogenase (EC 1.2.1.24). ADH : AleohoI dehydrogenase (EC 1.1.1.71). TCA : Triehloraeetie acid. SDS : sodium dodeeyl sulfate. To whom all eorrespondenee should be addressed.
column), w e r e p i p e t t e d into tubes c o n t a i n i n g cold buffer consisting of 100 mM Tris/HC1 p H 8.6, 50 mM KCI, 3 × 10 .4 M NAD, 0.1 mM EDTA a n d 20 mM m e r c a p t o e t h a n o l . T h e r e a c t i o n w a s started by r a p i d a d d i t i o n of SSA to a final c o n c e n t r a t i o n of 1 × 10 -4 M in a total v o l u m e of 1 ml, and the samples w e r e i n c u b a t e d at 37°C for five minutes. T h e r e a c t i o n w a s s t o p p e d by i m m e r s i n g the tubes in b o i l i n g w a t e r for five m i n u t e s and after cooling, the fluorescence was r e a d in a Zeiss f l u o r i m e t e r at e x c i t a t i o n 355 n m ; and emission 470 n m ( u n c o r r e c t e d values). F o r q u a n t i t a t i v e e n z y m e assays at each step of the p u r i f i c a t i o n p r o c e d u r e , the same i n c u b a t i o n m e d i u m as above w a s used w i t h the a d d i t i o n of T r i t o n X - 1 0 0 to a final c o n c e n t r a t i o n of 1 p e r cent. A d d i t i o n of T r i t o n i n c r e a s e d the a c t i v i t y y i e l d in p a r t i c u l a t e f r a c t i o n s but h a d no significant effect w h e n clear e n z y m e samples ~x'ere used. T h e i n c u b a t i o n m e d i u m w a s m a i n t a i n e d at 37°C in t h e r m o s t a t e d cuvettes in the f l u o r i m e t e r , and the r e a c t i o n w a s started by a d d i t i o n of 50 td of suitably d i l u t e d enzyme. T h e rate of i n c r e a s e of fluorescence was r e c o r d e d d i r e c t l y and the c h a n g e p e r m i n u t e w a s c o m p a r e d to the fluoresc e n c e u n d e r the same c o n d i t i o n s of a solution of NADH p r e v i o u s l y c a l i b r a t e d by its o p t i c a l density at 340 nm. F o r the p e r i o d of the m e a s u r e m e n t s the rate of i n c r e a s e of f l u o r e s c e n c e w a s constant.
Protein determinations. T h e m e t h o d of Lo~vry et al [9] w a s used for s a m p l e s o b t a i n e d b e f o r e D E A E cellulose c h r o m a t o g r a p h y . F o r the later stages, w h e r e the p r o t e i n c o n c e n t r a t i o n w a s v e r y low, the F l u o r e s c a m i n e
258
C. Cash, L. Ciesielski, M. Maitre and P. Mandel.
m e t h o d [10] w a s used. At the c o n c e n t r a t i o n s used, AMP and m e r c a p t o e t h a n o l did not interfere. Bovine s e r u m a l b u m i n 'was used as a s t a n d a r d .
Gel Electrophoresis. 7.5 p e r cent P o l y a c r y l a m i d e gels w e r e p r e p a r e d a c c o r d i n g to the t e c h n i q u e of J o i v i n et aI [ l l i . E l e c t r o p h o r e s i s w a s c a r r i e d out for about 12 h at 4°C and 3 m i l l i a m p s p e r gel. T h e b a n d s w e r e stained in Coomassie b r i l l a n t blue. ENZYME PURIFICATION.
Extraction. 40 W i s t a r rats w e r e killed by d e c a p i t a t i o n , the b r a i n s r a p i d l y r e m o v e d and s u s p e n d e d in a total v o l u m e of 240 ml of ice cold w a t e r c o n t a i n i n g 5 mM m e r c a p t o e t h a n o l a n d 0.1 mM EDTA. T h e suspension w a s h o m o g e n i z e d using a c o m m e r c i a l food b l e n d e r at m a x i m u m speed for a total of six minutes, a l l o w i n g i n t e r v a l s to p r e v e n t significant heating. T h e h o m o g e n a t e w a s c e n t r i f u g e d at 50,000 g for 30 m i n u t e s at 4°C, and the s¢lpernatants decanted. T h e pellets w e r e r e s u s p e n d e d in the above m e d i u m and the b l e n d i n g and c e n t r i fugation w a s r e p e a t e d . T h e s u p e r n a t a n t s w e r e pooled.
Ammonium sulphate fractionation. T h e p o o l e d s u p e r n a t a n t s w e r e b r o u g h t to 45 p e r cent s a t u r a t i o n at ice t e m p e r a t u r e using solid (NH~). SO~, the pH b e i n g m a i n t a i n e d at 7.2 by a d d i t i o n of 2M (NH4)OH. T h e suspension w a s cent r i f u g e d at 30,000 g for 30 mn ; the pellets w e r e d i s c a r d e d and the s u p e r n a t a n t b r o u g h t to 70 p e r cent (NHa) 2 SO 4 s a t u r a t i o n as above. After centrifugation the r e s u l t a n t pellets w e r e dissolved in the m i n i m u m v o l u m e (about 5 inl) of 2 mM p h o s p h a t e buffer c o n t a i n i n g 5 mM m e r c a p t o e t h a n o l and 0.1 mM EDTA.
Column chromatography. All c h r o m a t o g r a p h y 'was p e r f o r m e d in a cold r o o m at about 4°C and all e q u i l i b r a t i o n and elution buffers c o n t a i n e d 5 mM m e r c a p t o e t h a n o l and 0.1 mM EDTA. Columns w e r e e x t e n s i v e l y equilib r a t e d w i t h starting buffer b e f o r e use.
Gel filtration. This w a s p e r f o r m e d on a c o l u m n of Sephader, G200 (1 m length, 2.6 cm d i a m e t e r ) . T h e e n z y m e w a s eluted 'with 2 mM p h o s p h a t e buffer p H 7.2 and c o l l e c t e d in 5 ml fractions.
