BIOCHIMIE, 1977, n ° 59, 735-737.
Interaction of Blue Dextran and Cibacron Blue F3GA with calf spleen NAD+-glycohydrolase. F r a n c i s SCHUBErt 0 a n d M a r c PASCAL. I n s t i t u t de B o t a n i q u e de I ' U n i v e r s i t d L o u i s P a s t e u r , 28, r u e G o e t h e - - ~7~00 S t r a s b o u r g (France). (12-~-1977).
INTRODUCTION. S o m e p r o t e i n s a n d e n z y m e s h a v e b e e n r e p o r t e d to i n t e r a c t ' w i t h B l u e D e x t r a n to f o r m a c o m p l e x d i s s o e i a b l e b y s a l t [!1:-5]. Stel~wagen a n d c o w o r k e r s [69] h a v e e x t e n d e d t h i s o b s e r v a t i o n to a c e r t a i n n u m b e r of m o n o - a n d d i n u c l e o t i d e b i n d i n g e n z y m e s . T h e y proposed that the interaction of Blue Dextran or its e h r o m o p h o r e C i b a e r o n B l u e F3GA w i t h a p r o t e i n i n d i cates, u n d e r d e f i n e d c o n d i t i o n s , t h e p r e s e n c e of t h e d i n u e l e o t i d e f o l d b i n d i n g site, i.e. a s u p e r - s e c o n d a r y s t r u c t u r e of t h e n i c o t i n a m i d e a d e n i n e d i n n c l e o t i d e b i n d i n g d o m a i n i n NAD+-linked d e h y d r o g e n a s e s [1011]. ~ e h a v e s t u d i e d t h e i n t e r a c t i o n of B l u e D e x t r a n a n d C i b a e r o n B l u e F3GA ~vith N A D + - g l y e o h y d r o l a s e (EC 3.2.2.6), 'with t h e p u r p o s e to d e t e c t t h e e x i s t e n c e o f s t r u c t u r a l a n a l o g i e s bet'ween t h e N A D + - h i n d i n g d o m a i n of t h i s e n z y m e a n d t h e e n z y m e s p o s s e s s i n g t h e d i n u e l e o t i d e fold. M A T E R I A L S AND METHODS. Materials. C a l f s p l e e n , pig b r a i n (as a c e t o n e p o w d e r s ) a n d Neurospora crassa NAD+-glycohydrolases and ~-NAD + were purchased from S i g m a C h e m i c a l Co. B l u e D e x t r a n 2000 (M~vV = 2 × 106), S e p h a d e x G-50 a n d S e p h a r o s e 4B 'were o b t a i n e d f r o m P h a r m a e i a . C i b a c r o n B l u e F3GA (the c h r o m o p h o r e of B l u e D e x t r a n ) w a s a :kind g i f t of Drs. E. S u r y a n d K. Scheibli, (CibaGeigy, A. G. B a s e l ) . All o t h e r c h e m i c a l s w e r e Mercl~s products. Enzymes. Calf - spleen NAD+-glycohydrolase was solubilized f r o m a n a c e t o n e p ~ w d e r 'with p a n c r e a t i c l i p a s e a n d p a r t i a l l y p u r i f i e d (9 u n i t s / r a g p r o t e i n ) a c c o r d i n g to o u r p r e v i o u s l y p u b l i s h e d p r o c e d u r e [1'2]. T h i s s o l u b l e e n z y m e p r e p a r a t i o n 'was u s e d f o r t h e Ki d e t e r m i n a tions and affinity chromatography studies. Pig brain N A D ÷ - g l y c o h y d r o l a s e w a s s o l n b i l i z e d 'with p a n c r e a t i c l i p a s e [12], t h e s u p e r n a t a n t (100000 X g, 60 m i n ) h a d a specific a c t i v i t y o f 0.034 u n i t / m g p r o t e i n a n d w a s u s e d as s o l u b l e e n z y m e . Kinetic measurements and inhibition constants determination, I n i t i a l r a t e s of N A D + - g l y c o h y d r o l a s e c a t a l y s e d NAI) + hydrolysis were obtained titrimetrically [12]. T h e k i n e t i c s 'were p e r f o r m e d a t 37 °, p H 7.4 a t fixed i o n i c s t r e n g t h (NaC1) i n a final v o l u m e of 1.9 m l . A p p a r e n t i n h i b i t i o n c o n s t a n t s Ki w e r e d e t e r m i n e d f r o m t h e To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .
