Biochimiea et Biophysiea Acta, 11185( 1991) 322-328 ~ 1991ElsevierScience PublishersB.V. All rights reserved00115-2760/91/$03.50 ADONIS tttll15276091002578
322
BBALIP 53715
Inactivation of pancreatic and gastric lipases by THL and C I2:0-TNB: a kinetic study with emulsified tributyrin Y o u s s e f G a r g o u r i 2, H e n r i C h a h i n i a n 2, H e r v 6 M o r e a u and Robert Verger
t, S t ~ p h a n e R a n s a c i
t Centre de Biochimie et de Biologic Molt;culairc dr; CNRS. Marseille (France) and 2 Laboratoire de Biochimie, Ecole Nationale d'lngdnieurs de Sfa.r. Sfax (Tunisia)
(Received 20 December 1990) Key words: Pancreaticlipase;Gastric lipase; Enzymeinactivation THL is a potent inhibitor of pancreatic (PPL) and gastric (HGL, RGL) lipases. Inactivation occurs preferentially at the oil/water interface (method B~ C). In the aqueous phase (method A), the inhibition of HGL was accelerated by the presence of bile salts. C 12,0-TNB, a disulfide reagent, specifically inactivates gastric lipases and had no effect on the pancreatic lipase (in the presence of bile salts) whatever the method used. The capacity of THL and Ctz:0-TNB to inactivate lipases using Methods B and C was found to depend directly upon the interfacial area of the system used. Consequently, inactivation can be reduced or prevented by further addition of a water-insoluble substrate which reduces the surface density of inactivator molecules. With a heterogeneous system of this kind, typical of lipolysis, the use of a classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (Kin, KI, Vmx) are only apparent values.
Introduction Gastric lipases differ from pancreatic lipase in several major respects. Under acidic pH conditions, gastric lipases have been found to be remarkably stable and active, whereas pancreatic lipase irreversibly loses its enzymatic activity. The optimal pH for gastric lipase action is around 5.4 [1,2], whereas pancreatic lipase acts maximally at around pH 8 - 9 [3]. Some amphiphiles (bile salts, alimentary proteins and phospholipids) which prevent pure pancreatic lipase adsorption and hence lipase activity [4,5], are required for gastric lipases catalysis to occur because they prevent the irreversible denaturation of pure gastric Iipase at the substrate/water interface [1,6]. Unlike pancreatic lipases, human gastric lipase (HGL) and rabbit gastric lipasc (RGL) were found to Abbreviations: HGL, human gastric lipase; RGL, rabbit gastric Itpaso; PPL, porcine pancreatic lipase; BSA, bovine serum albumin; NaTDC, sodium taurodeoxycholate;Tributyrin, tributyroyl glycerol; Ct,_:q3-TNB, dodecyl ditbie-5-(2-nitrobenzoicacid); DTNB. 5.5t-dithiobis(2 nitrobenzoic acid): THL, tetrahydrolipstatin; E,c~3,diethyl p-nitrophenylphosphale. Correspondence: R. Verger, Centre de Biochimie et de Biologie Mol6culairc du CNRS, BP 71. 134112Marseille Cedex 9, France.
be inhibited by sulfhydryl reagents and by a new hydrophobic disulfide: dodecyl dithio-5-(2-nitrobenzoic acid) (CIz:u-TNB) [7,8]. More recently we reported that ajoene, derived from ethanolic garlic extracts, specifically inactivated gastric lipases (HGL and RGL) and had no effect on porcine pancreatic lipase [9]. This finding was consistent with the fact that ajoene is reactive towards sulfhydryl compounds. Hadvfiry et al. [10] and Borgstr6m [11] have convincingly shown that tetrahydrolipstatin (THL) derived from lipstatin, which is produced by S t r e p t o m y c e s toxytricini, acts in vitro as a potent inhibitor of pancreatic lipases as well as cholesterol ester hydrolase and human gastric lipase. These authors suggest that a st.oichiometric enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolyzed, with water as the final acceptor, leaving an intact enzyme and the inactive form of T H L [10,11]. The aim of the present study was to compare the effects of T H L and Ct2:n-TNB on pancreatic and gastric lipases. Materials and Methods Lipids
Tributyrin (puriss, 99%) was from Fluka (Buchs, Switzerland). Sodium taurodeoxycholate (NaTDC) was
323 from Sigma, Tributyrin and NaTDC were uscd without further purification and no thin-layer chromatography control.
Enzymes
diomcter) using a tributyrin emulsion as substrate: 0.2 ml of tributyrin added to 10 ml of 150 mM NaCI, 2 mM taurodeoxycholate and 1.5 p,M bovine serum albumin [1]. In the case of P P L colipase was added to the assay in a 20-fold molar excess.
PPL and porcine colipasc, HGL and RGL were purified at the laboratory using previously described procedures [3,12,13].
Methods to test lipase hzactit'ation
Sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Aldrich; l-dodecanethiol was from Jansen (Patin, France). Dodecyldithio-5-(2-nitrobenzoic acid) (Clz:0-TNB) was synthesized at the laboratory as recently described [7]. A stock solution of 20 mM in ethanol was used.
Tetrahydrolipstatin THL was a generous gift from professor A.E. Fischli (F. Hoffman La Roche, Basle, Switzerland). A stock solution (20 mM) in ethanol was prepared.
Determination of protein concentration Protein concentration was determined as described by Lowry et al. [14] or spectrophotomctrically at 280 nm using the absorption coefficient A~n%~= 15.8 for HGL, 11.3 for PPL.
Lipase actit'ity measurements All lipase activities were measured titrimetrically at pH 6.0 in the case of gastric lipases and pH 8.0 in the case of PPL at 37 ° C with a pH-stat (TTI" 80, Ra-
A
Substnlle _+In,activator / ~
M.