Ion exchange chromatography. T h e active f r a c t i o n f r o m S e p h a d e x G200 chrom a t o g r a p h y w a s absorbed onto a c o l u m n of D E A E Cellulose ( W h a t m a n DE52 ; 15 cm length, 2.6 cm
BIOCHIMIE, 1977, 59, n ° 3.
d i a m e t e r ) . T h e colunln was e x t e n s i v e l y r i n c e d w i t h s t a r t i n g buffer before elution w i t h 500 ml of a l i n e a r g r a d i e n t of 0 to 45 mM KC1 c o n t a i n i n g 2 mM p h o s p h a t e buffer p H 7.2. T h e eluate w a s c o l l e c t e d in 5 ml fractions.
Affinity chromatography. T h e active f r a c t i o n f r o m D E A E cellulose chrom a t o g r a p h y w a s a b s o r b e d onto a c o l u m n of 5' AMP substituted S e p h a r o s e ( P h a r m a c i a Sepharose 4B, 5 cm length, 0.5 cm d i a m e t e r ) . T h e c o l u m n w a s w a s h e d ~vith 10 mM p h o s p h a t e buffer p H 7.2 and eluted in 0.5 ml f r a c t i o n s w i t h 1 mM AMP in the same buffer. Glycerol to a c o n c e n t r a tion of 10 p e r cent w a s a d d e d to the e n z y m e fraction before storage a t - - 3 0 ° C . P H Y S I C A L P R O P E R T I E S AND S T R U C T U R E ,
Molecular weight of native enzyme. A c o l u m n of S e p h a d e x G200 (2.6 × 100 cnl) w a s e q u i l i b r a t e d w i t h 10 mM p h o s p h a t e buffer p H 7.2 c o n t a i n i n g 100 mM KC1. SSADH plus the f o l l o w i n g m o l e c u l a r w e i g h t m a r k e r s w e r e a p p l i e d to the c o l u m n in a total v o l u m e of 1 m l : o v a l b u m i n , human v-globulin, h o r s e h a e m o g l o b i n , yeast a l c o h o l d e h y d r o g e n a s e and d e x t r a n blue. T h e p r o t e i n s w e r e eluted 'with e q u i l i b r a t i o n buffer in 5 ml fractions. T h e peaks w e r e d e t e r m i n e d by t h e i r a b s o r b a n c e at 280 n m ; e x c e p t ADH a n d SSADH w h i c h w e r e d e t e r u l i n e d e n z y m a t i c a l l y .
Molecular ,weight of subunits. This w a s d e t e r m i n e d on 10 p e r cent p o l y a c r y l a m i d e gels in 0.1 p e r cent SDS and 6 M u r e a as d e s c r i b e d by W e b c r and Osborn [..12]. 20 ~g e n z y m e w a s i n c u b a t e d at 37°C in e l e c t r o p h o r e s i s buffer c o n t a i n i n g 0.1 p e r cent SDS, 5 M u r e a and 20 mM m e r c a p t o e t h a n o l . T h e f o l l o w i n g molecular weight markers were u s e d : myoglobin (17,000) c h y x n o t r y p s i n o g e n A (25,000) p e p s i n (35,000) o v a l b u m i n (45,000) and h u m a n a l b u m i n m o n o m e r (69,000). B r o m o p h e n o l blue w a s used to m a r k the front. The m o b i l i t i e s w e r e c a l c u l a t e d a c c o r d i n g to the m e t h o d of W e b e r and Osborn
[12]. Subunit binding. About 20 r~g of e n z y m e w a s b r o u g h t to pH 5 with KH2PQ ; urea and mercaptoethanol were a d d e d to final c o n c e n t r a t i o n s of 6 M and 60 mM r e s p e c t i v e l y . T h e e n z y m e w a s i n c u b a t e d at 40°C for I h, then b r o u g h t to p H 8 w i t h 0.1 M NaOH. l o d o a c e t a m i d e 'was a d d e d to a c o n c e n t r a t i o n of 200 mM and the sample i n c u b a t e d for 30' at 40 °. T h e t r e a t e d e n z y m e w a s s u b m i t t e d to e l e c t r o p h o rests on an SDS gel as d e s c r i b e d u n d e r <( Mole-
R a t brain succinic s e m i a l d e h y d e dehydrogenase. c u l a r w e i g h t of subunits >>. At the same time, a n o t h e r e n z y m e sample w a s run w h i c h had been t r e a t e d w i t h urea and SDS, but not m e r c a p t o ethanol.
Isoelectric focussing. This was p e r f o r m e d on c y l i n d r i c a l gels (5 m m by 7.3 cm) using the system devised by Alien E13]. The final c o n c e n t r a t i o n of a c r y l a m i d e in the gel was 6 p e r cent w i t h 2.5 p e r cent A m p h o l i n e pH 3 to 10. The e l e c t r o d e solutions 'were 0.1 M HC1 for the l o w e r bath (anode) and 0.15 M e t h a n o l a m i n e for the u p p e r bath (cathode) E l e c t r o p h o r e s i s was c a r r i e d out for about 8 hours at 4°C w i t h a c o n s t a n t c u r r e n t of 1 mA per gel, the voltage starting at 200 volts and r e a c h i n g 500 volts at the end of the run. Then, the gel was fixed for 2 h o u r s w i t h 2 changes of 12 p e r cent TCA, and afterw a r d s stained for 4 hours in a solution c o n t a i n i n g acetic acid, ethanol and 0.2 p e r cent aqueous Coomassie b r i l l i a n t blue in the p r o p o r t i o n s 1 : 2.5 : 2.5 respectively. The gel 'was destained w i t h several changes of acetic acid, ethanol, w a t e r , 1 : 2 . 5 : 6 . 5 by volume. An u n s t a i n e d gel focussed u n d e r i d e n t i c a l c o n d i t i o n s but ~without e n z y m e was cut into p o r t i o n s of about 2 mm. E a c h p o r tion w a s e x t r a c t e d o v e r n i g h t at 4°C w i t h one m l of w a t e r . The p H of each e x t r a c t Yeas m e a s u r e d to d e t e r m i n e the true p H gradient.
N-terminal analysis. The d a n s y l a t i o n m e t h o d of Zanetta was used E14] starting w'ith 100 :~g of p r o t e i n p r e c i p i t a t e d w i t h 10 p e r cent TCA and w a s h e d w i t h 1 ml M HC1 and t h e n t~vice w i t h 1 ml acetone. The chrom a t o g r a p h y 'was p e r f o r m e d both on silica gel and p o l y a m i d e sheets. The spots c o r r e s p o n d i n g to (x-dansyl-leucine and to e-dansyl-lysine w e r e eluted f r o m the silica gel and the fluorescence m e a s u r e d as d e s c r i b e d by Zanetta el at.