r e p l o t o f s l o p e s (in t h e c a s e of c o m p e t i t i v e i n h i b i t i o n ) a n d i n t e r c e p t s (in t h e c a s e of m i x e d i n h i b i t i o n ) o f t h e double reciprocal plots (Lineweaver-Bnrk) versus the c o r r e s p o n d i n g i n h i b i t o r c o n c e n t r a t i o n s [13]. C o n c e n t r a t i o n of B l u e D e x t r a n is e x p r e s s e d in t e r m s o f its e h r o m o p h o r e [8]Affinity chromatography. The Blue Dextran-Sepharose was prepared by deriv a t i z a t i o n of c y a n o g e n b r o m i d e a c t i v a t e d S e p h a r o s e - 4 B w i t h B l u e D e x t r a n [51. T h e a f f i n i t y c h r o m a t o g r a p h y gel w a s t e s t e d f o r i t s a b i l i t y to b i n d s o l u b i l i z e d c a l f spleen NAD+-glyeohydrolase. For analytical purposes, 30 ling of e n z y m e (0.3 u n i t ) in 10 m M Tris-HC1 b u f f e r (pH 7.4) w a s l o a d e d , at 4 ° , o n t o a m i c r o - c o l u m n c o n t a i n i n g B l u e D e x t r a n - S e p h a r o s e (1 m l ) p r e v i o u s l y e q u i l i b r a t e d 'with t h e c o r r e s p o n d i n g buffer. T h e col u m n w a s t h e n w a s h e d ' w i t h t h e s a m e b u f f e r (10 m l ) . T h e e l a t i o n of t h e e n z y m e w a s s t u d i e d b y a p p l y i n g to a c o l u m n a l i n e a r g r a d i e n t betxveen 0 a n d 1 M NaCI (in t h e T r i s b u f f e r ) , f r a c t i o n s o f 1 m l 'were c o l l e c t e d (fig. 2). F o r biospeeific e l u t i o n o f t h e NAD+glyeo h y d r o l a s e 10 m l of n u c l e o t i d e e o u n t e r l i g a n d , i n t h e T r i s - b u f f e r a d j u s t e d a t pH 7.4, w a s a p p l i e d f o l l o w e d b y 10 m l o f t h e T r i s - b u f f e r ( w a s h i n g ) . R e s i d u a l e n z y m e w a s d e t a c h e d ' w i t h a 1 M NaCI s o l u t i o n (5 m l ) . T h e e n z y m e a c t i v i t y of t h e d i f f e r e n t f r a c t i o n s ( e l u a t e s a n d washings) was measured using a modified KCN assay [12]. W h e n h i g h c o n c e n t r a t i o n s of n u e l e o t i d e s 'were u s e d , t h e a c t i v i t y in t h e e l u a t e s w a s a s s a y e d a f t e r dialysis against the Tris-buffer. RESULTS. Inhibilion studies. I n i t i a l r a t e s of NAD ÷ h y d r o l y s i s c a t a l y z e d b y s o l u bilized calf spleen NAD+-glycohydrolase are markedly a f f e c t e d b y C i b a c r o n B l u e F3GA a n d B l u e D e x t r a n . A representative Line'weaver-Bur.k plot for the inhibition b y B l u e D e x t r a n is s h o w n in figure IA ; a n a p p a r e n t i n h i b i t i o n c o n s t a n t o f 3.6 X 10-6 M w a s c a l c u l a t e d . T h e v a l u e s of e n z y m e - i n h i b i t o r d i s s o c i a t i o n c o n s t a n t Ki a r e v e r y d e p e n d e n t o n t h e i o n i c s t r e n g t h of t h e m e d i u m . W h e n I ~ 0.1 M B l u e D e x t r a n a n d C i b a e r o n B l u e F3GA a r e p u r e c o m p e t i t i v e i n h i b i t o r s , Ki i n c r e a s i n g nvith i n c r e a s i n g I (table I). W h e n t h e i o n i c s t r e n g t h is i n c r e a s e d f u r t h e r , in b o t h c a s e s t h e i n h i b i t i o n b e c o m e s m i x e d as i l l u s t r a t e d in figure 1B f o r C i b a c r o n B l u e F3GA (fig. 1B, t h e c a l c u l a t e d v a l u e s a r e Kit = 6.92 × 10-6 M a n d Kis ---- 1.28 × 10-6 M). F o r c o m p a r i s o n , ~ve h a v e a l s o i n v e s t i g a t e d t h e a c t i o n of C i b a c r o n B l u e F3GA o n t h e pig b r a i n a n d t h e N e u r o s p o r a c r a s s a N A D ~ - g i y c o h y d r o l a s e s . At I -: 0.1 M, u s i n g t h e D i x o n r e p r e s e n t a t i o n f o r d e t e r m i n i n g
F. Schuber and M. Pascal.
736
Ki [14], t h e f o l l o w i n g r e s u l t s 'were o b t a i n e d : pig b r a i n , b o u n d , 4.5 X 10-4 M ( c o m p e t i t i v e ) ; s o l u b i l i z e d , 5.,6 X 10-4 M ( c o m p e t i t i v e ) ; Neurospora crassa : 2,75 X 10-.3 M ( n o n c o m p e t i t i v e ) . T h e c a l f s p l e e n e n z y m e , i n t h e b o u n d f o r m ( a c e t o n e po'wder), w a s f o u n d to be
ned by the affinity column and could be released rap i d l y a n d q u a n t i t a t i v e l y , b y a p p l y i n g a NaC1 g r a d i e n t as s h o w n in figure 2. T h e m e a n v a l u e of NaC1 c o r r e s p o n d i n g to e n z y m e r e l e a s e :was 130 raM. T h e e n z y m e c o u l d n o t be e l u t e d b y l o w c o n c e n t r a t i o n s (1 m M
B
A
3
3
o
10(]
t50
2
E
'T "6
2
t00
..>
5O
J
1'0
:'0
1
I/NAO + [rnM- )
1~o 2o I/NAD ~ (raM -1 )
3FO
FI6. 1. - - Double reciprocal plot of calf spleen NAD÷-glycohydrolase i h h i b i t i o n
31o
by Blue Dexlran (,4) and
Cibacron Blue F3GA (B). I n i t i a l r a t e s of NAD h y d r o l y s i s 'wede d e t e r m i n e d a t 37°C, p H 7.4 a n d I = 0.05 M (plot A), I = (plot B). P r o t e i n c o n c e n t r a t i o n 3.5 i~tg/ml. T h e c o n c e n t r a t i o n s of i n h i b i t o r s w e r e : A - - B l u e D e x t r a n : 1, n o n e ; 2, 1.62 X 10-5 M a n d 3, 3.25 X 10-5 M. B - - C i b a e r o n B l u e F3GA : 1, n o n e ; 2, 1.25 X 10-6 M a n d 3, 4.9 X 1 0 ~ M.