//
I
1 I
O0
Method ( ~
Method
.~ 20
"~ 10
Method A: Lipase / inactit'ator preincubation method. This method was set up to test, in aqueous medium and in the absence of substrate, the possible reactions between lipases and THL or CL,:o-TNB (molar ratio 1/100). Residual lipase activity was measured using a tributyrin assay It]. Method B: hzacti~'atiorz during lipolysis. This method was designed to test whether any inactivation reaction occurred in the presence of a water-insoluble substrate during lipase hydrolysis. Lipase was injected into the thermostated (3 7 °C) reaction vessel of a Radiometer pH-stat, containing 10 ml NaCI (150 mM), NaTDC (2 raM), BSA (1.5 # M ) and 0.2 ml tributyrin emulsified by vigorous stirring. 2 min later, variable amounts of THL or CI.,:~-TNB were added. The lipase activity was continuously recorded. Method C: Poisoned interface method. Variable amounts of THL or C~z:,-TNB were injected into a tributyrin emulsion, prepared as in method B, I min before the enzyme addition.
(:~ it ± InacllvllO ///"./'r Llpase [//~""'~'~u~tnli~~te I
Me,,o® .m
"~
Three Methods, adapted from Moulin et al. [15] as depicted schematically in Fig. 1, were used to determine the inhibitory capacity of THL and CL,:o-TNB depending upon the order of addition of lipasc, substratc and inactivator.
2
Lipase inactivatorpreincubation
0
I 2 Tlme (arbitrary unlts) Inactlvalion during llpolysls
Llpase
/t
tl/;/
_4-Inacllvator bstrale
I
1
/
2
"poisoned Interface"
Fig. 1. Methods used to study the effectsof THL and C~2:.cTNBon lipaseactivities.Three methodsA. B and C were used. With each melhtld, arrows indicatethe order of successiveinjectionsof inhibitor(THL or Cu2:o-TNB20 mM in cthanolicsolution),lipases(HGL RGL or PPL) and substrate (tributyrin).Further detailsare givenin the text.
324 TABLE I
Results
Effect of pH on gastric lipase ,,,actication b)' THL (method A) Method A, (lipase / inactit'ator preincubation), to study the effect of THL or C]2:(~-TNB on gastric lipase acticity
HGL or RGL (26/zM) were incubated with THL (2.6 raM) at 37°C in a final volume of 150 /11 of either 5(I mM acetate buffer at pH ranging from 3 to 6 or Ill mM Tris-HCI buffer at pH 7 and 8, NaCI (150 mM). After 1 h of incubation, the residual lipase activity was measured at pH 6.0, 37 °C on tributyrin (0.2 ml) as substrate emulsified in 10 ml of NaCI (15(I mM). NaTDC (2 raM), BSA (1.5 ~M).
T h e incubation at p H 6.0 of H G L (Fig. 2A) and R G L (Fig. 2B) in the presence of T H L ( m o l a r ratio of 1 to 1001 was p e r f o r m e d and the residual lipase activities were m e a s u r e d . In the absence of substrate (Method A), it can be seen from Fig. 2 that T H L partially inactivates H G L and R G L . A f t e r 240 rain of incubation, 50% and 25% of residual H G L and R G L activity were m e a s u r e d , respectively. T h e presence of N a T D C (final concentration 4 raM) in the incubation m e d i u m did not affect R G L inhibition, w h e r e a s the remaining H G L activity d e c r e a s e d from 50% to 20% when T H L was used in the absence or p r e s e n c e of N a T D C , respectively. A rapid and complete inhibition of gastric lipases was observed after 10 rain of incubation with C]2:n-TNB (molar excess of 100, see Fig. 2), w h e n e n z y m e s w e r e incubated u n d e r the s a m e experimental conditions as those used with T H L , in the absence of N a T D C .
pH
Remaining activity(%)
3 4 6 7 8
90 80 60 52 48
80 50 30 26 20
Effect of p H on gastric lipase inactivation by THL (Method A) W e m e a s u r e d the r e m a i n i n g H G L and R G L activities after 1 h of incubation with T H L at various p H values. Control e x p e r i m e n t s with no T H L run for each
Method A / pH 6.0 (Lipase - inactivator preincubation) 100 I
B! • THL
| HGL ; o --'-
II Ik
A
/
THL+4 mM Fig TDC
THL
•
RGL ~o THL, 4mMN, TDC
' • C~2. 0 -TNB
\ A Ct2:o -TNB
== a ~ . . e . . ~ . . ~
OI
0
"z~.-_
•
I
t
30
60
90
I
.
t
=~..L_=
120 240 0 30 Time ( min I
o
,~._~.
I
I
I ..# I
60
90
120 240
Fig. 2. Variations in HGL (A) and RGL (B) activity during incubation with C!2:!~-TNR( ~_) or with THL in the absence ( B ) or presence tel ) of 4 mM NaTDC, Lipases (26 # M) were incubated at 37 o C, in a final volume of 150 #1 of 50 mM acetate buffer (pH 6.0); NaCI (150 raM) with THL or Ct2:=~-TNB(2.6 mM). Residual lipase activity was measured at pH 6.0, 37°C by tributyrin (0.2 ml) hydrolysis using 10 ml of NaCI (150 raM), NaTDC (2 raM), BSA (1.5 .u,M). Each kinetics represents one experiment,
325 assay s h o w e d n o significant d e c r e a s e in gastric lipase activity. T a b l e ! shows t h a t the highest inactivation levels w e r e o b t a i n e d in the n e u t r a l a n d aikaliiie p H r a n g e . A t p H 3.0, 5.0, 6.0 or 8.0, i n c u b a t i o n o f H G L with C~2:0-TNB ( m o l a r ratio: 30) i n d u c e d r a p i d a n d c o m p l e t e e n z y m e inactivation [7].
with H G L a n d R G L a n d p H 8.0 with PPL. 2 min a f t e r e n z y m e addition, T H L was injected at variable final c o n c e n t r a t i o n s . As c a n be seen f r o m the kinetic curves in Fig. 3, the rate of tributyrin hydrolysis by P F L (Fig. 3A), R G L (Fig. 3B) a n d H G L (Fig. 3(2) d e c r e a s e d to different extents. T h e lipase hydrolysis rate d e c r e a s e d m o r e rapidly at the h i g h e r T H L c o n c e n t r a t i o n s . W i t h all the lipases tested m o r e t h a n 9 0 % of the e n z y m e inactivation was o b s e r v e d to o c c u r with final T H L c o n c e n t r a t i o n of 200 p.M. Fig. 3 D shows t h a t C~2:oT N B acts as an H G L inhibitor in the s a m e c o n c e n t r a -
Method B, (inactiration during lipolysis), for studying the kinetics of tributyrin hydrolysis by gastric and pancreatic lipases in the presence of THL or CI,.:(cTNB T h e hydrolysis of tributyrin w a s r e c o r d e d at p H 6.0
Method B (Inactivation during lipolysis)
®
01|M
20pM
//.,
/,.
