Eur. J. Biohem. 23, 892898 (1995) FEBS 1995 Kinetis of the twostep hydrolysis of triaylglyerol by panreati lipases Athanasios LYKDS, Vassilis MOUGOS and Pantelis ARZOGLOU Laboratory of Biohemistry, Department of Chemistry, Aristotle University of Thessaloniki, Greee Laboratory of Exerise Biohemistry, Department of Physial Eduation and Sports Siene, Aristotle University of Thessaloniki, Greee (Reeived 25 January/l6 Marh 1995) EJB 94 172/4 Panreati lipases atalyze the hydrolysis of triaylglyerol in a sequential manner. First, triaylglyerol is hydrolyzed to 1,2diaylglyerol, whih is subsequently onverted to 2monoaylglyerol. We studied the kinetis of trioleoylglyerol hydrolysis by rabbit and human panreati lipases. The produts (aylglyerols and fatty aid) were analyzed by extration from the reation mixture, separation by thinlayer hromatography, and quantifiation by apillary gas hromatography. The firstorder rate onstants of trioleoylglyerol and dioleoylglyerol hydrolysis were alulated showing that both enzymes hydrolyze dioleoylglyerol faster than trioleoylglyerol. Using rabbit panreati lipase, we found that deoxyholate enhaned dioleoylglyerol hydrolysis to a higher degree than trioleoylglyerol hydrolysis. Colipase inreased both rate onstants similarly at high deoxyholate onentrations (35 mm), while at low onentrations (5 mm) a seletivity toward trioleoylglyerol was observed. From the variation of the rate onstants with respet to temperature, we alulated the apparent ativation energies of trioleoylglyerol and dioleoylglyerol hydrolysis to be 59.8 kj. mol and 53.5 kj. mol, respetively. Upon storage, both rabbit and human panreati lipases showed a greater loss of ativity toward dioleoylglyerol as ompared to trioleoylglyerol, suggesting that different onformational elements of the enzyme moleule are responsible for the interation with eah substrate. Keywords. Panreati lipase ; kinetis ; rate onstants ; triaylglyerol ; diaylglyerol. Panreati lipases atalyze the hydrolysis of triaylglyerol produing 1,2diayglyerol, 2monoaylglyerol, and fatty aids. Lipases are ative at the oil/water interfae in heterogeneous reation systems (Brokman, 1984). One of the main harateristis of lipolysis by panreati lipases is that it takes plae in a sequential manner. First, a moleule of triaylglyerol is hydrolyzed yielding one moleule of fatty aid and one moleule of 1,2diaylglyerol, whih is subsequently hydrolyzed to 2monoaylglyerol and fatty aid. Therefore, 1,2diaylglyerol onstitutes both the initial produt of lipolysis and the substrate for the seond reation. Using porine panreati lipase, Constantin et al. (196) reported a transient aumulation of 1,2diaylglyerol and a late prodution of glyerol resulting from the leavage of 1monoaylglyerol after isomerization of 2monoaylglyerol. A matter of great interest for understanding the whole mehanism of lipolysis, is the kinetis of the hydrolysis of diaylglyerol subsequent to the degradation of triaylglyerol. Despite intense researh in the general field of lipases in reent years, the kinetis of this reation remain unlear. Using triotanoylglyerol and 1,2diotanoylglyero1 monolayers, Lagoki et al. (1 973) reported that the hydrolysis of the diester to 2monootanoylglyerol by porine panreati lipase was slower than hydrolysis of the triester. The latter study was made in the absene Correspondene to P. Arzoglou, Laboratory of Biohemistry, Department of Chemistry, Aristotle University of Thessaloniki, GR546 Thessaloniki, Greee Fax: +31 997689. Abbreviations. RPL, rabbit panreati lipase; HPL, human panreati lipase. Enzyme. Panreati lipase (EC 3.1.1.3). of any ofators and the rates of diaylglyerol and triaylglyerol hydrolysis were measured in separate experiments. n the present study, we have onsidered lipolysis to be a ommon hemial sequential reation aording to the sheme : kl k2 (ayl),gro(ayl),gro aylgro, (1) where (ayl),gro, (ayl),gro and aylgro represent triaylglyerol, diaylglyerol, and monoaylglyerol, respetively, and k, and k, are rate onstants. By determining the onentrations of the reatants at different time points we were able to alulate k, and k2. This approah has already been applied to the study of kinetis of human milk bilesaltativated lipase (Wang et al., 1988). We have also investigated the effet of bile salt and olipase (two main ofators of intestinal lipolysis) on the rates of triaylglyerol and diaylglyerol hydrolysis. MATEKALS AND METHODS Materials. Rabbit panreas aetone powder, trioleoylglyerol, dioleoylglyerol, triheptadeanoylglyerol, heptadeanoi aid, sodium deoxyholate, sodium taurodeoxyholate and siliagel thinlayerhromatography (TLC) plates were purhased from Sigma. Colipase was from Boehringer Mannheim. Organi solvents were from Riedelde Haen. 1,2Diheptadeanoyglyerol, 1,3diheptadeanoylglyerol and monoheptadeanoylglyerol were from Larodan. Lipases. Rabbit panreati lipase (RPL) was extrated from 1 g rabbit panreas aetone powder by ontinuous stirring in 3 ml 2 mm sodium aetate, ph 4., at 4 C for 6 min. After entrifugation at 1 g for 1 min, the ph of the supernatant was adjusted to 5. with NaOH. The preparation was loaded onto an 8 mx2.5 m CM 52 olumn already equilibrated at