c o m p e t i t i v e l y i n h i b i t e d (Ki = 5 X 10-.5 M at I = 0.1 M). A s i m i l a r r e s u l t 'was o b t a i n e d 'with a n e n d o p l a s m i c r e t i c u l u m b o u n d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e (P. T r a v o a n d F. S c h u b e r , to be p u b l i s h e d ) . TABLE I.
E f f e c t of ionic strength on i n h i b i t i o n constants.
50.-
E /s E
NaCI
Cibaeron Blue F3GA
Blue Dextran
M
Ki (~M)
Ki (~M)
/
i !
0.100
0.11 0.39 0.54
//
s- 1 A
// .~
25-
/
ta 0.050 0.075
0Ago M
3.6 7.0 11.75
- 0.5
i
T h e Ki (cOmpetitive) v a l u e s w e r e c a l c u l a t e d as i n d i caked u n d e r M a t e r i a l s a n d M e t h o d s .
•
f~o'"
s "°
""
•/
A f f i n i t y chromatography studies. W e w a n t e d to e x p l o r e t h e c o n d i t i o n s of b i n d i n g of t h e s o i u b i l i z e d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e to a n a f f i n i t y gel a n d o f i t s r e l e a s e . T h i s 'work 'was c o n d u c t e d avith a B l u e D e x t r a n - S e p h a r o s e g e l ; t h e d y e is l i n k e d to D e x t r a n b y i t s t r i a z i n e r i n g a n d to t h e matrix by its anthraquinone amino group. T h e Calf s p l e e n N A D + - g l y c o h y d r o l a s e in 10 m M T r i s HC1 b u f f e r (pH 7.4) w a s f o u n d to be c o m p l e t e l y r e t a i -
BIOCHIMIE, 1977, 59, n ° 8-9.
0
I
10
I
20
3w0
ml
Fro. 2. - - E l u t i o n profile of calf spleen NAD+-gly cohydrolase f r o m a Blue Dextran-Sepharose $B affin i t y column. 30 ~xg of e n z y m e (0.3 u n i t s ) in 10 m M T r i s - H C l b u f f e r (pH 7.4) w a s l o a d e d o n t o a c o l u m n c o n t a i n i n g 1 m l of gel. T h e c o l u m n 'was w a s h e d ~with l0 m l o f t h e s a m e buffer. A l i n e a r NaC1 g r a d i e n t bet~veen 0 a n d 1 M (2 X 20 m l ) 'was t h e n a p p l i e d . E n z y m e a c t i v i t y o f t h e f r a c t i o n s (1 m l ) w a s m e a s u r e d u s i n g t h e c y a n i d e m e t h o d .
NAD+-glycohydrolase
interaction
,with Blue
Dextran.
737
r a n g e ) of NAD% N A D H o r AMP ; i n t h i s c a s e t h e a c t i v i t y ~was r e c o v e r e d i n t h e final 1 M NaCI w a s h (see Methods). Ho'wever t h e N A D + g l y c o h y d r o l a s e 'was e l u t e d , ' w i t h 80-100 p e r c e n t r e c o v e r y , u s i n g 10 m M solution of these nucleotides. In a control experiment r e p l a c e m e n t o f t h e b i o s p e c i f i c l i g a n d s b y 50 m M NaCI did n o t e l u t e t h e e n z y m e .
l a r i t i e s vcith i t s n u c l e o t i d e b i n d i n g d o m a i n . T h e c a l f s p l e e n N A D + - g l y c o h y d r o l y s e is t h e first e x a m p l e of a N A D * - t r a n s f o r m i n g e n z y m e (e. 9. h y d r o l y s i s , t r a n s g l y c o s i d a t i o n , A D P - r i b o s y l a t i o n , . . . ) xvhieh s h a r e s s u c h f e a t u r e s w i t h e n z y m e s w h e r e NAD ÷ is a c t i n g a s a c o e n z y m e . I n c o n t r a s t Neurospora erassa NAD+-gly eohydrolase would not possess such a structural characteristic.
DISCUSSION.
T h e f i n d i n g t h a t t h e a f f i n i t y of t h e d y e v a r i e s w i t h t h e f o r m o f t h e N A D + - g l y c o h y d r o l a s e i.e. m e m b r a n e b o u n d or s o l u b i l i z e d (by t w o o r d e r s o f m a g n i t u d e f o r t h e c a l f s p l e e n e n z y m e s ) , a n d w i t h t h e s o u r c e of t h e e n z y m e is i n t e r e s t i n g . T h e d i f f e r e n t i n t e r a c t i o n of C i b a c r o n B l u e F3GA w i t h t h e N A D + - g l y e o h y d r o l a s e f o r m s , as d e t e c t e d k i n e t i e a l l y , c o u l d c o n c e i v a b l y reflect s t r u c t u r a l a n d c o n f o r m a t i o n a l c h a n g e s of t h e i r active sites but also differences in secondary interactions between the dye molecule and the enzyme surface independently from the catalytic properties ot t h e e n z y m e .