Amino acid composition. About 100 ~tg of e n z y m e 'was p r e c i p i t a t e d w i t h 10 p e r cent TCA acid and after c e n t r i f u g a t i o n , the p r o t e i n was h y d r o l y s e d in a sealed tube in 500 ~xl 6 M HC1 for 18 h o u r s at l l 0 ° C . T h e a m i n o - a c i d composition was determined with a Technicon a m i n o acid analyser. T r y p t o p h a n ~ . h i c h is d e s t r o y e d by this m e t h o d 'was not d e t e r m i n e d . KINETIC STUDIES.
Determination of Michaelis constants. The w e r e p e r f o r m e d at (a) 25°C and (b) 37°C. The i n c u b a t i o n m e d i u m consisted of 100 mM Tris HC1 c o n t a i n i n g 50 mM KC1, 20 mM m e r c a p t o ethanol and 0.1 mM EDTA. Various substrate con-
BIOCHIMIE, 1977, 59, n" 3.
259
c e n t r a t i o n s w e r e a d d e d at c o n s l a n t volume. The r e a c t i o n w a s started by a d d i t i o n of p u r e e n z y m e and the rate of i n c r e a s e of fluorescence m e a s u r e d directly. The slopes vcere a l w a y s o b t a i n e d f r o m the l i n e a r part of the r e c o r d i n g and w e r e thus initial rates.
Reverse reaction. The rate of the r e v e r s e r e a c t i o n ~vas m e a s u r e d by d e t e r m i n i n g the a m o u n t of NAD f o r m e d by the strong alkali m e t h o d of L o w r y et al [15] at various time intervals up to 7 h o u r s at 37°C in the usual i n c u b a t i o n m e d i u m but c o n t a i n i n g the reverse substrate p a i r i.e. s u c c i n i c acid and NADH at a c o n c e n t r a t i o n each of 1.75 × 10-~ M. This rate was c o m p a r e d to the f o r w a r d r e a c t i o n rate u n d e r i d e n t i c a l c o n d i t i o n s and e n z y m e c o n c e n tration but w i t h the f o r w a r d r e a c t i o n substrate p a i r at 1.75 X 10 -4 M. Controls w e r e run w i t h o u t enzyme.
Reaction equilibrium. The usual i n c u b a t i o n m e d i u m w a s used, but the substrates NAD and SSA w e r e e a c h at 1.75 × 10 4 M. The r e a c t i o n was started by a d d i t i o n of purified e n z y m e to the m e d i u m m a i n t a i n e d at 37°C in f l u o r i m e t e r cuvettes. R e a d i n g w e r e taken until no f u r t h e r i n c r e a s e in fluorescence could be detected. The f l u o r i m e t e r w a s then r e c a l i b r a t e d and the NADH p r e s e n t d e t e r m i n e d .
Determination of pH optimum. F o r p H ' s b e l o w 9, 100 mM s o d i u m p y r o p h o s phate buffer w a s u s e d ; above p H 9, g l y e i n e / NaOH buffers. The p H m e d i a all c o n t a i n e d NAD 3 × 10 -~M, SSA 1 × 10 -4 M, KC150 mM, m e r c a p t o e t h a n o l 20 mM and E D T A 0.1 raM. The rales of f l u o r e s c e n c e i n c r e a s e 'were r e c o r d e d d i r e c t l y (initial rates).
Substrate specificity. The initial r e a c t i o n rates w i t h various aldeh y d e s at 1 × 10 -~ M c o n c e n t r a t i o n s and NAD at 3 X 10 .4 M veere c o m p a r e d to a c o n t r o l w i t h SSA at 1 × 10 -~ M. S i m i l a r l y the r e a c t i o n rate using 3 × 10 -4 M NADP yeas c o m p a r e d to that o b t a i n e d using 3 × 10 -4 M NAD.
Inhibition studies. 1) Aldehydes. R e a c t i o n rates w e r e m e a s u r e d in the p r e s e n c e of v a r i o u s a l d e h y d e s and a range of para-substitared b e n z a l d e h y d e s at 1 X 10 -3 M, veith 3 × 10 -4 M NAD and 1 × 10 -~ M SSA. The m e c h a n i s m of i n h i b i t i o n by p - h y d r o x y b e n z a l d e h y d e was det e r m i n e d by m e a s u r i n g the initial rates at several i n h i b i t o r c o n c e n t r a t i o n s w i t h v a r i o u s fixed con-
C. Cash, L . C i e s i e l s k i , M. M a i t r e a n d P . M a n d e i .
260
c e n t r a t i o n s of o n e s u b s t r a t e a n d t h e n v a r i o u s fixed c o n c e n t r a t i o n s of the o t h e r substrate. 2) Substrate inhibition by SSA. I n h i b i t i o n b y h i g h c o n c e n t r a t i o n s o f SSA w a s m e a s u r e d u s i n g v a r i a b l e c o n c e n t r a t i o n s o f NAD. 3) AMP. T h e m e c h a n i s m o f i n h i b i t i o n b y AMP w a s stud i e d u s i n g s e v e r a l c o n c e n t r a t i o n s o f AMP, f i r s t u s i n g v a r i o u s f i x e d c o n c e n t r a t i o n s of NAD, t h e n v a r i o u s f i x e d c o n c e n t r a t i o n s o f SSA.
rious c o n c e n t r a t i o n s of m e r c a p t o e t h a n o l b e t w e e n 0 a n d 10 raM. T h e r e a c t i o n ~,as s t a r t e d b y a d d i tion of e n z y m e .