®
~
-!
A
o= :::L
I
_i -g_ M
!
®
!
/
30pM
/ - / 1 , ~ p M
l
OpM
i Ot~M
I
;0
;5
J
.z-
~
~pM
100pM
~~________~1..~ ~M
/ o
Time ( min )
~
Fig. 3. Effect of variable THL concentrations (A, B, C) or C~::o-TNB concentrations (D) on the rate of hydrolysis of tributyrin by (A): PPL (5 nM) + colipase ( I00 nM); (B): RGL (24 nM), (C, D): HGL (24 nM). Assays were cm tied o it at 37 ° C, pH 8.0 with PPL and pH 6.0 with HGL and RGL using 0.2 ml tributyrin emulsified in tO ml of NaCI (150 raM), NaTDC (2 raM), BSA (1.5 p.M). Each kinetics represents one typical experiment.
326
h,
cllolate up to a final concentr~!tiolt of 200 IzM (data not shown).
i Method C ("poisoned interface") I
J
100
Met/zod C, (poisoned #lterface), for studying the effect of inactit'ator concentration on the rate of tributyrin hydrolysis by gastric attd pancreatic lipases
g
.::_
00
25
50
0
200
400
600
THL concentration (p M) C12:9-TNB concentration (~ M) Fig. 4, Effect of increasing concentrations of THL (A) or C I2:u-TNB (B) on the rate of hydrolysis of tributyrin by PPL (~ nM) in the presence of ¢olipase (100 nM) (El), or RGL (24 nM) (e) or HGL (24 nM) (©). Experiments were performed at 37°C (pH 8.0), with PPL and pH 6.0 with HGL and RGL using 0.2 ml tributyrin emulsified in 10 ml of NaCI (150 mM), NaTDC (2 raM), BSA (I.5 /,tM). In all cases. THL or C~.,:~cTNB. from a 20 mM ethanolic solution, were injected 1 rain prior to the enzyme additien. Initial velocity measurements are given. Each point represents an independent assay.
tion r a n g e as T H L . Similar results w e r e obtained with R G L u n d e r the s a m e e x p e r i m e n t a l conditions ( d a t a not shown). By contrast, PPL was not affected by the prer, ence of C~z:,-TNB in the p r e s e n c e of tauaodeoxy-
T h e rate of tributyrin hydrolysis by H G L , R G L or PPL was d e t e r m i n e d as a function of the T H L concentration (Fig. 4 A t or C~2:u-TNB concentration (Fig. 4B). in all cases, T H L or CL,:{j-TNB was injected into a tributyrin emulsion 1 min prior to the e n z y m e addition and the initial lipase hydrolysis rate was m e a s u r e d . As shown by the curve in Fig. 4A, the rate of tributyrin hydrolysis by H G L , R G L or P P L d e c r e a s e d according to a similar pattern, with increasing T H L c o n c e n t r a tions. T h e inactivator c o n c e n t r a t i o n which r e d u c e d the lipase activity to 50% (Is(0 of its initial value was
Method C (,,poisoned interface") o NO inactivat0r C12 : 0-TNB ( 400 P.M ) HGL I ° .rHL(60p. M) 100
#
-
-
o
o
~ 50
TC4/pH 8.01PPL o No inaetivat0r
(,~
PPL I = THL(60P. M)
100
/~
~
N 0 o
10
20
i 30
i 40
Time ( min ) Fig. 5. Kinetics of tributyrin hydrolysis at pH 8.0 by PPL in the presence of TILL. The arrows show the successive injections of trihutyrin (0.2 ml)l: TIlL (10 p_M); PPL (5 nM) with eolipase (1(}() nM) followed by a second injection of PPL (5 nM); tributyrin (1 ml): PPL (5 nM), Assays were performed at 37°C (pH 8.0), using tributyrin ((I.2 ml) as substrate emulsified in I(1 ml Naf'l (150 raM), NaTDC (2 raM) and BSA (1.5/zM). This experiment was performed three times and no significant difference was observed.
0.5
t
I
1
1.5
Tributvrin ( rnl Fig. 6. Effect of increasing amounts of tributyrin on the rate of its hydrolysis by (A): HGL (24 nM) or (B): PPL (5 nM) with colipase (I(X) nM) in the absence ([3) or presence (,=) of THL (60 pM) or (©) C=z:II-TNB (40() pM). Assays were carri=,d out at 37°C (pH 8.0) with PPL and pH 6.0 with HGL, in a medlum containing 10 ml NaCI (150 mM), NaTDC (2 raM), BSA (1,5 p,M) and variable amounts of tributyrin. Each point represents an independent assay.
327 around 10 #M. Fig. 4B shows that C~,:,~-TNB acts as an inhibitor with both HGL (15.: 110 /.tM) and RGL (150:250/.tM), in contrast to PPL ~;'hich is insensitive to CI2:0-TNB, in the presence of bile salts. Furthermore, we studied the effect of the surface density of the inactivator on the rate of PPL inhibition by T H E Using Method C, THL (l(I/.tM final concentration) was injected into the bulk phase (10 ml) containing 0.2 ml of tributyrin and 1 min later 10 units of PPL (with a 20-fold molar excess of colipase) wcrc added and the enzyme kinetics were recorded. As can be seen from Fig. 5, the PPL activity was severely reduced several min after the PPI., injection. A second addition of PPL (10 units) did not further ennance the tributyrin hydrolysis, indicating that THL was still available for lipase inactivation. The addition of excess tributyrin (1 ml) failed to reactivate the enzyme, thus showing that an irreversible lipase inactivation had taken place. A third addition of PPL (ltl units) immediately led to 10 units of PPL activity (Fig. 5). The addition of excess tributyrin therefore probably reduced the interracial THL concentration and decreased its inhibition capacity. Similar results were obtained when gastric lipases (at pH 6.1)) were used instead of PPL (data not shown). in order to evaluate more exactly the influence of the surface concentration of the inactivator on the level of lipase inhibition using method C. we used a fixed amount of THL (60/.tM) or C~::.-TNB ~400/zM) and varied the amount of tributyrin added (Fig. 6). Under these experimental eoaditions, 50% lipase mactivation by TIlL was observed in the presence of i.8 ml and 1.4 ml of tnbutyrin with HGL (Fig. 6A) and PPL (Fig. 6B), respectively. Fig. 6A further shows that I ml of tributyrin was required for HGL activity to reach 50% of its maximal value in the presence of 400 ~M C~2:o-TNBDiscussion
It has been established that the catalytic properties of gastric lipases differ markedly from those of porcine pancreatic lipase [2,3]. These differences may be related to differences in structure between these two enzyme classes. We have reported that gastric lipases, unlike pancreatic ones, were inactivated by sulfhydryl reagents and by ajoene [7,8]. This inactivation results from to the modification of one free sulfhydryl group, which was shown to be essential for the expression of enzymatic activity and not for the lipid binding step [161. In the present study, we compared the effect., of THL and CI2:~-TNB, which arc both water-insoluble, on the gastric and pancreatic lipase inactivation, using three different methods (Fig. l).