T h i s 1kinetic s t u d y i n d i c a t e s t h a t a t r e l a t i v e l y l o w i o n i c s t r e n g t h , b o t h C i b a c r o n B l u e F3GA a n d B l u e Dextran present high affinities for the substrate bind i n g site of s o l u b i l i z e d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e . C i b a c r o n B l u e F3GA is t h e m o s t p o t e n t i n h i b i t o r yet described for this enzyme. The inhibition constants found for the chromophore and its conjugated form (table I) a n d t h e i r r a t i o s a r e c o m p a r a b l e to t h e v a l u e s r e p o r t e d i n t h e e a s e of N A D +- d e p e n d e n t d e h y d r o g e n a s e s [8, 9]. T h e h i g h d e p e n d e n c e of Ki o n i o n i c strength indicates that the dye presumbably interacts vcith p o s i t i v e l y c h a r g e d r e s i d u e s o n t h e e n z y m e s u r face. I n t e r e s t i n g l y , W i l s o n r e c e n t l y s h o w e d t h a t hexokinase, an enzyme which does not contain the d i n u c l e o t i d e fold, i n t e r a c t s w i t h C i b a c r o n B l u e F3GA b u t o n l y ~veakly ~vith B l u e D e x t r a n [15]. I n o u r c a s e c o v a l e n t b i n d i n g of t h e d y e to D e x t r a n r e d u c e s o n l y b y o n e o r d e r o f m a g n i t u d e its i n h i b i t o r y p o w e r . T h e k i n e t i e a l l y d e t e r m i n e d a f f i n i t y of C i b a e r o n B l u e F3GA f o r t h e active site of s o l n b i l i z e d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e m a d e it a p o t e n t i a l l y u s e f u l l i g a n d to i m m o b i l i z e o n a n a f f i n i t y c o l u m n . T h e e n z y m e 'was f o u n d to be q u a n t i t a t i v e l y a d s o r b e d b y a Blue Dextran-Sepharose c o l u m n , at a o H v a l u e (pH 7.4) close to i t s i s o e l e c t r o n i c p o i n t [12] ; u n d e r t h e s e e x p e r i m e n t a l c o n d i t i o n s n o n specific a d s o r p t i o n , i.e. i o n e x c h a n g e , w a s b e i n g m i n i m i z e d . As d e s c r i b e d f o r o t h e r b i o s p e c i f l c a l l y a d s o r b e d e n z y m e s [6], a c o n c e n t r a t i o n of NaC1 h i g h e r t h a n 100 m M 'was n e c e s s a r y i n o r d e r to e l u t e t h e N A D + - g l y e o h y d r o l a s e : its r e l e a s e c o u l d a l s o be a c h i e v e d b y u s e o f i t s s u b s t r a t e or c o m p e t i t i v e n u c l e o t i d e s (NADH a n d AMP) at f a i r l y high concentrations. A c c o r d i n g to t h e p r e s e n t d a t a t h e s o l u b i l i z e d c a l f s p l e e n N A D * - g l y e o h y d r o l a s e b e h a v e s e o m l a a r a b l y to t h e d i n u e l e o t i d e f o l d p o s s e s s i n g e n z y m e s i.e. s t r o n g i n h i b i t i o n b y b o t h C i b a e r o n B l u e F3GA a n d i t s c o n j u g a t e d f o r m B l u e D e x t r a n , a d s o r p t i o n to B l u e D e x t r a n Sepharose column and biospecifie elution. This would s u g g e s t , i f t h e c r i t e r i a of T h o m p s o n a n d S t e l l w a g e n [6] p r o v e correct, t h a t t h i s e n z y m e c o n t a i n s t h e d i n u eleotide fold or at least shares large structural simi-
BIOCHIMIE, 1977, 59, n ° 8 - 9
REFERENCES. 1. Staal, G. E. J., V i s s e r , J. ~ Veeger, C. (1969) Biochim. Biophys. Acta, 185, 39-48. 2. S w a r t , A. C. W . ~ H e m k e r , H. C. (1970) Biochim. Bioph[ls. Acta, 222, 692-695. 3. Staal, G. E. J., K o s t e r , J. F., K a m p , H., V a n Millig e n - B o e r s m a , L. & Veeger, C. (1971) Biochim. Biophys. Acta, 227, 86-96. 4. B S h m e , H.-J., K o p p e r s c h l h g e r , G., S c h u l z , J. & H o f m a n n , E. (1972) J. Chromatogr., 69, 209-214. 5. R y a n , L. D. ~ V e s t l i n g , C. S. (1974) Arch. Biochem. Biophys., 160, 279-284. 6. T h o m p s o n , S. T., C a s s , K. H. S t e l l w a g e n , E. (1975) Proe. Nat. Acad. Sci. USA, 72, 669-672. 7. Stell~vagen, E., C a s s , R., T h o m p s o n , S. T. & W o o d y , M. (1975) Nature, 257, 716-718. 8. T h o m p s o n , S. T. & Stell'wagen, E. (1976) Proc. Nat. Acad. Sci. USA, 73, 3,61-365. 9. S t e l l w a g e n , E. (1977) Acc. Chem. Res., 10, 92-98. 10. R o s s m a n , M. G., L i l j a s , A., B r h n d e n , C.-I. & B a n a s z a k , L. J. (1975) in (Boye.r, P. D. ed.) 3 r d Ed., Vol. !11, pp. 61-102. A c a d e m i c Press, New-York. 11. R o s s m a n , M. G., M o r a s , D. & O l s e n , K. Mr. (1974) Nature, 250, 194-199. 12. S c h u b e r , F. ~ T r a v o , P. (1976) Eur. J. Biochem., 65, 247-~55. 13. C l e l a n d , W . W . (1963) Riochim. Biophys. Acta, 67, 173-187. 14. D i x o n , M. ~ W e b b , E. C. (I964) in E n z y m e s , 2 ~ Ed., D. 315-359, L o n g m a n s , G r e e n a n d Co., L o n d o n . 15. W i l s o n , J. E. (1976) Biochcm. Biophys. Res. Commun., 72, 816-823.