Lykidis et al. (Em J. Biohern. 23) 893 ph 5.. Lipase ativity was eluted from the olumn with a NaCl gradient (.3 M) and was preipitated with (NH4),S4; the 25% pellet was olleted and suspended in 1 ml 3 mm Tris/HC, ph 9. (buffer A). All subsequent steps were arried out with the aid of this buffer. The enzymeontaining preparation was loaded on a 55 mxl m gelfiltration olumn (Ultrogel AA 44). The eluate was passed through two hydrophobi olumns : a 4 mx 1.4 m phenylsepharose olumn, whih did not retain the lipolyti ativity, and a 3 mx1.4 m otylsepharose olumn, from whih the enzyme ativity was eluted with 4 mm sodium taurodeoxyholate in buffer A. Finally, an anionexhange hromatography olumn (5 mx 1.4 m, DEAE Trisaryl), previously equilibrated with 4 mm taurodeoxyholate in buffer A, was employed. The lipolyti ativity was eluted by applying a NaCl gradient (.3 M). Protein was determined aording to Bradford (1976). Human panreati lipase (HPL) was purified aording to a modified version of De Caro's proedure (Tavridou et al., 1992). The purified enzymes were stored at 4 C. Lipase assay. A rapid turbidimetri method (Arzoglou et al., 1989) was employed to assess the lipolyti ativity of frations during the purifiation proedure. The standard reation mixture (1 ml) ontained.4 mm emulsified trioleoylglyerol, 3 mm Tris/HC, ph 9.,.3 mg/l olipase and 35 mm sodium deoxyholate. The derease in absorbane was measured at 365 nm. 1 U lipase is defined as the amount of enzyme liberating 1 pmol fatty aid/min. Preparation of emulsions. The proess for obtaining trioleoylglyerol emulsions has already been desribed (Tavridou et al., 1992). This method produes emulsions of homogeneous partile size (average droplet diameter 2.7 pm with less than 2% of droplets larger than 1 pm). Briefly, a stok emulsion of 1 mm trioleoylglyerol was prepared by adding an appropriate amount to a 12 g/l hydroxypropylmethylellulose. After shaking by hand, the suspension was soniated twie for 3 s with a 3s interval in a Minisoni 4 ultrasoni laboratory homogenizer. A stok emulsion of dioleoylglyerol was prepared in the same way exept that it was soniated in a different apparatus, Soniprep 15, beause of the smaller volume needed. Lipid analysis. Unless otherwise indiated, the reation mixture for analysis of the omponents of lipolysis (5 ml) ontained 2 mm trioleoylglyerol, 35 mm sodium deoxyholate,.3 mgfl olipase, and 3 mm 3[(l,ldimethyl2hydroxyethyl) amino]2hydroxypropanesulfoni aid, ph 9., at 37 "C. The reation was initiated by the addition of 1 pg RPL or 2 pg HPL. To quantify trioleoylglyerol, 1,2dioleoylglyerol, 1,3dioleoylglyerol, monooleoylglyerol, and olei aid we followed a proedure onsisting of four steps: extration of lipids from the reation mixture, separation of lipid lasses by TLC, preparation of fattyaid methyl esters and apillary gas hromatography. Lipid extration was based on the proedure of Dole (1956)..25ml aliquots were removed from the reation mixture at several time points and were mixed with 1.25 ml 4: 1O:l (by vol.) isopropanol/heptane/os M sulfuri aid ontaining triheptadea noylglyerol, 1,2diheptadeanoylglyerol, 1,3diheptadeanoylglyerol, monoheptadeanoylglyerol, and heptadeanoi aid as internal standards. After 1 min,.5 ml heptane and.75 ml water were added for phase separation. The two phases were mixed vigorously for 1 min and the upper phase was removed. Heptane was evaporated under nitrogen and the residue was redissolved in 5 pl 2: 1 (by vol.) hlorofodmethanol. 