Iodoacetamide inactivation. I n c u b a t i o n s w e r e c a r r i e d o u t at 37°C w i t h SSA at 1 × 10 -4 M a n d NAD at 3 × 10 -4, 1.5 × 10 -4 a n d 0.5 × 10 -4 M. T h e m e d i u m w a s 100 m M T r i s HCI b u f f e r c o n t a i n i n g 50 m M KC1, 0.1 m M E D T A a n d 5 m M m e r c a p t o e t h a n o l . T h e p H w a s 7.5 as a t t h i s p H , t h e c a r b o x y m e t h y l a t i o n r e a c t i o n is s p e cific to c y s t e i n r e s i d u e s . T h e r e a c t i o n w a s s t a r t e d
TABLE I,
Purification of s u c c i n i c s e m i a l d e h y d e dehgdrogenase f r o m rat brain. Fraction
Volume (ml)
Units/ml
Homogenate . . . . . . . . . . . . . . . . . . Pooled supernatants . . . . . . . . . . J Sephadex G200 eluate . . . . . . . . . DEAE cellulose eluate . . . . . . . . . A M P affinity column eluate . . . . I
240 306 94 116 2.2
0.48
0.3 0.2 0.08 3.07
! [ I I
mg/protein/ml
Units/rag protein
Yield
Purification
30 15 2.5 0.1 0.11
0.016 0. 021 0.08 0.87 27
100 80 16 8 6
1 1.3 5 55 1710
1 unit = 1 ~mol NADH f o r m e d per minute.
Mercaptoethanol reactivation. Mercaptoethanol-free enzyme was prepared by r e a b s o r p t i o n of diluted e n z y m e on an affinity c o l u m n a n d e l u t i o n w i t h 1 m M AMP i n b u f f e r containing no mercaptoethanol. Reactivation curves w e r e o b t a i n e d by fluorescence m e a s u r e m e n t i n t h e u s u a l m e d i u m at 37°C b u t c o n t a i n i n g va-
M I
5
2
8
4
I0'_ - - r - - - v - IOO
150
FIG. 2. - - Molecular pweight determination of native enzyme by Sephadex G200 filtration : l. ~/ Globulin ; 2. Alcohol dehydrogenase ; 3. H a e m o g l o b i n ; 4. Ovalb u m i n ; 5. SSADH.
FIo. 1. - - SDS-Polyacrylamide gel electrophoresis of purified enzyme. BIOCHIMIE, 1977, 59, n ° 3.
b y a d d i t i o n of e n z y m e to t h e m e d i u m c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of i o d o a c e t a m i d e . T h e inactivation period was measured by direct record i n g of the f l u o r e s c e n c e change.
Bat brain succinic semialdehyde dehydrogenase. RESULTS,
Puri[ieation. T h e e n z y m e w a s p u r i f i e d 1,76(} f o l d c ~ p a r e d t o t h e i n i t i a l h o m o g e n a l e ' w i t h a y i e l d of a b o u t 6 p e r c e n t . T h e f i n a l e l u a l e f r o m t h e a f f i n i t y col u m n (2.2 ml) c o n t a i n e d 250 ~g p r o t e i n a n d h a d a s p e c i f i c a c t i v i t y o f 27 u n i t s p e r m i l l i g r a m , (ta-
~o~
261
ble I), 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 u s i n g non-denaturing conditions gave a single band cont a i n i n g all t h e e n z y m e a c t i v i t y , SDS gel e l e c t r o p h o r e s i s a l s o r e v e a l e d a s i n g l e b a n d (see fig. i ) .
31olecular weights and subunits.
of the native enzyme
S e p h a d e x G 200 gel f i l t r a t i o n u s i n g m o l e c u l a r w e i g h t m a r k e r s g a v e a m o l e c u l a r w e i g h t of a b o u t 140,000 (see fig. 2). F r o m SDS gel e l e c t r o p h o r e s i s , a m o l e c u l a r w e i g h t of a b o u t 68,000 w a s c a l c u -
5 0
04
M-W
1@ ~b~ x ~-"~
~1
i
t
1
I
I
2
3
~rfm
Fro. 3. - - Molecular ~meiyht delerrainalion af enzgme s n b u n i t s by SDS 9el electrophoresis. 1. Myoglobin ; 2, C h y m o t r y p s i n o g e n A ; 3, P e p s i n ; 4, Ovalbumin ; 5, Serum Albumin. SSADH subunits have the same m o b i l i t y as s e r u m albumin. TABLF. lI.
A m i n o a c i d c o m p o s i t i o n o[ r a t b r a i n succinic semialdehyde dehydrogenase.
,> *
i
I
[;'to. 4, - - Lin~weaver-BurJ~ rates at ~5~C as a f u n c t i o n of variable concentrations of the 8 × 10-s M ; 2. SSA 4 X 10-o and 4. SSA 1,5 X 10-5 M.
plot of initial reaclion NAD concentration, and second substrale. 1. SSA M ; 3. SSA 2.5 × 10-5 M
l u t e d (fig. 3). P r e t r e a t m e n t o f t h e e n z y m e w i t h o r without mercaptoethanol and iodoacetamide did n o t c h a n g e t h e r.f. o n SDS gels.
Isoelectric point. Amino acid
Glutamate . . . . . . . . . . . . . . Aspartate . . . . . . . . . . . . . . . Threonine . . . . . . . . . . . . . . . Serine . . . . . . . . . . . . . . . . . . Glyeine . . . . . . . . . . . . . . . . . Cysteine . . . . . . . . . . . . . . . . Methionine . . . . . . . . . . . . . . Valine . . . . . . . . . . . . . . . . . . Praline . . . . . . . . . . . . . . . . . Arginine . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . Lysine . . . . . . . . . . . . . . . . . . Phenylalanine . . . . . . . . . . . Tyrosine . . . . . . . . . . . . . . . . Leueine . . . . . . . . . . . . . . . . . lsoleueine . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . . Tryptaphan
Residues per Mol
100 80 5~ 78 120 16 14 88 38 90 14 70 46 30 86 40 116
. . . . . . . . . . . . .
Total . . . . . . . . . . . . . . . . . . .
BIOCHIMIE, 1977, 59, n ° 3.
1078
T h e i s o e l e c t r i c p o i n t is a b o u t 7,2.
104 KNAD
40 30
10 i
///
t
2
L
4
i
d
! 8
FIG, ft. - - Replot f r o m figure 1~ o f intercepts on :e axis (~s' NAD) against reciprocal SSA eoneenlralions.
C. Cash, L. C i e s i e l s k i , M. M a i t r e a n d P. M a n d e l .
262
N - T e r m i n a l analysis.
Sabstrate s p e c i f i c i t y .
A single N-Terminal amino-acid corresponding to l e u c i n e w a s f o u n d , t h u s s u g g e s t i n g t w o i d e n tical subunits.