Interaction of Blue Dextran and Cibacron Blue F3GA with calf spleen NAD+-glycohydrolase. F r a n c i s SCHUBErt 0 a n d M a r c PASCAL. I n s t i t u t de B o t a n i q u e de I ' U n i v e r s i t d L o u i s P a s t e u r , 28, r u e G o e t h e - - ~7~00 S t r a s b o u r g (France). (12-~-1977).
INTRODUCTION. S o m e p r o t e i n s a n d e n z y m e s h a v e b e e n r e p o r t e d to i n t e r a c t ' w i t h B l u e D e x t r a n to f o r m a c o m p l e x d i s s o e i a b l e b y s a l t [!1:-5]. Stel~wagen a n d c o w o r k e r s [69] h a v e e x t e n d e d t h i s o b s e r v a t i o n to a c e r t a i n n u m b e r of m o n o - a n d d i n u c l e o t i d e b i n d i n g e n z y m e s . T h e y proposed that the interaction of Blue Dextran or its e h r o m o p h o r e C i b a e r o n B l u e F3GA w i t h a p r o t e i n i n d i cates, u n d e r d e f i n e d c o n d i t i o n s , t h e p r e s e n c e of t h e d i n u e l e o t i d e f o l d b i n d i n g site, i.e. a s u p e r - s e c o n d a r y s t r u c t u r e of t h e n i c o t i n a m i d e a d e n i n e d i n n c l e o t i d e b i n d i n g d o m a i n i n NAD+-linked d e h y d r o g e n a s e s [1011]. ~ e h a v e s t u d i e d t h e i n t e r a c t i o n of B l u e D e x t r a n a n d C i b a e r o n B l u e F3GA ~vith N A D + - g l y e o h y d r o l a s e (EC 3.2.2.6), 'with t h e p u r p o s e to d e t e c t t h e e x i s t e n c e o f s t r u c t u r a l a n a l o g i e s bet'ween t h e N A D + - h i n d i n g d o m a i n of t h i s e n z y m e a n d t h e e n z y m e s p o s s e s s i n g t h e d i n u e l e o t i d e fold. M A T E R I A L S AND METHODS. Materials. C a l f s p l e e n , pig b r a i n (as a c e t o n e p o w d e r s ) a n d Neurospora crassa NAD+-glycohydrolases and ~-NAD + were purchased from S i g m a C h e m i c a l Co. B l u e D e x t r a n 2000 (M~vV = 2 × 106), S e p h a d e x G-50 a n d S e p h a r o s e 4B 'were o b t a i n e d f r o m P h a r m a e i a . C i b a c r o n B l u e F3GA (the c h r o m o p h o r e of B l u e D e x t r a n ) w a s a :kind g i f t of Drs. E. S u r y a n d K. Scheibli, (CibaGeigy, A. G. B a s e l ) . All o t h e r c h e m i c a l s w e r e Mercl~s products. Enzymes. Calf - spleen NAD+-glycohydrolase was solubilized f r o m a n a c e t o n e p ~ w d e r 'with p a n c r e a t i c l i p a s e a n d p a r t i a l l y p u r i f i e d (9 u n i t s / r a g p r o t e i n ) a c c o r d i n g to o u r p r e v i o u s l y p u b l i s h e d p r o c e d u r e [1'2]. T h i s s o l u b l e e n z y m e p r e p a r a t i o n 'was u s e d f o r t h e Ki d e t e r m i n a tions and affinity chromatography studies. Pig brain N A D ÷ - g l y c o h y d r o l a s e w a s s o l n b i l i z e d 'with p a n c r e a t i c l i p a s e [12], t h e s u p e r n a t a n t (100000 X g, 60 m i n ) h a d a specific a c t i v i t y o f 0.034 u n i t / m g p r o t e i n a n d w a s u s e d as s o l u b l e e n z y m e . Kinetic measurements and inhibition constants determination, I n i t i a l r a t e s of N A D + - g l y c o h y d r o l a s e c a t a l y s e d NAI) + hydrolysis were obtained titrimetrically [12]. T h e k i n e t i c s 'were p e r f o r m e d a t 37 °, p H 7.4 a t fixed i o n i c s t r e n g t h (NaC1) i n a final v o l u m e of 1.9 m l . A p p a r e n t i n h i b i t i o n c o n s t a n t s Ki w e r e d e t e r m i n e d f r o m t h e To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .
