Inhibition of sheep pancreatic lipase activity against emulsified tributyrin by non-ionic detergents

BIOCHIMIE, 1976, 58, 751-753.

Inhibition of sheep pancreatic lipase activity against emulsified tributyrin by non-ionic detergents. P. C A N m N L R . JULIEN, J. RATHELOT a n d L, SABDA <~. l n s t i l u t de Chimie Biologique, Facultd Saint-Charles, Place Victor Hugo, 13003 Marseillc, France. (9-2-1976).

INTRODUCTION. L i p a s e - c a t a l y z e d h y d r o l y s i s of e m u l s i f i e d t r i b u t y r i n is s t r o n g l y i n h i b i t e d b y c o n j u g a t e d bile s a l t s at c o n centration near their critical micellar concentration (CMC). A d d i t i o n o f a n e x c e s s of c o l i p a s e , t h e p r o t e i n c o f a c t o r of l i p a s e , r e a c t i v a t e s t h e l i p o l y s i s r e a c t i o n . B o r g s t r o m et al. [1] h a v e s h o w n t h a t t h e d i h y d r o x y bile s a l t s w h i c h h a v e a CMC l o w e r t h a n t h e t r i h y d r o x y c o m p o u n d s a r e b e t t e r i n h i b i t o r s of t h e e n z y m e r e a c t i o n . H o w e v e r , i n h i b i t i o n b y n o n c o n j u g a t e d bile s a l t s w a s m u c h less c o m p l e t e a l t h o u g h t h e i r CMC is i n t h e s a m e r a n g e as t h e c o r r e s p o n d i n g c o n j u g a t e d d e r i v a tives. I n a d d i t i o n , a n i o n i c d e t e r g e n t s o t h e r t h a n bile s a l t s w e r e f o u n d to i n h i b i t t h e e n z y m e r e a c t i o n in t h e c o n c e n t r a t i o n r a n g e o f t h e i r CMC. A s s u m p t i o n w a s m a d e [1, 2] t h a t bile s a l t i n h i b i t i o n of t h e e n z y m a t i c h y d r o l y s i s of t r i g l y e e r i d e s at t h e lipid-water interface might result from the building of a n e g a t i v e l y c h a r g e d m o n o l a y e r of a n i o n i c d e t e r gent molecules on the surface snbstrate that hinders l i p a s e a d s o r p t i o n . C h a p u s el al. [3] g a v e e v i d e n c e t h a t i n t h e p r e s e n c e o f bile salts, t h e e n z y m e f a i l s to a d s o r b to a n h y d r o p h i l i c i n t e r f a c e . It is l i k e l y t h a t colipase which has a high binding capacity for mieelles of bile s a l t s as r e c e n t l y d e m o n s t r a t e d b y Sari, C h a r l e s a n d c o w o r k e r s [4, 5] a n d b y B o r g s t r o m a n d D o n n e r [6], r e c o g n i z e s t h e d e t e r g e n t m o n o l a y e r a n d a d s o r b s to it, t h u s e n a b l i n g l i p a s e to f o r m a n active enzyme-substrate complex. I n vie'w o f a b e t t e r u n d e r s t a n d i n g of t h e m e c h a n i s m b y w h i c h d e t e r g e n t m o l e c u l e s p r e s e n t at t h e l i p i d water interface inhibit the lipase-catalyzed hydrolysis of ' w a t e r - i n s o l u b l e s u b s t r a t e s , We h a v e s t u d i e d t h e effect of n o n - i o n i c s u r f a c t a n t s o n e n z y m e a c t i v i t y . T h e present communication d e s c r i b e s t h e i n f l u e n c e of Brij 35 a n d T r i t o n X - I 0 0 o n t h e a c t i v i t y of s h e e p p a n c r e a t i c l i p a s e t o ' w a r d e m u l s i f i e d t r i b u t y r i n in t h e a b s e n c e a n d in t h e p r e s e n c e of c o l i p a s e . M A T E R I A L S AND METHODS. S h e e p p a n c r e a t i c l i p a s e , free o f c o l i p a s e , w a s p r e p a r e d as p r e v i o u s l y d e s c r i b e d [7]. T h e specific a c t i v i t y of t h e e n z y m e m e a s u r e d a g a i n s t t r i o l e i n in s t a n d a r d c o n d i t i o n s [8] w a s 3500 !~x eq. f a t t y acid m i n - I m g - l . P o r c i n e l i p a s e w a s p r e p a r e d b y t h e m e t h o d of V e r g e r et al. [9]. S h e e p p a n c r e a t i c c o l i p a s e w a s o b t a i n e d i n a partially purified form as follows : proteins of the acetone precipitate obtained during enzyme preparat i o n [7] 'were d i s s o l v e d in a m i n i m u m v o l u m e of 6) 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 .