Other aldehydes. T h e a l d e h y d e s tested c a n n o t be c o n s i d e r e d as significant s u b s t r a t e s for SSADH in v i e w of t h e i r
A m i n o acid composition. T h e q u a n t i t i e s a r e r e p r e s e n t e d o n t a b l e II. T a k i n g a c c o u n t t h e 100 I~g o f p r o t e i n h y d r o l y s e d a n d t h e m o l e c u l a r w e i g h t f o u n d o f a b o u t 136,000, w e c a l c u l a t e d 539 a m i n o a c i d r e s i d u e s p e r c h a i n . T h e m a i n 3 a m i n o a c i d p r e s e n t a r e Glu, A s p , Ala a n d Gly. T h e y r e p r e s e n t a b o u t 30 p e r c e n t o f t h e t o t a l a m i n o a c i d s of t h e m o l e c u l e .
1
3
1
I
,/
I
I
I
I
I
6
7
8
9
10
h
pH
2
I 5
I 10 .M.SSA
I 15
•
I , 20
FIG. 7. - - Subslrate inhibition by SSA Plot of reciprocal velocity against SSA concentration. t. NAD 3 X 10-4 M ; 2. NAD 1 X 10-4 M.
FIG. 6. - - Relative activity of SSADH
as a function of pH.
f e e b l e r a t e of o x i d a t i o n i n c o m p a r i s o n w i t h SSA o x i d a t i o n (see t a b l e III). KINETIC
STUDIES.
TABLE IV.
K m determinations. By m e a s u r i n g i n i t i a l r a t e s at v a r i o u s f i x e d c o n c e n t r a t i o n s of one s u b s t r a t e w h i l s t v a r y i n g the c o n c e n t r a t i o n s o f t h e o t h e r , K m ' s ( l i m i t ) ~vere o b t a i n e d f r o m s e c o n d a r y plots of the initial Linew e a v e r - B u r k p l o t s . T h e s e a r e a b o u t 2 X 10-~ lVl f o r NAD a n d 1 X 10 -4 M f o r SSA (see fig. 4, fig. 5).
I n h i b i t i o n by aldehydes. Aldehyde (t 0-:aM)
Per cent residual activity
Valeraldehyde . . . . . . . . . . . Benzaldehyde . . . . . . . . . . . Chloral hydrate . . . . . . . . . . p-hydroxyhenzaldehyde . . .
84 70 60 0
pH optimum. I n h i b i t i o n by a series of para-sabstituted benzaldehydes (10-.3 M).
(see fig. 6). T h e r e is a b r o a d p H o p t i m u m w i t h a m a x i m u m at a b o u t p H 8.6.
R group
Per cent residual activity
TABLE III.
Relative rates of oxidation of some other aldehydes.
OH
(Rate Related to SSA = 100 per cent).
Br
F
.................... ......................
Cl ...................... .....................
I ......................
Aldehyde (t0-4M)
Per cent activity
Valeraldehyde . . . . . . . . . . . Chloral hydrate . . . . . . . . . . Acetaldehyde . . . . . . . . . . . . Benzaldehyde . . . . . . . . . . . p-hydroxybenzaldehyde...
2.6
BIOCH1M1E, 1977, 59 n ° 3.
1.0 0.5 0.5 0
NO:~ . . . . . . . . . . . . . . . . . . . . NH 2 . . . . . . . . . . . . . . . . . . . . C~N ................... CH:~ . . . . . . . . . . . . . . . . . . . .
! I
CH(CH~)~ . . . . . . . . . . . . . . . O-CH:~ . . . . . . . . . . . . . . . . . . O-C2H.,, . . . . . . . . . . . . . . . . . .
! , ,
C._,H:, . . . . . . . . . . . . . . . . . . . .
0 81 62 62 48 80 100 82 44
49 27 58 58
Rat brain succinic semialdehyde dehydrogenase.
263
NADP. W i t h N A D P a s s u b s t r a t e a b o u t 13 p e r c e n t o f the activity was observed compared to a control using equimolar NAD as substrate.
8 slope
6
,°t
/
3O-
_t Vo
2O0
2
4 6 8 4 H B X 106 M
10
Fro. 9. - - Secondary plot of slopes versus
inhibitor concentration from [igure 8.
I0-
o
1
20
X 104M
Flo. 8. - - Double reciprocal plot of inhibition o[ SSADH by #HB at 3 concentrations of SSA. NAD c o n c e n t r a t i o n is 3 X 10-4 M. 1. No i n h i b i t o r ; 2. 4 HB 2 X 10-6 M ; 3. 4HB 5 × 10-6 M a n d 4. 4HB 1 X 10-5 M. TABLE V.
Reactivation of suecinic s e m i a l d e h y d e dehydrogenase by ~-mercaptoethanol. ~-mercaptoethanol concentration 3 1.5 4.5 3 1.5 0
X X X X ×
10-eM 10-UM 10-3M 10-aM 10-aM
Per cent ol m a x i m u m rate achieved 100 100 78 72 32 0
Reactivation period
30" 30" 1'30" 2'30" *~
7' 0
Inhibition. 1) Substrate inhibition by SSA. SSA b e c o m e s i n h i b i t o r y at c o n c e n t r a t i o n s a b o v e 10 -3 M (fig. 7). At i n h i b i t o r S S A c o n c e n t r a t i o n s ,
BIOCHIMIE, 1977, 59, n ° 3.
i 2
I 4
i 6
I 6
10
Fta. 10. - - Double reciprocal plot o[ SSADH inhibition
by ~HB at 3 concentrations o[ NAD. SSA c o n c e n t r a t i o n is 1 × 10-4 M. 1. No i n h i b i t o r ; 2. 4HB 2 X 10-6 M ; 3. 4HB 5 × 10-G M ; 4. 4HB 1 × 10-5 M. 20
C. Cash, L . C i e s i e l s k i , ill. M a i t r e a n d P . M a n d e l .
264
t h e r e is, as has been p r e v i o u s l y d e m o n s t r a t e d by K a m m e r a t and Veldstra [5], a l i n e a r i t y b e t w e e n the SSA c o n c e n t r a t i o n a n d the r e c i p r o c a l value of the i n i t i a l r e a c t i o n v e l o c i t y (see fig. 7). F o r d i f f e r e n t c o n c e n t r a t i o n s of NAD, a plot of 1/(SSA) ( i n h i b i t o r y c o n c e n t r a t i o n s ) versus 1 / V 0 gives p a r a l l e l lines s h o w i n g that the i n h i b i t i o n is u n e o m p e t i t i v e w i t h r e s p e c t to NAD, suggesting a r e a c t i o n b e t w e e n one SSA m o l e c u l e a n d a substrate e n z y m e c o m p l e x .