r e p l o t o f s l o p e s (in t h e c a s e of c o m p e t i t i v e i n h i b i t i o n ) a n d i n t e r c e p t s (in t h e c a s e of m i x e d i n h i b i t i o n ) o f t h e double reciprocal plots (Lineweaver-Bnrk) versus the c o r r e s p o n d i n g i n h i b i t o r c o n c e n t r a t i o n s [13]. C o n c e n t r a t i o n of B l u e D e x t r a n is e x p r e s s e d in t e r m s o f its e h r o m o p h o r e [8]Affinity chromatography. The Blue Dextran-Sepharose was prepared by deriv a t i z a t i o n of c y a n o g e n b r o m i d e a c t i v a t e d S e p h a r o s e - 4 B w i t h B l u e D e x t r a n [51. T h e a f f i n i t y c h r o m a t o g r a p h y gel w a s t e s t e d f o r i t s a b i l i t y to b i n d s o l u b i l i z e d c a l f spleen NAD+-glyeohydrolase. For analytical purposes, 30 ling of e n z y m e (0.3 u n i t ) in 10 m M Tris-HC1 b u f f e r (pH 7.4) w a s l o a d e d , at 4 ° , o n t o a m i c r o - c o l u m n c o n t a i n i n g B l u e D e x t r a n - S e p h a r o s e (1 m l ) p r e v i o u s l y e q u i l i b r a t e d 'with t h e c o r r e s p o n d i n g buffer. T h e col u m n w a s t h e n w a s h e d ' w i t h t h e s a m e b u f f e r (10 m l ) . T h e e l a t i o n of t h e e n z y m e w a s s t u d i e d b y a p p l y i n g to a c o l u m n a l i n e a r g r a d i e n t betxveen 0 a n d 1 M NaCI (in t h e T r i s b u f f e r ) , f r a c t i o n s o f 1 m l 'were c o l l e c t e d (fig. 2). F o r biospeeific e l u t i o n o f t h e NAD+glyeo h y d r o l a s e 10 m l of n u c l e o t i d e e o u n t e r l i g a n d , i n t h e T r i s - b u f f e r a d j u s t e d a t pH 7.4, w a s a p p l i e d f o l l o w e d b y 10 m l o f t h e T r i s - b u f f e r ( w a s h i n g ) . R e s i d u a l e n z y m e w a s d e t a c h e d ' w i t h a 1 M NaCI s o l u t i o n (5 m l ) . T h e e n z y m e a c t i v i t y of t h e d i f f e r e n t f r a c t i o n s ( e l u a t e s a n d washings) was measured using a modified KCN assay [12]. W h e n h i g h c o n c e n t r a t i o n s of n u e l e o t i d e s 'were u s e d , t h e a c t i v i t y in t h e e l u a t e s w a s a s s a y e d a f t e r dialysis against the Tris-buffer. RESULTS. Inhibilion studies. I n i t i a l r a t e s of NAD ÷ h y d r o l y s i s c a t a l y z e d b y s o l u bilized calf spleen NAD+-glycohydrolase are markedly a f f e c t e d b y C i b a c r o n B l u e F3GA a n d B l u e D e x t r a n . A representative Line'weaver-Bur.k plot for the inhibition b y B l u e D e x t r a n is s h o w n in figure IA ; a n a p p a r e n t i n h i b i t i o n c o n s t a n t o f 3.6 X 10-6 M w a s c a l c u l a t e d . T h e v a l u e s of e n z y m e - i n h i b i t o r d i s s o c i a t i o n c o n s t a n t Ki a r e v e r y d e p e n d e n t o n t h e i o n i c s t r e n g t h of t h e m e d i u m . W h e n I ~ 0.1 M B l u e D e x t r a n a n d C i b a e r o n B l u e F3GA a r e p u r e c o m p e t i t i v e i n h i b i t o r s , Ki i n c r e a s i n g nvith i n c r e a s i n g I (table I). W h e n t h e i o n i c s t r e n g t h is i n c r e a s e d f u r t h e r , in b o t h c a s e s t h e i n h i b i t i o n b e c o m e s m i x e d as i l l u s t r a t e d in figure 1B f o r C i b a c r o n B l u e F3GA (fig. 1B, t h e c a l c u l a t e d v a l u e s a r e Kit = 6.92 × 10-6 M a n d Kis ---- 1.28 × 10-6 M). F o r c o m p a r i s o n , ~ve h a v e a l s o i n v e s t i g a t e d t h e a c t i o n of C i b a c r o n B l u e F3GA o n t h e pig b r a i n a n d t h e N e u r o s p o r a c r a s s a N A D ~ - g i y c o h y d r o l a s e s . At I -: 0.1 M, u s i n g t h e D i x o n r e p r e s e n t a t i o n f o r d e t e r m i n i n g