0.4 NaC1 c o n t a i n i n g 1 m M b e n z a m i d i n e , a n d a n a l i q u o t of tiffs s o l u t i o n c o n t a i n i n g 13 m g of p r o t e i n s w a s filtered t h r o u g h a c o l u m n of S e p h a d e x G 100 (2.5 c m × 90 era) e q u i l i b r a t e d i n 0.4 M NaCI. L i p a s e a n d colip a s e e m e r g e d a t 1.7 Vo a n d 2.3 Vo, r e s p e c t i v e l y . T h e colipase containing fractions were pooled and concent r a t e d b y p r e c i p i t a t i o n o f p r o t e i n s w i t h 0.8 sat. a m m o n i u m s u l f a t e . T h e p r e c i p i t a t e w a s d i s s o l v e d in w a t e r a n d t h e p H of t h e s o l u t i o n w a s b r o u g h t to 2.0. I n s o l u ble m a t e r i a l w a s d i s c a r d e d a n d t h e pH w a s r a i s e d to 7.0. C o l i p a s e p r e p a r a t i o n w e r e f r o z e n a n d k e p t at --20 °. L i p a s e a c t i v i t y a g a i n s t t r i b u t y r i n w a s a s s a y e d pot e n t i o m e t r i c a l l y a t p H 8.0 a n d 25 ° in t h e f o l l o w i n g c o n d i t i o n s : 1 m l o f t r i b u t y r i n ( S i g m a ) w a s a d d e d to 19 m l of 10 m M Tris-HC1 b u f f e r p H 8.0 c o n t a i n i n g 4 m M CaCI~ a n d 10 m M NaC1, a n d e m u l s i f i e d b y stirring in carefully standardized conditions under a s t r e a m of n i t r o g e n [10]. A l i q u o t s of t h e l i p a s e s o h l t i o n s c o n t a i n i n g 3 to 4 e n z y m e u n i t s were u s e d i n t h e a s s a y s . L i p a s e a c t i v i t y w a s d e r i v e d f r o m t h e s l o p e of t h e zero o r d e r k i n e t i c s a n d e x p r e s s e d as e n z y m e u n i t s p e r m g o f p r o t e i n s . D e t e r m i n a t i o n of l i p a s e a c t i v i t y w a s c a r r i e d o u t in t h e a b s e n c e o f e o l i p a s e or in t h e p r e s e n c e o f a s u f f i c i e n t a m o u n t o f c o f a e t o r (20 eolip a s e u n i t s ) [8] to o b t a i n m a x i m a l a c t i v i t y u n d e r t h e assay conditions. T h e p r o t e i n c o n t e n t of s o l u t i o n s of l i p a s e a n d colip a s e w a s d e t e r m i n e d b y t h e c o l o r i m e t r i c m e t h o d of Lo'wry u s i n g b o v i n e s e r u m a l b u m i n as s t a n d a r d . T h e n o n - i o n i c s u r f a c t a n t s , Brij 35 a n d T r i t o n X-100 w e r e f r o m BDH. T h e i r c r i t i c a l m i c e l l a r c o n c e n t r a t i o n w a s e s t i m a t e d b y t h e s p e c t r a l m e t h o d in a q u e o u s s o l u t i o n s o f i o d i n e (40 ,~g p e r m l ) . T h e a b s o r b a n c e w a s d e t e r m i n e d a t 370 n m at c o n c e n t r a t i o n s of d e t e r g e n t v a r y i n g f r o m 0 to 0.3 m g p e r m l (figure 1). As s h o w n o n figure 1, t h e p l o t o f t h e a b s o r b a n c e o f s o l u t i o n s o f Brij 35 v e r s u s t h e c o n c e n t r a t i o n of d e t e r g e n t g i v e s t w o s t r a i g h t l i n e s . T h e l i n e c o r r e s p o n d i n g to t h e s l o p e p r i o r to t h e f o r m a t i o n of m i c e l l e s i n d i c a t e s t h e p r e s e n c e o f a g g r e g a t e s at a c o n c e n t r a t i o n of Brij 35 as l o w as 30 ,~g p e r m l ( p r e m i c e l l e s ) . T h e c o n c e n t r a t i o n of Brij 35 c o r r e s p o n d i n g to t h e t r a n s i t i o n p o i n t (160 f~g p e r m l ) b e t w e e n t h e p r e m i c e l l a r l i m b a n d t h e m i c e l l a r l i m b o f t h e c u r v e is g e n e r a l l y r e f e r r e d as t h e critical micellar concentration. However, an apparent CMC o f 75 ~xg p e r m l ~ ' a s e s t i m a t e d f r o m t h e a b s c i s s a of t h e i n t e r c e p t of t h e m i c e l l a r l i m b slope. T h i s v a l u e is close to t h a t p r e v i o u s l y d e t e r m i n e d (95 ~xg p e r m l ) b y A l b r o et al. [11]. W i t h t h e s a m e m e t h o d , a n a p p a r e n t CMC of 130 I~g p e r m l f o r T r i t o n X-100 w a s d e t e r m i n e d , a v a l u e w h i c h is i n good a c c o r d a n c e w i t h t h a t f o u n d b y A l b r o et al. [11] a n d b y R i b e i r o a n d D e n n i s [12].