In the absence of water-insoluble substrate (method A), our results show that CI2:~-TNB i~ a more potent inactivator of gastric lipases than THL. it is worth noting that gastric lipases are only partially inactivated by THL, even after a long incubation period (4 h) (Fig. 2) or with a large molar excess of T H E After incubation of C~,:.-TNB for 10 min with gastric lipase at a molar excess of 11~0, a complete loss of activity was observed, whereas 50c~ and 25% of HGL and RGL activity, respectively, wcre still detectable when THL was used under the same experimental conditions. Adding bile salt to the incubation medium (method A~ accelerated the inactivation rate of HGL by THL due to the fact that THL probably formed mixed bile salt/THL micelles. As in the case of lipase substrates, the presence of mixed THL/bile saR micelles may give rise to a better "interracial quality', thus improving the lipase adsorption a n d / o r increasing in THL accessibility. This finding is reminiscent of the effect of diethyl p-nitrophcnyl phosphate ( E , ~ ) on porcine pancreatic lipasc. In the latter case, Desnuelle et al. [17] showed that emulsified EuH~ irreversibly inactivates PPL, whereas aqueous solutions of this organo-phosphorous compound do not. Mayli6 et al. [18] and Rouard et al. [19] dcscribed the covalent modification of a serinc rcsiduc of PPL (in the presence of colipase) induced by mixed E¢,,./bile salt micelles. Moreau et al. [20] demonstrated that the essential serine residue which was stoichiometrically labelled by this organophosphorus reagent is involved in catalysis and not in lipid binding. In the presence of an emulsified substrate, gastric and pancreatic lipases were completely inactivated when THL was injected before or after the enzyme addition, which is in agreement with previous data by Borgstr6m [1 l] and Hadvfiry et al. [10]. However, it is worth noting from the data presented in Figs. 5 and 6 that the THL and C~2:.-TNB inactivation capacity is directly dependent upon the interfacial area of the system used. Consequently, the lipase inactivation can be reduced or prevented by further addition of a water-in,w+.~b!e substrate which reduce,', the surface density of the inactivator molecules. With a hetcrogeneous system of this kind, typical of lipolytic systems, the use of the classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (K~, K., Irmax, IC5o) represent apparent values [21]. Consequently, the IC~. data are only empirical values and cannot be significantly and directly compared from one set of experimental conditions to another. Borgstr6m ([11] and Basle, Workshop on Lipases, April 27, 1989) using human pancreatic lipase, human pancreatic carboxyl ester lipase, human gastric lipase, bile-salt-stimulated tipase from human milk, hormonesensitive lipase and lipoprotein lipase showed that all
328 these s e r i n e / h i s t i d i n e hydrolases are inactivated at different levels by T H L T a k e n as a whole, these resuits support the view that T H L is a fairly general l i p a s e / e s t e r a s e inactivator. T h e situation s e e m s to be different with C~2:()-TNB, which specifically inactivates gastric lipases and had no effect on the pancreatic lipasc (in the presence of bile salts), w h a t e v e r the m e t h o d used (A, B or C).
Acknowledgements T h e authors are grateful to Professor A.E. Fischli (F. H o f f m a n La Roche, Basle, Switzerland) for the generous gift of T H L . We acknowledge the help of F. F e r r a t o (Marseille) with the PPL purification and of D~" J. Blanc (Marseille) for correcting the manuscript. This research was partly carried out with the financial support of Bridge-T-Lipase p r o g r a m m e of the European C o m m u n i t i e s u n d e r contract No. B I O T - C T g l 0274 ( D T E E ) .
References 1 Gargouri. Y., Pit~roni. G.. Rivi~re, C., Sauni,~re. J.F.. Lowe. P.A.. 8arda, L. and Verger, R. 119861 Gastroenterology 91,919-925. 2 Hamosh, M. 119841 in Lipases (BorgstrSm, B. and Brockman, H.L.. eds.J, pp. 49-81, Elsevier, Amsferdam. 3 Velger. R. 119841in Lipases (BorgstrSm. B. and Brockman. H.L., eds.), pp. 83-150, Elsevier, Amsterdam.