3) I n h i b i t i o n by 5' AMP. As can be seen f r o m a competitive inhibitor a K i of about 7,5 10 -5 inhibitor with respect
figs 12, 13, 14, 15, AMP is w i t h r e s p e c t to NAD w i t h M and a non c o m p e t i t i v e to SSA.
4
40
2{3
1
Vo
30 3 I
15 intercept 2E
10 10
0
I 2
I 4
I 6
I 8
I 10
N A D X 10-4M
1
I 0
2
I
I
4 6 4HB ~ 106M
I 8
10
Fro. 11. - - Secondary plot of intercepts versus
FIG. 12. - - Double reciprocal plot of SSADH inhibition by AMP at 3 concentrations of NAD. SSA is 1 X 10-4 M. 1. No i n h i b i t o r ; 2. AMP 0.1 m M ; 3. AMP 0.2 mM and 4. AMP 0.5 raM.
inhibitor concentration from figure 10.
It should be noted that s u e e i n i c acid, the product of SSA o x i d a t i o n , was not i n h i b i t o r y even at 100 mM. 2) I n h i b i t i o n by aldehydes. p - h y d r o x y b e n z a l d e h y d e (4 HB) is the most p o w e r f u l of the i n h i b i t o r y a l d e h y d e s studied. No e n z y m e a c t i v i t y could be d e t e c t e d ~vith 4 HB at 10 ~a M and SSA and NAD at 1 × 10 -4 and 3 × 10-~ M r e s p e c t i v e l y . A series of p-substitued benz a l d e h y d e s w e r e in g e n e r a l m o r e i n h i b i t o r y as the substituent group b e c a m e less e l e c t r o n e g a t i v e (see table 4). It can be seen (Figs 8, 9, 10, 11) thai 4 HB is a c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t to SSA ",vith a K i of about 3 × 10 -6 M and is an u n c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t to NAD.
BIOCHIMIE, 1977, 59, n ° 3.
ADP and ATP but not a d e n i n e or a d e n o s i n e i n h i b i t s SSADH to a s i m i l a r extent to AMP.
Mercaptoethanol reactivation. A p u r e p r e p a r a t i o n of SSADH is c o m p l e t e l y i n a c t i v e in the absence of m e r c a p t o e t h a n o l . By a d d i n g v a r i o u s l o w c o n c e n t r a t i o n s of m e r c a p t o ethanol to the i n c u b a t i o n m e d i u m c o n t a i n i n g the enzyme, a d i m i n i s h i n g p e r i o d of r e a c t i v a t i o n ~vith i n c r e a s i n g m e r c a p t o e t h a n o l c o n c e n t r a t i o n s ~vas o b s e r v e d (table V). About 10 -2 M m e r c a p t o e t h a n o l w a s n e c e s s a r y for full e n z y m e activity. An analysis of the r e a c t i v a t i o n c u r v e s (see a p p e n d i x ) i n d i c a t e s that the r e a c t i v a t i o n m e c h a n i s m follows pseudo-first o r d e r kinetics.
Iodoacelamide inactivation. With 10 -2 M i o d o a c e t a m i d e , no e n z y m e a c t i v i t y was detected, u n d e r the c o n d i t i o n s e m p l o y e d .
265
Rat brain succinic semialdehyde dehydrogenase. W i t h l o w e r c o n c e n t r a t i o n s , a d e c r e a s e in r e a c t i o n rate could be o b s e r v e d , l e a d i n g a l w a y s to total
i n a c t i v a t i o n . A n a l y s i s of t h e i n c u b a t i o n c u r v e (see a p p e n d i x ) s h o w s t h a t i n a c t i v a t i o n is p s e u d o first o r d e r . It c a n b e s e e n f r o m t a b l e VI t h a t t h e i n a c t i v a t i o n p e r i o d i n c r e a s e s w i t h NAD c o n c e n tration. TABLE ¥ I .
Inactivation of succinic semialdehyde dehydrogenase by iodoacetamide.
slope
3 3
X 1D-4M X 10--~M
1.5
X
Inactivation period
lodoacetamide concentration
NAD concentration
4 2 2 2
10 -~ M
0.75 X 10 -4 M
1'30"
mM mM mM mM
13'45" 3'10" 10"
10
I
,/
I 0.1
I 0.2
I 03
I 04
I 0.5
MM. A M P
FI~. 13. - - Secondary plot of slopes uersus inhibitor
concentration from figure 12.
t a w
1 O
10
I 1
I 2
I 3
I 4
I 5
~M. AMP
FI(~. 15. - - Secondary plot of intercepts versus
inhibitor concentration from figure 1-~,
Reverse reaction.
,,-f//
I
t
I
1
I
0
1
2
3
4
~AX
10"4 M
Under identical reaction conditions save for the s u b s t i t u t i o n of t h e f o r w a r d s u b s t r a t e b y e q u i m o l a r c o n c e n t r a t i o n s of t h e r e v e r s e s u b s t r a t e p a i r , t h e r e v e r s e r e a c t i o n p r o c e e d e d at 1/1230 o f t h e r a t e of the f o r w a r d r e a c t i o n .
FI(L 14. - - Double reciprocal plot of SSADH inhibition
by AMP at 3 concentrations of SSA.
NAD is 3 X 10-4 M. 1. No i n h i b i t o r ; 2. AMP 1 mM ; 3. AMP 2 mM and 4. AMP 5 mM.
BIOCHIMIE, 1977, 59, n ° 3.