F. Schuber and M. Pascal.
736
Ki [14], t h e f o l l o w i n g r e s u l t s 'were o b t a i n e d : pig b r a i n , b o u n d , 4.5 X 10-4 M ( c o m p e t i t i v e ) ; s o l u b i l i z e d , 5.,6 X 10-4 M ( c o m p e t i t i v e ) ; Neurospora crassa : 2,75 X 10-.3 M ( n o n c o m p e t i t i v e ) . T h e c a l f s p l e e n e n z y m e , i n t h e b o u n d f o r m ( a c e t o n e po'wder), w a s f o u n d to be
ned by the affinity column and could be released rap i d l y a n d q u a n t i t a t i v e l y , b y a p p l y i n g a NaC1 g r a d i e n t as s h o w n in figure 2. T h e m e a n v a l u e of NaC1 c o r r e s p o n d i n g to e n z y m e r e l e a s e :was 130 raM. T h e e n z y m e c o u l d n o t be e l u t e d b y l o w c o n c e n t r a t i o n s (1 m M
B
A
3
3
o
10(]
t50
2
E
'T "6
2
t00
..>
5O
J
1'0
:'0
1
I/NAO + [rnM- )
1~o 2o I/NAD ~ (raM -1 )
3FO
FI6. 1. - - Double reciprocal plot of calf spleen NAD÷-glycohydrolase i h h i b i t i o n
31o
by Blue Dexlran (,4) and
Cibacron Blue F3GA (B). I n i t i a l r a t e s of NAD h y d r o l y s i s 'wede d e t e r m i n e d a t 37°C, p H 7.4 a n d I = 0.05 M (plot A), I = (plot B). P r o t e i n c o n c e n t r a t i o n 3.5 i~tg/ml. T h e c o n c e n t r a t i o n s of i n h i b i t o r s w e r e : A - - B l u e D e x t r a n : 1, n o n e ; 2, 1.62 X 10-5 M a n d 3, 3.25 X 10-5 M. B - - C i b a e r o n B l u e F3GA : 1, n o n e ; 2, 1.25 X 10-6 M a n d 3, 4.9 X 1 0 ~ M.
c o m p e t i t i v e l y i n h i b i t e d (Ki = 5 X 10-.5 M at I = 0.1 M). A s i m i l a r r e s u l t 'was o b t a i n e d 'with a n e n d o p l a s m i c r e t i c u l u m b o u n d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e (P. T r a v o a n d F. S c h u b e r , to be p u b l i s h e d ) . TABLE I.
E f f e c t of ionic strength on i n h i b i t i o n constants.
50.-
E /s E
NaCI
Cibaeron Blue F3GA
Blue Dextran
M
Ki (~M)
Ki (~M)
/
i !
0.100
0.11 0.39 0.54
//
s- 1 A
// .~
25-
/
ta 0.050 0.075
0Ago M
3.6 7.0 11.75
- 0.5
i
T h e Ki (cOmpetitive) v a l u e s w e r e c a l c u l a t e d as i n d i caked u n d e r M a t e r i a l s a n d M e t h o d s .
•
f~o'"
s "°
""
•/
A f f i n i t y chromatography studies. W e w a n t e d to e x p l o r e t h e c o n d i t i o n s of b i n d i n g of t h e s o i u b i l i z e d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e to a n a f f i n i t y gel a n d o f i t s r e l e a s e . T h i s 'work 'was c o n d u c t e d avith a B l u e D e x t r a n - S e p h a r o s e g e l ; t h e d y e is l i n k e d to D e x t r a n b y i t s t r i a z i n e r i n g a n d to t h e matrix by its anthraquinone amino group. T h e Calf s p l e e n N A D + - g l y c o h y d r o l a s e in 10 m M T r i s HC1 b u f f e r (pH 7.4) w a s f o u n d to be c o m p l e t e l y r e t a i -
BIOCHIMIE, 1977, 59, n ° 8-9.
0
I
10
I
20
3w0
ml
Fro. 2. - - E l u t i o n profile of calf spleen NAD+-gly cohydrolase f r o m a Blue Dextran-Sepharose $B affin i t y column. 30 ~xg of e n z y m e (0.3 u n i t s ) in 10 m M T r i s - H C l b u f f e r (pH 7.4) w a s l o a d e d o n t o a c o l u m n c o n t a i n i n g 1 m l of gel. T h e c o l u m n 'was w a s h e d ~with l0 m l o f t h e s a m e buffer. A l i n e a r NaC1 g r a d i e n t bet~veen 0 a n d 1 M (2 X 20 m l ) 'was t h e n a p p l i e d . E n z y m e a c t i v i t y o f t h e f r a c t i o n s (1 m l ) w a s m e a s u r e d u s i n g t h e c y a n i d e m e t h o d .
NAD+-glycohydrolase
interaction
,with Blue
Dextran.
737
r a n g e ) of NAD% N A D H o r AMP ; i n t h i s c a s e t h e a c t i v i t y ~was r e c o v e r e d i n t h e final 1 M NaCI w a s h (see Methods). Ho'wever t h e N A D + g l y c o h y d r o l a s e 'was e l u t e d , ' w i t h 80-100 p e r c e n t r e c o v e r y , u s i n g 10 m M solution of these nucleotides. In a control experiment r e p l a c e m e n t o f t h e b i o s p e c i f i c l i g a n d s b y 50 m M NaCI did n o t e l u t e t h e e n z y m e .
l a r i t i e s vcith i t s n u c l e o t i d e b i n d i n g d o m a i n . T h e c a l f s p l e e n N A D + - g l y c o h y d r o l y s e is t h e first e x a m p l e of a N A D * - t r a n s f o r m i n g e n z y m e (e. 9. h y d r o l y s i s , t r a n s g l y c o s i d a t i o n , A D P - r i b o s y l a t i o n , . . . ) xvhieh s h a r e s s u c h f e a t u r e s w i t h e n z y m e s w h e r e NAD ÷ is a c t i n g a s a c o e n z y m e . I n c o n t r a s t Neurospora erassa NAD+-gly eohydrolase would not possess such a structural characteristic.