4 Gargouri, Y., Pi~roni, G.. Rivi;zre,C., Sugihara, A. Sarda, L. and Verger, R. 119851J. Biol. Chem. 260. 2268-2273. 5 Gargouri, Y., Pi6roni, G.. Rivi~re, C, Sarda, L. anti Verger, R. 119861 Biochemistry 25, 1733-1738. 6 Gargouri. Y,. Pi~roni. G.. Lowe. P.A.. Sarda. L. and Verger. R. 119861 Eur. J. Biochem. 156, 305-3111. 7 Gargouri, Y., Moreau, H., Pi6roni, G. and Verger, R. 119881 J. Biol. Chem. 263, 2159-2162. 8 Moreau, H., Gargouri, Y.. Pi6roni. G. and Verger, R. 119881 FEBS Lett. 23f,, 383-387. 9 Gargouri, Y,. Moreau, H.. Jain. M.K.. de Haas. G.H. and Verger. R. 11989) Biochim. Biophys. Acta 111116,137-139. 10 Hadv~iry. P., Lengsfeld, H. and Wolfer, H. (',988) Biochem. J. 256, 357-361. 11 BorgstrSm, B. 119881 Biochim. Biophys. Aeta 962, 308-316. 12 Tiruppathi. C. and Balasubramanian, K.A. 119821 Bioehim. Biophys. Acta 712, 692-697. 13 Moreau. H., Gargouri. Y., Leeat, D., Junien. J.L. and Verger, R. 119881 Bioehim. Biophys. Aeta 960, 286-293. 14 Lowry, O.H.. Rosebrough. N.J., Farr, A.L. and Randall, R.J. 119511 J. Biol. Chem. 193, 265-275. 15 Moulin. A.. Fourneron. J.D.. Pi~roni. G. and Verger, R. 119891 Biochemistry 28, 6340-6346. 16 Gargouri, Y., Moreau. H., Pi~roni. G. and Ve'ger, R. 119891Eur. J. Biochem. 180, 367-371. 17 Desnuelle, P., Sarda. L. and Ailhaud, G. 119601 Biochim. Biophys. Acta 37, 570-571. 18 Mayli6, M.F., Charles. M. and Desnue0e, P. 119721 Biochim. Biophys. Aeta 276, 162-175. 19 Rouard. M., Sari, H.. Nurit, S., Entressangles. B. and Desnuelle, P. 119781 Biochim. Biophys. Aeta 530, 227-235. 20 Moreau, H., Moulin, A., Gargouri, Y., No~l, J.P. and Verger, R. (1991) Biochemistry 30, 1037-1041. 21 Verger, R. 119801Methods Enzymol. 64. 340-392.
322
BBALIP 53715
Inactivation of pancreatic and gastric lipases by THL and C I2:0-TNB: a kinetic study with emulsified tributyrin Y o u s s e f G a r g o u r i 2, H e n r i C h a h i n i a n 2, H e r v 6 M o r e a u and Robert Verger
t, S t ~ p h a n e R a n s a c i
t Centre de Biochimie et de Biologic Molt;culairc dr; CNRS. Marseille (France) and 2 Laboratoire de Biochimie, Ecole Nationale d'lngdnieurs de Sfa.r. Sfax (Tunisia)
(Received 20 December 1990) Key words: Pancreaticlipase;Gastric lipase; Enzymeinactivation THL is a potent inhibitor of pancreatic (PPL) and gastric (HGL, RGL) lipases. Inactivation occurs preferentially at the oil/water interface (method B~ C). In the aqueous phase (method A), the inhibition of HGL was accelerated by the presence of bile salts. C 12,0-TNB, a disulfide reagent, specifically inactivates gastric lipases and had no effect on the pancreatic lipase (in the presence of bile salts) whatever the method used. The capacity of THL and Ctz:0-TNB to inactivate lipases using Methods B and C was found to depend directly upon the interfacial area of the system used. Consequently, inactivation can be reduced or prevented by further addition of a water-insoluble substrate which reduces the surface density of inactivator molecules. With a heterogeneous system of this kind, typical of lipolysis, the use of a classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (Kin, KI, Vmx) are only apparent values.
Introduction Gastric lipases differ from pancreatic lipase in several major respects. Under acidic pH conditions, gastric lipases have been found to be remarkably stable and active, whereas pancreatic lipase irreversibly loses its enzymatic activity. The optimal pH for gastric lipase action is around 5.4 [1,2], whereas pancreatic lipase acts maximally at around pH 8 - 9 [3]. Some amphiphiles (bile salts, alimentary proteins and phospholipids) which prevent pure pancreatic lipase adsorption and hence lipase activity [4,5], are required for gastric lipases catalysis to occur because they prevent the irreversible denaturation of pure gastric Iipase at the substrate/water interface [1,6]. Unlike pancreatic lipases, human gastric lipase (HGL) and rabbit gastric lipasc (RGL) were found to Abbreviations: HGL, human gastric lipase; RGL, rabbit gastric Itpaso; PPL, porcine pancreatic lipase; BSA, bovine serum albumin; NaTDC, sodium taurodeoxycholate;Tributyrin, tributyroyl glycerol; Ct,_:q3-TNB, dodecyl ditbie-5-(2-nitrobenzoicacid); DTNB. 5.5t-dithiobis(2 nitrobenzoic acid): THL, tetrahydrolipstatin; E,c~3,diethyl p-nitrophenylphosphale. Correspondence: R. Verger, Centre de Biochimie et de Biologie Mol6culairc du CNRS, BP 71. 134112Marseille Cedex 9, France.
be inhibited by sulfhydryl reagents and by a new hydrophobic disulfide: dodecyl dithio-5-(2-nitrobenzoic acid) (CIz:u-TNB) [7,8]. More recently we reported that ajoene, derived from ethanolic garlic extracts, specifically inactivated gastric lipases (HGL and RGL) and had no effect on porcine pancreatic lipase [9]. This finding was consistent with the fact that ajoene is reactive towards sulfhydryl compounds. Hadvfiry et al. [10] and Borgstr6m [11] have convincingly shown that tetrahydrolipstatin (THL) derived from lipstatin, which is produced by S t r e p t o m y c e s toxytricini, acts in vitro as a potent inhibitor of pancreatic lipases as well as cholesterol ester hydrolase and human gastric lipase. These authors suggest that a st.oichiometric enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolyzed, with water as the final acceptor, leaving an intact enzyme and the inactive form of T H L [10,11]. The aim of the present study was to compare the effects of T H L and Ct2:n-TNB on pancreatic and gastric lipases. Materials and Methods Lipids
Tributyrin (puriss, 99%) was from Fluka (Buchs, Switzerland). Sodium taurodeoxycholate (NaTDC) was
323 from Sigma, Tributyrin and NaTDC were uscd without further purification and no thin-layer chromatography control.
Enzymes
diomcter) using a tributyrin emulsion as substrate: 0.2 ml of tributyrin added to 10 ml of 150 mM NaCI, 2 mM taurodeoxycholate and 1.5 p,M bovine serum albumin [1]. In the case of P P L colipase was added to the assay in a 20-fold molar excess.