Equilibrium. At e q u i l i b r i u m , 92 p e r c e n t o f t h e NAD w a s r e d u c e d to N A D H . T h u s t h e e q u i l i b r i u m c o n s t a n t
C. Cash, L. Ciesielski, M. Maitre and P. Mandel.
266 K ----
[NAD]
[Succinate]
= 132. and the h G ° [NAD] [SSA] for the f o r w a r d r e a c t i o n is - - 3,000 c a l / m o l . DISCUSSION. Using affinity c h r o m a t o g r a p h y as a final step, we o b t a i n e d SSADH w h i c h was h o m o g e n o u s a c c o r d i n g to the c r i t e r i a of both n o n - d e n a t u r i n g and SDS 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 a n d by N - t e r m i n a l analysis. An a p p a r e n t p u r i f i c a t i o n f a c t o r of 1,700 fold w i t h r e s p e c t to the i n i t i a l h o m o g e n a t e w a s obtained, the specific a c t i v i t y w a s 27 units p e r m i l l i g r a m of p r o t e i n . No isoenzymes w e r e detected. To study the k i n e t i c p a r a m e t e r s a n d m e c h a nism, w e ~vere obliged to w o r k w i t h i n a s o m e w h a t l i m i t e d r a n g e of substrate c o n c e n t r a t i o n s ; the u p p e r limits b e i n g fixed by the r e l a t i v e l y l o w Km's and the p h e n o m e n o n of substrate i n h i b i tion ; the l o w e r limits b e i n g fixed by the need to h a v e an a c c u r a t l y m e a s u r a b l e i n i t i a l r e a c t i o n rate for s e v e r a l minutes. If the e n z y m e c o n c e n t r a t i o n w e r e r e d u c e d to a l l o w l o n g e r i n c u b a t i o n p e r i o d s , its stability b e c a m e a l i m i t i n g factor. L i n e w e a v e r - B u r k plots of i n i t i a l rates w h e n one substrate 'was v a r i e d at s e v e r a l fixed conc e n t r a t i o n s of the o t h e r gave lines w i t h slopes w h i c h are not significantly different. This is unusual for d e h y d r o g e n a s e , but has been observed with g l y c e r a l d e h y d e - p h o s p h a t e dehydrogenase f r o m Pisum sativum [16]. F r o m the m e c h a nistic p o i n t of v i e w , this can be e x p l a i n e d in one of the f o l l o w i n g w a y s : 1) T h e i u e c h a n i s m is ping-pong, i.e., a p r o d u c t is released b e f o r e the a d d i t i o n of the s e c o n d substrate. 2) T h e m e c h a n i s m is sequential, but i n v o l v e s a t h i r d s a t u r a t i n g substrate [17] a d d i n g in the s e c o n d position. 3) T h e e n z y m e substrate d i s s o c i a t i o n c o n s t a n t is m u c h s m a l l e r than the Kin. In this case, the c h a n g e in slope is v e r y small in c o m p a r i s o n to the c h a n g e in i n t e r c e p t for a l i m i t e d range of substrate c o n c e n t r a t i o n s [18]. Thus, in o r d e r to f u r t h e r d e l i n e a t e the m e c h a n i s m , w e h a v e studied d e a d - e n d i n h i b i t i o n patterns. AMP, an analogue of NAD, w a s f o u n d to be a c o m p e t i t i v e i n h i b i t o r vis h vis NAD, but a non c o m p e t i t i v e i n h i b i t o r vis h vis SSA. H o w e v e r , p-hydroxybenzaldehyde, a competitive inhibitor w i t h r e s p e c t to SSA, gave u n c o m p e t i t i v e i n h i b i tion w h e n NAD w a s the v a r i a b l e substrate. A
BIOCHIMIE, 1977, 59, n ° 3.
s e c o n d a r y plot of the i n t e r c e p t versus i n h i b i t o r c o n c e n t r a t i o n s is linear. This is i n d i c a t i v e of an o r d e r e d bi-bi or ter-bi m e c h a n i s m w h e r e NAD must b i n d first to the enzyme, f o l l o w e d by SSA, before release of products. That is, p - h y d r o x y b e n z a l d e h y d e b i n d s only w i t h the enzyme-NAD c o m p l e x and h e n c e competes w i t h the s e c o n d substrate SSA, but is u n c o m p e t i t i v e s vis ~ vis NAD as it has no affinity for the free enzyme. T h e possible r e a c t i o n m e c h a n i s m is s u m m a r i z e d in the s c h e m e : E-AMP
hMl~
E
E-NADH
NAD'
.
,
E-NAO
" E-NAD-H20-SSA
5ucclr~te
E NAD-H20 SSA - SSA -
-
T h e m a i n i n d i c a t i o n s for an o r d e r e d ter-bi m e c h a n i s m w i t h w a t e r as a substrate are that : 1) W a t e r is n e c e s s a r y for the s t o e c h i o m e t r y of the c h e m i c a l r e a c t i o n R-C-H q- NAD + H20 ~-~ R-C-OH + NADH ~- H ~ I[ 0 O 2) A ter-bi o r d e r e d m e c h a n i s m w i t h w a t e r as the second substrate w o u l d e x p l a i n the p a r a l l e l lines of the L i n e w e a v e r - B u r k plot. O r d e r e d ter-bi m e c h a n i s m w i t h the s e c o n d substrate at s a t u r a t i n g level can be d i s t i n g u i s e d f r o m o r d e r e d bi-bi m e c h a n i s m by p r o d u c t i n h i b i t i o n . I n h i b i t i o n w i t h NADH w i t h r e s p e c t to SSA w o u l d be u n c o m p e t i t i v e in the ease of a ter-bi m e c h a nism, non c o m p e t i t i v e in the ease of a bi-bi mechanism. E x p e r i m e n t s h a v e been c o n d u c t e d but are i n c o n c l u s i v e o'wing to the love i n h i b i t i o n constants. P r o d u c t s are ~xeak i n h i b i t o r s w h e n the f o r w a r d r e a c t i o n is s t r o n g l y favoured. To distinguish b e t w e e n these t'wo o r d e r e d m e c h a n i s m o t h e r i n v e s t i g a t i o n s should be c o n d u c t e d .
APPENDIX.
D e t e r m i n a t i o n of a c t i v a t i o n and i n a c t i v a t i o n p e r i o d s f r o m the f l u o r e s c e n c e r e c o r d i n g s u s i n g 1st o r d e r r e a c t i o n kinetics.
Rat brain succinic semialdehyde dehydrogenase. 1) I n a c t i v a t i o n b y I o d o a c e t a m i d e c e n t p r o d u c t (fig. 16).