P. Canioni, R. Julien, J. Rathelot and L. Sarda.

752

Mixed b o v i n e bile s a l t s 'W~l'e p r e p a r e d f r o m ox bile as d e s c r i b e d b y M o n t e t a n d D e r v i e h i a n [13]. T h e i r CMC is i n t h e c o n c e n t r a t i o n r a n g e of 1.5-2 mM in 0.1 M NaC1.

The influence of v a r y i n g c o n c e n t r a t i o n of Brij 35 o n t h e a c t i v i t y of s h e e p p a n c r e a t i c l i p a s e a g a i n s t trib u t y r i n at p H 8.0 is s h o w n on figure 2. T w o series of e x p e r i m e n t s 'were p e r f o r m e d in the a b s e n c e a n d in

lb

la

O'.9

c

0,8

o

.Q <

Fit;. 1. - - Determination of the critical micellar concentration of B r i j 35 (polyoxyethylene lauryl elher). Figure l a : Difference s p e c t r u m of

0.7

0.6

a n a q u e o u s s o l u t i o n of i o d i n e (40 u g p e r ml) i n t h e p r e s e n c e of 0.2 p e r cent delea'gen~. Figure l b : V a r i a t i o n o f t h e a b s o r h anee a t 370 n m as a f u n c t i o n of detergent concentration.

//

0.5 / / /

0.4 300

//

, , J r 11 i f ] = 350

100

400 nm

200 Detergent Concentration

R E S U L T S AND DISCUSSION. S o l u t i o n s of s h e e p p a n c r e a t i c l i p a s e (0.3 m g / m l ) a d d e d w i t h n o n - i o n i c d e t e r g e n t s a t c o n c e n t r a t i o n above t h e i r a p p a r e n t critical m i c e l l a r c o n c e n t r a t i o n (Brij 35 : 0.16 m g / m l ; T r i t o n X-100 : 0.2 m g / m l ) r e t a i n s its a c t i v i t y a f t e r one h o u r i n c u b a t i o n at 25 °.

.E E

2a

~01.0

the p r e s e n c e of a n excess of sheep colipase. I n the a b s e n c e of colipase, c o m p l e t e i n h i b i t i o n of the e n z y m e r e a c t i o n 'was a t t a i n e d at a d e t e r g e n t c o n c e n t r a t i o n of 15 ;.ttg p e r m l t h a t is to s a y 'well b e l o w t h e a p p a r e n t CMC of the detergent. W h e n Brij 35 a n d c o l i p a s e w e r e added to t h e a s s a y s y s t e m , the r a t e of i n h i b i t i o n of the e n z y m e r e a c t i o n 'was m a r k e d l y decreased b y the

oc

..~- - . . a .

,e°

300 pg per ml

2b

100

~

A

\\

0

5

10

/1/

15

20

25 0 Detergent

5 concentration

10 Hg p e r ml

15

20

25

Fro. 2. - - Influence of non ionic detergent on lipase actioity against emulsified tribu-

lyrin at pH 8.0. Figure 2a : Effect of Brij 35 on the a c t i v i t y of sheep a n d p o r c i n e p a n c r e a t i c l i p a s e in the a b s e n c e an~d in t h e p r e s e n c e of colipase. Figure 2b : Relative r a t e of i n h i b i t i o n of sheep a n d p o r c i n e p a n c r e a t i c l i p a s e in the a b s e n c e a n d in t h e p r e s e n c e of colipase. 0--@ : s h e e p lipase, no c o l i p a s e added. O--O s h e e p lipase in t h e p r e s e n c e of colipase. • ....... • : p o r c i n e lipase, no c o l i p a s e added. ...... A p o r c i n e l i p a s e in the p r e s e n c e of colipase. The critical m i c e l l a r c o n c e n t r a t i o n of Brij 35 is 75 Itg per nil. S i m i l a r r e s u l t s w e r e o b t a i n e d in i n h i b i t i o n s t u d i e s w i t h T r i t o n X-100 (see text).