PPL and porcine colipasc, HGL and RGL were purified at the laboratory using previously described procedures [3,12,13].
Methods to test lipase hzactit'ation
Sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Aldrich; l-dodecanethiol was from Jansen (Patin, France). Dodecyldithio-5-(2-nitrobenzoic acid) (Clz:0-TNB) was synthesized at the laboratory as recently described [7]. A stock solution of 20 mM in ethanol was used.
Tetrahydrolipstatin THL was a generous gift from professor A.E. Fischli (F. Hoffman La Roche, Basle, Switzerland). A stock solution (20 mM) in ethanol was prepared.
Determination of protein concentration Protein concentration was determined as described by Lowry et al. [14] or spectrophotomctrically at 280 nm using the absorption coefficient A~n%~= 15.8 for HGL, 11.3 for PPL.
Lipase actit'ity measurements All lipase activities were measured titrimetrically at pH 6.0 in the case of gastric lipases and pH 8.0 in the case of PPL at 37 ° C with a pH-stat (TTI" 80, Ra-
A
Substnlle _+In,activator / ~
M.
//
I
1 I
O0
Method ( ~
Method
.~ 20
"~ 10
Method A: Lipase / inactit'ator preincubation method. This method was set up to test, in aqueous medium and in the absence of substrate, the possible reactions between lipases and THL or CL,:o-TNB (molar ratio 1/100). Residual lipase activity was measured using a tributyrin assay It]. Method B: hzacti~'atiorz during lipolysis. This method was designed to test whether any inactivation reaction occurred in the presence of a water-insoluble substrate during lipase hydrolysis. Lipase was injected into the thermostated (3 7 °C) reaction vessel of a Radiometer pH-stat, containing 10 ml NaCI (150 mM), NaTDC (2 raM), BSA (1.5 # M ) and 0.2 ml tributyrin emulsified by vigorous stirring. 2 min later, variable amounts of THL or CI.,:~-TNB were added. The lipase activity was continuously recorded. Method C: Poisoned interface method. Variable amounts of THL or C~z:,-TNB were injected into a tributyrin emulsion, prepared as in method B, I min before the enzyme addition.
(:~ it ± InacllvllO ///"./'r Llpase [//~""'~'~u~tnli~~te I
Me,,o® .m
"~
Three Methods, adapted from Moulin et al. [15] as depicted schematically in Fig. 1, were used to determine the inhibitory capacity of THL and CL,:o-TNB depending upon the order of addition of lipasc, substratc and inactivator.
2
Lipase inactivatorpreincubation
0
I 2 Tlme (arbitrary unlts) Inactlvalion during llpolysls
Llpase
/t
tl/;/
_4-Inacllvator bstrale
I
1
/
2
"poisoned Interface"
Fig. 1. Methods used to study the effectsof THL and C~2:.cTNBon lipaseactivities.Three methodsA. B and C were used. With each melhtld, arrows indicatethe order of successiveinjectionsof inhibitor(THL or Cu2:o-TNB20 mM in cthanolicsolution),lipases(HGL RGL or PPL) and substrate (tributyrin).Further detailsare givenin the text.
324 TABLE I
Results
Effect of pH on gastric lipase ,,,actication b)' THL (method A) Method A, (lipase / inactit'ator preincubation), to study the effect of THL or C]2:(~-TNB on gastric lipase acticity
HGL or RGL (26/zM) were incubated with THL (2.6 raM) at 37°C in a final volume of 150 /11 of either 5(I mM acetate buffer at pH ranging from 3 to 6 or Ill mM Tris-HCI buffer at pH 7 and 8, NaCI (150 mM). After 1 h of incubation, the residual lipase activity was measured at pH 6.0, 37 °C on tributyrin (0.2 ml) as substrate emulsified in 10 ml of NaCI (15(I mM). NaTDC (2 raM), BSA (1.5 ~M).
T h e incubation at p H 6.0 of H G L (Fig. 2A) and R G L (Fig. 2B) in the presence of T H L ( m o l a r ratio of 1 to 1001 was p e r f o r m e d and the residual lipase activities were m e a s u r e d . In the absence of substrate (Method A), it can be seen from Fig. 2 that T H L partially inactivates H G L and R G L . A f t e r 240 rain of incubation, 50% and 25% of residual H G L and R G L activity were m e a s u r e d , respectively. T h e presence of N a T D C (final concentration 4 raM) in the incubation m e d i u m did not affect R G L inhibition, w h e r e a s the remaining H G L activity d e c r e a s e d from 50% to 20% when T H L was used in the absence or p r e s e n c e of N a T D C , respectively. A rapid and complete inhibition of gastric lipases was observed after 10 rain of incubation with C]2:n-TNB (molar excess of 100, see Fig. 2), w h e n e n z y m e s w e r e incubated u n d e r the s a m e experimental conditions as those used with T H L , in the absence of N a T D C .
pH
Remaining activity(%)
3 4 6 7 8
90 80 60 52 48
80 50 30 26 20
Effect of p H on gastric lipase inactivation by THL (Method A) W e m e a s u r e d the r e m a i n i n g H G L and R G L activities after 1 h of incubation with T H L at various p H values. Control e x p e r i m e n t s with no T H L run for each
Method A / pH 6.0 (Lipase - inactivator preincubation) 100 I
B! • THL
| HGL ; o --'-
II Ik
A
/
THL+4 mM Fig TDC
THL
•
RGL ~o THL, 4mMN, TDC
' • C~2. 0 -TNB
\ A Ct2:o -TNB
== a ~ . . e . . ~ . . ~
OI
0
"z~.-_
•
I
t
30
60
90
I
.
t
=~..L_=
120 240 0 30 Time ( min I
o
,~._~.