--
Fluores-
2) R e a c t i v a t i o n b y m e r c a p t o e t h a n o l . Eo Initial enzyme inactive E A c t i v e e n z y m e at t i m e t T Reactivation period
E o A c t i v e e n z y m e at z e r o t i m e E A c t i v e e n z y m e at t i m e t
t E = E o (1-e - - Log2) T dF v --- K E (2) dt
v Reaction rate T Inactivation period F Fluorescence (Arbitrary Units) F o F l u o r e s c e n c e at z e r o t i m e F~
267
t x all o t h e r s y m b o l s ~ as a b o v e (8)
F l u o r e s c e n c e at i n f i n i t e t i m e / /
F
/
%" ,'
//
///
///
/ / /
//
/
//
~///
///
>t
',. )//
,t FIG.
16.
/
/
/
/
/
/
(1) E : (2)
v --
Eo.e dF
t
---
T
Log2 Fro, 17.
KE
--
dt
F r o m (8) a n d (2) F :
F r o m 1) a n d 2) KT
KT
+ Fo
F~ -- F . . . . . e Log2
(4)
t
__ ~t Log2
T
= a constant --
T - - - - Log2 __ 1)) F = K E o (t + - e T Log2
(5)
t Log2 T
(6)
F~--F are the values experimentaly determined from the fluorescence recordings; Log (F~--F) is t r e a t e d b y t h e least m e a n s q u a r e m e t h o d . If e x p e r i m e n t a l l y a s t r a i g h l i n e is o b t a i n e d ~ i t h a g o o d c o e f f i c i e n t of c o r r e l a t i o n t h e i n a c t i v a t i o n p e r i o d c a n be e x p r e s s e d b y t h e relation T
Log2 slope
BIOCHIMIE, 1977, 59, n ° 3.
constant
T
T h e c o n s t a n t is d e t e r m i n e d f r o m F o = T constant = Log2
Log2 KT
t Log2) +
e
(3)
Log2
Log (F~--F)
Log2
F = t (1-e - - -T L o g 2 ) + F o
Foo --
T
K E o (t +
(7)
(9) O
(9)
U s i n g a s u p p l e m e n t a r y f u n c t i o n F u w h i c h is the F l u o r e s c e n c e w h i c h ~vould be o b s e r v e d in all t h e e n z y m e s w e r e a c t i v e at z e r o t i m e F u = K E o t , we can write : t
X = Fu-F : Xoo :
KEo (1--e
KEoXoo--X
L o g (Xoo - - X) : - - - -
T
t
---
t
(10)
Log2
(11)
Log2 + a c o n s t a n t
(12)
= KEoe T
Log2) T
C. Cash, L. C i e s i e l s k i , M. M a i t r e a n d P. M a n d e l .
268
( X ~ - - X) is a n e x p e r i m e n t a l v a l u e d e t e r m i n e d f r o m t h e f l u o r e s c e n c e r e c o r d i n g . L o g ( X ~ - - X) is t r e a t e d as a b o v e . T h e r e a c t i v a t i o n p e r i o d of t h e e n z y m e is g i v e n b e t h e r e l a t i o n : Log2
T
slope R~su~i~. La semi aldt~hyde succinique d6shydrog6nase de cerveau de rat a 6t6 purifi6e j n s q u ' h homog6n6it6 selon les crit6res d'Slectrophor6se. Son poids mol6culaire est d ' e n v i r o n 140.000 Daltons et elle est formde de deux sous-unitSs a p p a r e m m e n t identiques. La r~action catalys~e par la prot~ine pure est enti~r e m e n t d6pendante de la pr6sence de groupes sulfhydriles endogbnes. Les Km limites pour ]e NAD et le semi ald6hyde succinique sont r e s p e c t i v e m e n t de 2 × 10-5 M e t 1 X 10-4 M a u pH o p t i m u m de 8,6. Des 6tudes cin6tiques et d ' i n h i b i t i o n m o n t r e n t que le m6canisme de la r6action est u n m6canisme ordonn6, h ordre obligatoire d ' a d d i t i o n des substrats : le NAD dolt se fixer le premier, le semi ald6hyde succinique le dernier. REFERENCES. 1. Schousboe, A., Wfl, J. Y. & Roberts, E. (1973) Biochem., 17, 2868-2873. 2. Maitre, M., Ciesielski, L., Cash, C. a Mande], P. (1975) Eur. J. Biochem., 57, 15.7-169.
BIOCHIMIE, 1977, 59, n ° 3.
3. Cash, C., Maitre, M., Ciesielski, L. ,~ Mandel, P. 0974) FEBS Letters, 47, 199-203. 4. Bloch-Tardy, M., Rolland, B. ~ Gonnard, P. (1974) Biochimie, 5.6, 823-837. 5. K a m m e r a a t , C. ,~ Veldstra, H. (1967) Biochem. Biophys. Acla, 15'1, 1-10. 6. Embree, L. J. ~ Albers, R. W. (1963) Biochem. Pharmacol., 13, 1209-1217. 7. Albers, R. W. ~ Koval, G. J. (1961) Biochem. Biophys. Acta, 57, 29-35. 8. Cash, C., Ciesielsld, L., Maitre, M. ~ Mandel, P. (1975) C. R. Soc. Biol. (Paris), 169, 884-887. 9. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. ,~ Randel, R. J. (1951) J. Biol. Chem., 11~3, 265-275. 10. Udenfried, S., Stein, S., Bohlen, P., Dairman, W'., Leimgruber, W. ,~ Weigele, M. (1972) Science, 178, 871-872. 1I. Joivin~ T., Chrombaeh, A..~ Naughton, M. A. (1964) Anal. Biochem., 9, 351-369. 12. Weber, K..a Osborn, M. (1969) J. Biol., 244, 44064412. 13. Allen in <( Disc Electrophoresis >>. Ind. Ed. Mauer (1971) p. 134, W a l t e r de Gruyter, Berlin. 14. Zanetta, J. P., Vincendon, G., Mandel, P. ~ Gombos, G. (19'70) J. Chromatog., 5, 441-548. 15. Lowry, O. ~ P a s s o n n e a u , J. in (( A Flexible System of Enzymic Analysis >> (1972) Academic Press, p. 10-11. 16. Duggelby, R. ~ Dennis, D. (1973) J. Biol. Chem., 249, 167-174. 17. Cleland, W. (1967) Annual Review of Biochemistry, ~6, 77-117. 18. Dalziel, K. (< The enzymes >>. Third Edition Academic Press, Vol. II, Ed. Boyer, p. 1-60.