BIOCHIMIE, 1976, 58, n ° 6.

Inhibition of lipase by non-ionic detergents. p r e s e n c e of t h e p r o t e i n c o f a c t o r u p to a d e t e r g e n t c o n c e n t r a t i o n of 25 ,~g p e r m l . At h i g h e r c o n c e n t r a t i o n s , h o w e v e r , c o l i p a s e f a i l e d to r e s t o r e e n z y m e a c t i vity. As s h o w n c n figures 2a a n d 2b, s i m i l a r r e s u l t s were o b t a i n e d in i n h i b i t i o n s t u d i e s of p o r c i n e l i p a s e w i t h Brij 35. It m u s t be o b s e r v e d t h a t t h e h i g h e r specific a c t i v i t y of t h e p o r c i n e e n z y m e as d e t e r m i n e d in t h e a b s e n c e of a d d e d c o l i p a s e , m e r e l y reflects t h e p r e s e n c e of c o n t a m i n a t i n g c o l i p a s e in t h e p o r c i n e e n z y m e p r e p a r a t i o n . As e x p e c t e d , t h i s d i f f e r e n c e w a s no l o n g e r o b s e r v e d w h e n b o t h e n z y m e s were a s s a y e d w i t h a n e x c e s s of c o f a c t o r . I n t h e a b s e n c e of d e t e r gent, e o l i p a s e m a r k e d l y i n c r e a s e d s h e e p l i p a s e a c l i vity a s p r e v i o u s l y o b s e r v e d 'with e n z y m e p r e p a r a t i o n s f r o m o t h e r s p e c i e s [1, 14]. Very similar results were obtained by using an o t h e r n o n - i o n i c d e t e r g e n t , T r i t o n X-100 ( n o t s h o w n ) . In t h i s l a t e r case, f u l l i n h i b i t i o n of t h e e n z y m e r e a c t i o n 'was a c h i e v e d at d e t e r g e n t c o n c e n t r a t i o n s of 40 ,ug p e r m l a n d 60 I~g p e r m l i n t h e a b s e n c e a n d in t h e p r e s e n c e o f col i p a s e , r e s p e c t i v e l y ( c o r r e s p o n d i n g m o lar c o n c e n t r a t i o n s of T r i t o n X-100 a r e 0.065 m M a n d 0.1 m M ' w h e n e x p r e s s e d in t e r m s of a n a v e r a g e m o l e c u l a r w e i g h t of 624 d a l t o n s r12]). T h e r e f o r e , it is a g a i n o b s e r v e d t h a t c o l i p a s e f a i l s to r e a c t i v a t e l i p a s e in t h e p r e s e n c e of c o n c e n t r a t i o n s of n o n - i o n i c d e t e r g e n t b e l o w or a r o u n d t h e CMC.

753

d i h y d r o x y c o n j u g a t e d bile s a l t s a n d p o r c i n e l i p a s e (Ref. 1, figure 11). T h e r e f o r e , b y c o m p a r i n g t h e effect of n o n - i o n i c d e t e r g e n t s a n d bile s a l t s o n t h e a c t i v i t y of l i p a s e a g a i n s t e m u l s i f i e d t r i b u t y r i n , it a p p e a r s t h a t t h e y b o t h i n h i b i t l i p o l y s i s i n t h e a b s e n c e of c o l i p a s e at c o n c e n t r a t i o n s well b e l o w t h e i r CMC. F u r t h e r m o r e , n o n - i o n i c d e t e r g e n t s w h i c h h a v e a CMC lovcer t h a n t h a t of bile s a l t s a r e i n h i b i t o r s at m u c h l o w e r c o n centration than the anionic detergents. Results reported here are consistent with the hypothesis that the f a i l u r e of l i p a s e to i n t e r a c t w i t h s u b s t r a t e s u c h a s e m u l s i f i e d t r i b u t y r i n is m a i n l y d u e to t h e e x c e s s of d e t e r g e n t m o l e c u l e s t h a t a c c u m u l a t e at t h e i n t e r f a c e at c o n c e n t r a t i o n belo~v t h e CMC. It is t h u s r e a s o n a b l e *o a s s u m e t h a t e l e c t r o s t a t i c r e p u l s i o n p l a y a l i m i t e d ro!e in t h e i n h i b i t i o n of l i p a s e by d e t e r g e n t s at i n t e r face w i t h r e g a r d to t h e c o m p e t i t i o n bet~veen e n z y m e a n d d e t e r g e n t m o l e c u l e s I31. C o l i p a s e ~ ' h i c h p a r t i a l l y r e a c t i v a t e s l i p a s e at v e r y l o w c o n c e n t r a t i o n of n o n - i o n i c d e t e r g e n t s , c a n n o t p r e v e n t i n h i b i t i o n of t h e e n z y m e a t d e t e r g e n t c o n c e n t r a t i o n i n t h e r a n g e of t h e CMC in c o n t r a s t to ~vhat is o b s e r v e d i n t h e c a s e of bile salts. A l t h o u g h it is l i k e l y t h a t c o l i p a s e m a y i n t e r a c t w i t h m i c e l l e s of n o n - i o n i c 13rij 35 or T r i t o n X-100, b e c a u s e of t h e h i g h a f f i n i t y of t h e s e d e t e r g e n t s f o r h y d r o p h o b i c i n t e r f a c e s , t h e p r o t e i n c o f a c t o r m i g h t n o t be a b l e to d i s p l a c e d e t e r gent monomers from the substrate-water interface.