I
I
I ..# I
60
90
120 240
Fig. 2. Variations in HGL (A) and RGL (B) activity during incubation with C!2:!~-TNR( ~_) or with THL in the absence ( B ) or presence tel ) of 4 mM NaTDC, Lipases (26 # M) were incubated at 37 o C, in a final volume of 150 #1 of 50 mM acetate buffer (pH 6.0); NaCI (150 raM) with THL or Ct2:=~-TNB(2.6 mM). Residual lipase activity was measured at pH 6.0, 37°C by tributyrin (0.2 ml) hydrolysis using 10 ml of NaCI (150 raM), NaTDC (2 raM), BSA (1.5 .u,M). Each kinetics represents one experiment,
325 assay s h o w e d n o significant d e c r e a s e in gastric lipase activity. T a b l e ! shows t h a t the highest inactivation levels w e r e o b t a i n e d in the n e u t r a l a n d aikaliiie p H r a n g e . A t p H 3.0, 5.0, 6.0 or 8.0, i n c u b a t i o n o f H G L with C~2:0-TNB ( m o l a r ratio: 30) i n d u c e d r a p i d a n d c o m p l e t e e n z y m e inactivation [7].
with H G L a n d R G L a n d p H 8.0 with PPL. 2 min a f t e r e n z y m e addition, T H L was injected at variable final c o n c e n t r a t i o n s . As c a n be seen f r o m the kinetic curves in Fig. 3, the rate of tributyrin hydrolysis by P F L (Fig. 3A), R G L (Fig. 3B) a n d H G L (Fig. 3(2) d e c r e a s e d to different extents. T h e lipase hydrolysis rate d e c r e a s e d m o r e rapidly at the h i g h e r T H L c o n c e n t r a t i o n s . W i t h all the lipases tested m o r e t h a n 9 0 % of the e n z y m e inactivation was o b s e r v e d to o c c u r with final T H L c o n c e n t r a t i o n of 200 p.M. Fig. 3 D shows t h a t C~2:oT N B acts as an H G L inhibitor in the s a m e c o n c e n t r a -
Method B, (inactiration during lipolysis), for studying the kinetics of tributyrin hydrolysis by gastric and pancreatic lipases in the presence of THL or CI,.:(cTNB T h e hydrolysis of tributyrin w a s r e c o r d e d at p H 6.0
Method B (Inactivation during lipolysis)
®
01|M
20pM
//.,
/,.
®
~
-!
A
o= :::L
I
_i -g_ M
!
®
!
/
30pM
/ - / 1 , ~ p M
l
OpM
i Ot~M
I
;0
;5
J
.z-
~
~pM
100pM
~~________~1..~ ~M
/ o
Time ( min )
~
Fig. 3. Effect of variable THL concentrations (A, B, C) or C~::o-TNB concentrations (D) on the rate of hydrolysis of tributyrin by (A): PPL (5 nM) + colipase ( I00 nM); (B): RGL (24 nM), (C, D): HGL (24 nM). Assays were cm tied o it at 37 ° C, pH 8.0 with PPL and pH 6.0 with HGL and RGL using 0.2 ml tributyrin emulsified in tO ml of NaCI (150 raM), NaTDC (2 raM), BSA (1.5 p.M). Each kinetics represents one typical experiment.
326
h,
cllolate up to a final concentr~!tiolt of 200 IzM (data not shown).
i Method C ("poisoned interface") I
J
100
Met/zod C, (poisoned #lterface), for studying the effect of inactit'ator concentration on the rate of tributyrin hydrolysis by gastric attd pancreatic lipases
g
.::_
00
25
50
0
200
400
600
THL concentration (p M) C12:9-TNB concentration (~ M) Fig. 4, Effect of increasing concentrations of THL (A) or C I2:u-TNB (B) on the rate of hydrolysis of tributyrin by PPL (~ nM) in the presence of ¢olipase (100 nM) (El), or RGL (24 nM) (e) or HGL (24 nM) (©). Experiments were performed at 37°C (pH 8.0), with PPL and pH 6.0 with HGL and RGL using 0.2 ml tributyrin emulsified in 10 ml of NaCI (150 mM), NaTDC (2 raM), BSA (I.5 /,tM). In all cases. THL or C~.,:~cTNB. from a 20 mM ethanolic solution, were injected 1 rain prior to the enzyme additien. Initial velocity measurements are given. Each point represents an independent assay.
tion r a n g e as T H L . Similar results w e r e obtained with R G L u n d e r the s a m e e x p e r i m e n t a l conditions ( d a t a not shown). By contrast, PPL was not affected by the prer, ence of C~z:,-TNB in the p r e s e n c e of tauaodeoxy-
T h e rate of tributyrin hydrolysis by H G L , R G L or PPL was d e t e r m i n e d as a function of the T H L concentration (Fig. 4 A t or C~2:u-TNB concentration (Fig. 4B). in all cases, T H L or CL,:{j-TNB was injected into a tributyrin emulsion 1 min prior to the e n z y m e addition and the initial lipase hydrolysis rate was m e a s u r e d . As shown by the curve in Fig. 4A, the rate of tributyrin hydrolysis by H G L , R G L or P P L d e c r e a s e d according to a similar pattern, with increasing T H L c o n c e n t r a tions. T h e inactivator c o n c e n t r a t i o n which r e d u c e d the lipase activity to 50% (Is(0 of its initial value was
Method C (,,poisoned interface") o NO inactivat0r C12 : 0-TNB ( 400 P.M ) HGL I ° .rHL(60p. M) 100
#
-
-
o
o
~ 50
TC4/pH 8.01PPL o No inaetivat0r
(,~
PPL I = THL(60P. M)
100
/~
~
N 0 o
10
20
i 30
i 40
Time ( min ) Fig. 5. Kinetics of tributyrin hydrolysis at pH 8.0 by PPL in the presence of TILL. The arrows show the successive injections of trihutyrin (0.2 ml)l: TIlL (10 p_M); PPL (5 nM) with eolipase (1(}() nM) followed by a second injection of PPL (5 nM); tributyrin (1 ml): PPL (5 nM), Assays were performed at 37°C (pH 8.0), using tributyrin ((I.2 ml) as substrate emulsified in I(1 ml Naf'l (150 raM), NaTDC (2 raM) and BSA (1.5/zM). This experiment was performed three times and no significant difference was observed.
0.5
t
I
1
1.5
Tributvrin ( rnl Fig. 6. Effect of increasing amounts of tributyrin on the rate of its hydrolysis by (A): HGL (24 nM) or (B): PPL (5 nM) with colipase (I(X) nM) in the absence ([3) or presence (,=) of THL (60 pM) or (©) C=z:II-TNB (40() pM). Assays were carri=,d out at 37°C (pH 8.0) with PPL and pH 6.0 with HGL, in a medlum containing 10 ml NaCI (150 mM), NaTDC (2 raM), BSA (1,5 p,M) and variable amounts of tributyrin. Each point represents an independent assay.