DISCUSSION.
T h e f i n d i n g t h a t t h e a f f i n i t y of t h e d y e v a r i e s w i t h t h e f o r m o f t h e N A D + - g l y c o h y d r o l a s e i.e. m e m b r a n e b o u n d or s o l u b i l i z e d (by t w o o r d e r s o f m a g n i t u d e f o r t h e c a l f s p l e e n e n z y m e s ) , a n d w i t h t h e s o u r c e of t h e e n z y m e is i n t e r e s t i n g . T h e d i f f e r e n t i n t e r a c t i o n of C i b a c r o n B l u e F3GA w i t h t h e N A D + - g l y e o h y d r o l a s e f o r m s , as d e t e c t e d k i n e t i e a l l y , c o u l d c o n c e i v a b l y reflect s t r u c t u r a l a n d c o n f o r m a t i o n a l c h a n g e s of t h e i r active sites but also differences in secondary interactions between the dye molecule and the enzyme surface independently from the catalytic properties ot t h e e n z y m e .
T h i s 1kinetic s t u d y i n d i c a t e s t h a t a t r e l a t i v e l y l o w i o n i c s t r e n g t h , b o t h C i b a c r o n B l u e F3GA a n d B l u e Dextran present high affinities for the substrate bind i n g site of s o l u b i l i z e d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e . C i b a c r o n B l u e F3GA is t h e m o s t p o t e n t i n h i b i t o r yet described for this enzyme. The inhibition constants found for the chromophore and its conjugated form (table I) a n d t h e i r r a t i o s a r e c o m p a r a b l e to t h e v a l u e s r e p o r t e d i n t h e e a s e of N A D +- d e p e n d e n t d e h y d r o g e n a s e s [8, 9]. T h e h i g h d e p e n d e n c e of Ki o n i o n i c strength indicates that the dye presumbably interacts vcith p o s i t i v e l y c h a r g e d r e s i d u e s o n t h e e n z y m e s u r face. I n t e r e s t i n g l y , W i l s o n r e c e n t l y s h o w e d t h a t hexokinase, an enzyme which does not contain the d i n u c l e o t i d e fold, i n t e r a c t s w i t h C i b a c r o n B l u e F3GA b u t o n l y ~veakly ~vith B l u e D e x t r a n [15]. I n o u r c a s e c o v a l e n t b i n d i n g of t h e d y e to D e x t r a n r e d u c e s o n l y b y o n e o r d e r o f m a g n i t u d e its i n h i b i t o r y p o w e r . T h e k i n e t i e a l l y d e t e r m i n e d a f f i n i t y of C i b a e r o n B l u e F3GA f o r t h e active site of s o l n b i l i z e d c a l f s p l e e n N A D + - g l y c o h y d r o l a s e m a d e it a p o t e n t i a l l y u s e f u l l i g a n d to i m m o b i l i z e o n a n a f f i n i t y c o l u m n . T h e e n z y m e 'was f o u n d to be q u a n t i t a t i v e l y a d s o r b e d b y a Blue Dextran-Sepharose c o l u m n , at a o H v a l u e (pH 7.4) close to i t s i s o e l e c t r o n i c p o i n t [12] ; u n d e r t h e s e e x p e r i m e n t a l c o n d i t i o n s n o n specific a d s o r p t i o n , i.e. i o n e x c h a n g e , w a s b e i n g m i n i m i z e d . As d e s c r i b e d f o r o t h e r b i o s p e c i f l c a l l y a d s o r b e d e n z y m e s [6], a c o n c e n t r a t i o n of NaC1 h i g h e r t h a n 100 m M 'was n e c e s s a r y i n o r d e r to e l u t e t h e N A D + - g l y e o h y d r o l a s e : its r e l e a s e c o u l d a l s o be a c h i e v e d b y u s e o f i t s s u b s t r a t e or c o m p e t i t i v e n u c l e o t i d e s (NADH a n d AMP) at f a i r l y high concentrations. A c c o r d i n g to t h e p r e s e n t d a t a t h e s o l u b i l i z e d c a l f s p l e e n N A D * - g l y e o h y d r o l a s e b e h a v e s e o m l a a r a b l y to t h e d i n u e l e o t i d e f o l d p o s s e s s i n g e n z y m e s i.e. s t r o n g i n h i b i t i o n b y b o t h C i b a e r o n B l u e F3GA a n d i t s c o n j u g a t e d f o r m B l u e D e x t r a n , a d s o r p t i o n to B l u e D e x t r a n Sepharose column and biospecifie elution. This would s u g g e s t , i f t h e c r i t e r i a of T h o m p s o n a n d S t e l l w a g e n [6] p r o v e correct, t h a t t h i s e n z y m e c o n t a i n s t h e d i n u eleotide fold or at least shares large structural simi-
BIOCHIMIE, 1977, 59, n ° 8 - 9
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