Acknowledgements.

"7

S t i m u l a t i n g d i s c u s s i o n s w i t h Dr. S 6 m 6 r i v a a n d Dr. S a v a r y ~Tere m o s t h e l p f u l d u r i n g t h e p r e p a r a t i o n of t h i s m a n u s c r i p t .

7

.E E -o

REFERENCES. g :a.o.5

I 1

2

~

I 3

l

I

i

4 Bile salts

mM

Fro. 3. - - Effect of m i x e d bovine bile salts on the activity of sheep pancreatic lipase against e m u l s i f i e d t r i b u t y r i n at pH 8.0 in the absence (0--@) and in the presence (0 ©) of colipase.

I n figure 3, a r e p r e s e n t e d t h e bile s a l t ( b o v i n e bile s a l t s ) a c t i v i t y d e p e n d e n c e c u r v e s of s h e e p p a n c r e a t i c l i p a s e i n t h e a b s e n c e a n d in t h e p r e s e n c e of c o l i p a s e . P,~esults i n d i c a t i n g f u l l e n z y m e i n h i b i t i o n at 1 m M bile s a l t s c o n c e n t r a t i o n a r e i n good a c c o r d a n c e w i t h t h o s e previously obtained by Borgstrom and Erlanson with

BIOCHIMIE, 1976. 58, n ° 6.

1. B o r g s t r o m , B. a E r l a n s o n , C. (1973) Europ. J. Biochem., 37, 60-68. 2. B o r g s t r o m , B. (1975) J. Lip. Res., 14, 411-417. 3. C h a p u s , C., Sari, H., S ~ m 6 r i v a , M. a D e s n u e l l e , P. (1975) FEBS Letters, 58, 155-158. 4. C h a r l e s , M., A s t i e r , M., S a u v e , P. a D e s n u e l l e , P. (1975) Europ J. Biochem., 58, 555-559. 5. Sari, H., E n t r e s s a n g l e s , B. ~ D e s n u e l l e , P. (1975) Europ. J. Biochem., 58, 561-565. 6. B o r g s t r o m , B. a D o n n e r , J. (1975) J. Lip. Res., 16, 287-292. 7. C a n i o u i , P., B e n a j i h a , A., J u l i e n R., R a t h e l o t , J., B e n a b d e l j l i l , A. a S a r d a , L. '(1975) Biochimie, 57, 35-41. 8. R a t h e l o t , J., J u l i e n , R., Coeroli, C., C a n i o n i , P. -~ S a r d a , L. (1975) Biochimie, 57, 1117-1122. 9. V e r g e r , R., De H a a s , G. H., S a r d a , L. ~ D e s n u e l l e , P. (1969) Biochim. Biophys. Acta, 188, 272-282. 10. S 6 m 6 r i v a , M., D u f o u r , C. ~ D e s n u e l l e , P. (1971) Biochemistry, 10, 2143-2149. 11. Albro, P. ~v~. ~ L a t i m e r , A. D. (1974) Biochemistry, 13, 1431-1439. 12. R i b e i r o , A. A. ~ D e n n i s , E. A. (1975) Biochemistry, 14, 3746-3755. 13. M o n t e t , J. C. ~ D e r v i c h i a n , D. G. (1971) Biochimie, 53, 751-754. 14. J u l i e n , R , C a n i o n i , P., R a t h e l o t , J. 8, S a r d a , L. (1972) Biochim. Biophys. Acta, 280, 215-224.