327 around 10 #M. Fig. 4B shows that C~,:,~-TNB acts as an inhibitor with both HGL (15.: 110 /.tM) and RGL (150:250/.tM), in contrast to PPL ~;'hich is insensitive to CI2:0-TNB, in the presence of bile salts. Furthermore, we studied the effect of the surface density of the inactivator on the rate of PPL inhibition by T H E Using Method C, THL (l(I/.tM final concentration) was injected into the bulk phase (10 ml) containing 0.2 ml of tributyrin and 1 min later 10 units of PPL (with a 20-fold molar excess of colipase) wcrc added and the enzyme kinetics were recorded. As can be seen from Fig. 5, the PPL activity was severely reduced several min after the PPI., injection. A second addition of PPL (10 units) did not further ennance the tributyrin hydrolysis, indicating that THL was still available for lipase inactivation. The addition of excess tributyrin (1 ml) failed to reactivate the enzyme, thus showing that an irreversible lipase inactivation had taken place. A third addition of PPL (ltl units) immediately led to 10 units of PPL activity (Fig. 5). The addition of excess tributyrin therefore probably reduced the interracial THL concentration and decreased its inhibition capacity. Similar results were obtained when gastric lipases (at pH 6.1)) were used instead of PPL (data not shown). in order to evaluate more exactly the influence of the surface concentration of the inactivator on the level of lipase inhibition using method C. we used a fixed amount of THL (60/.tM) or C~::.-TNB ~400/zM) and varied the amount of tributyrin added (Fig. 6). Under these experimental eoaditions, 50% lipase mactivation by TIlL was observed in the presence of i.8 ml and 1.4 ml of tnbutyrin with HGL (Fig. 6A) and PPL (Fig. 6B), respectively. Fig. 6A further shows that I ml of tributyrin was required for HGL activity to reach 50% of its maximal value in the presence of 400 ~M C~2:o-TNBDiscussion
It has been established that the catalytic properties of gastric lipases differ markedly from those of porcine pancreatic lipase [2,3]. These differences may be related to differences in structure between these two enzyme classes. We have reported that gastric lipases, unlike pancreatic ones, were inactivated by sulfhydryl reagents and by ajoene [7,8]. This inactivation results from to the modification of one free sulfhydryl group, which was shown to be essential for the expression of enzymatic activity and not for the lipid binding step [161. In the present study, we compared the effect., of THL and CI2:~-TNB, which arc both water-insoluble, on the gastric and pancreatic lipase inactivation, using three different methods (Fig. l).
In the absence of water-insoluble substrate (method A), our results show that CI2:~-TNB i~ a more potent inactivator of gastric lipases than THL. it is worth noting that gastric lipases are only partially inactivated by THL, even after a long incubation period (4 h) (Fig. 2) or with a large molar excess of T H E After incubation of C~,:.-TNB for 10 min with gastric lipase at a molar excess of 11~0, a complete loss of activity was observed, whereas 50c~ and 25% of HGL and RGL activity, respectively, wcre still detectable when THL was used under the same experimental conditions. Adding bile salt to the incubation medium (method A~ accelerated the inactivation rate of HGL by THL due to the fact that THL probably formed mixed bile salt/THL micelles. As in the case of lipase substrates, the presence of mixed THL/bile saR micelles may give rise to a better "interracial quality', thus improving the lipase adsorption a n d / o r increasing in THL accessibility. This finding is reminiscent of the effect of diethyl p-nitrophcnyl phosphate ( E , ~ ) on porcine pancreatic lipasc. In the latter case, Desnuelle et al. [17] showed that emulsified EuH~ irreversibly inactivates PPL, whereas aqueous solutions of this organo-phosphorous compound do not. Mayli6 et al. [18] and Rouard et al. [19] dcscribed the covalent modification of a serinc rcsiduc of PPL (in the presence of colipase) induced by mixed E¢,,./bile salt micelles. Moreau et al. [20] demonstrated that the essential serine residue which was stoichiometrically labelled by this organophosphorus reagent is involved in catalysis and not in lipid binding. In the presence of an emulsified substrate, gastric and pancreatic lipases were completely inactivated when THL was injected before or after the enzyme addition, which is in agreement with previous data by Borgstr6m [1 l] and Hadvfiry et al. [10]. However, it is worth noting from the data presented in Figs. 5 and 6 that the THL and C~2:.-TNB inactivation capacity is directly dependent upon the interfacial area of the system used. Consequently, the lipase inactivation can be reduced or prevented by further addition of a water-in,w+.~b!e substrate which reduce,', the surface density of the inactivator molecules. With a hetcrogeneous system of this kind, typical of lipolytic systems, the use of the classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (K~, K., Irmax, IC5o) represent apparent values [21]. Consequently, the IC~. data are only empirical values and cannot be significantly and directly compared from one set of experimental conditions to another. Borgstr6m ([11] and Basle, Workshop on Lipases, April 27, 1989) using human pancreatic lipase, human pancreatic carboxyl ester lipase, human gastric lipase, bile-salt-stimulated tipase from human milk, hormonesensitive lipase and lipoprotein lipase showed that all
328 these s e r i n e / h i s t i d i n e hydrolases are inactivated at different levels by T H L T a k e n as a whole, these resuits support the view that T H L is a fairly general l i p a s e / e s t e r a s e inactivator. T h e situation s e e m s to be different with C~2:()-TNB, which specifically inactivates gastric lipases and had no effect on the pancreatic lipasc (in the presence of bile salts), w h a t e v e r the m e t h o d used (A, B or C).
Acknowledgements T h e authors are grateful to Professor A.E. Fischli (F. H o f f m a n La Roche, Basle, Switzerland) for the generous gift of T H L . We acknowledge the help of F. F e r r a t o (Marseille) with the PPL purification and of D~" J. Blanc (Marseille) for correcting the manuscript. This research was partly carried out with the financial support of Bridge-T-Lipase p r o g r a m m e of the European C o m m u n i t i e s u n d e r contract No. B I O T - C T g l 0274 ( D T E E ) .
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