7 p1 were spotted onto a TLC plate whih was then developed with 8: 2 :1 (by vol.) petroleum etheddiethyl ethedaeti aid. Lipids were made visible under ultraviolet light after spraying with dihlorofluoresein. The retention fator of triaylglyerols was.87, fatty aids.71, 1,3diaylglyerols.25, 1,2diaylglyer Table 1. Purifiation steps of rabbit panreati lipase. Step Ativ Protein Speifi Purifi Reovity ativity ation ery U mg U/mg fold % Extration 814 54 15 1 1 CM 52 592 12 49 3 12 Ultrogel AA 44 493 1.6 38 2 6 PhenylSepharose 358.8 448 3 44 OtylSepharose 255.4 631 42 31 DEAETrisaryl 2.3 661 44 24 1s.16, and monoaylglyerols.2. ndividual spots were sraped off and inubated in 96:4 (by vol.) methanollsulfuri aid at 6 C overnight. The fattyaid methyl esters thus produed were extrated with petroleum ether. After evaporation under nitrogen, the residue was redissolved in arbon disulfide and 1 pl was injeted into a Hewlett Pakard 589 series 1 gas hromatograph equipped with a 3m long Carbowax apillary olumn from Allteh and a flameionization detetor. The olumn temperature was 21 C and the arrier gas was helium at a flow rate of 1.5 dmin. The retention times of methyl heptadeanoate and methyl oleate were 3.3 min and 4.1 min, respetively. The mass of methyl oleate was alulated by omparing the area under its peak in the hromatogram to that of methyl heptadeanoate. Finally, from the amount of methyl oleate orresponding to eah TLC spot, we alulated the amount of trioleoylglyerol, dioleoylglyerol, monooleoylglyerol, and olei aid in eah sample removed from the reation mixture. n addition, we alulated any amount of glyerol produed by applying the following reasoning : the initial amount of trioleoylglyerol in eah sample equals the sum of trioleoylglyerol, dioleoylglyerol, and monooleoylglyerol determined plus any glyerol produed. t also equals one third the sum of all oleoyl groups and olei aid determined. Therefore, the amount of glyerol an be alulated as the differene between the latter and the sum of trioleoylglyerol, dioleoylglyerol, and monooleoylglyerol. Determination of rate onstants. Assuming firstorder kinetis for the sequential reation 1, the equations providing the onentrations of (ayl),gro, (ayl),gro, and aylgro during the ourse of the reation are (Roberts, 1977) : [(ayl),gro] = [(ayl),gro], e'l', (2) [aylgro] = [(ayl),gro],., [(ayl),gro], is the initial onentration of (ayl),gro, and the initial onentrations of (ayl),gro and aylgro are zero. k, was determined by fitting the experimentally determined onentrations of trioleoylglyerol to Eqn (2) with the aid of the MiroCal Origin program (MiroCal Software, Northampton, MA). Using this value, and fitting the experimentally determined onentrations of dioleoylglyerol to Eqn (3), we alulated k,. All results reported are representative of at least two experiments. RESULTS Data on the purifiation of RPL are summarized in Table 1. The enzyme preparation obtained appears as one band after SDSPAGE (Fig. 1); its moleular mass was estimated to be
894 Lykidis et al. (Eur. J. Biohern. 23) 1 2 3 4 5 6 7 8 kda t 66 t 45 t 36 t 29 t 2 t 14 Fig. 1. SDSRAGE (1 % polyarylamide) patterns (silver staining) of rabbit panreati lipase at various purifiation steps. Lane 1, initial extrat; lane 2, preparation after adjusting to ph 5.; lane 3, frations ontaining lipolyti ativity after CM 52; lane 4, ammonium sulfate preipitate; lanes 5 8, frations after Ultrogel AA 44, phenylsepharose, otylsepharose, and DEAETrisaryl hromatographies. 46 kda. The proedure desribed results in a 24% yield and a 44fold enrihment ompared to the initial extrat. Kineti analysis of lipolysis by panreati lipases. Fig. 2A and B presents the time ourse of trioleoylglyerol hydrolysis by RPL and HPL, respetively. Trioleoylglyerol, 1,2dioleoylglyerol and monooleoylglyerol are expressed as molar frations of the initial trioleoylglyerol onentration. No prodution of 1,3 dioleoylglyerol or glyerol was observed. With both enzymes one an note that, as the onentration of trioleoylglyerol dereased, there was a transient aumulation of 1,2dioleoylglyerol and a ontinuous inrease in monooleoylglyerol onentration. This profile is typial of sequential reations with rate onstants of the same order of magnitude. Plots of ln[(ayl),gro] versus time (data not shown) were linear for both RPL and HPL (Y >.99), indiating that trioleoylglyerol hydrolysis is first order with respet to trioleoylglyerol. The slopes of these urves yield the experimental rate onstant, kl, for RPL and HPL. However, this methodology is not appliable to the determination of k, sine the onentration of 1,2dioleoylglyerol throughout lipolysis depends on both its hydrolysis and prodution rate. Therefore, another approah is neessary. Based on the firstorder dependene of trioleoylglyerol hydrolysis on trioleoylglyerol onentration, we made the assumption that hydrolysis of 1,2dioleoylglyerol also obeyed firstorder kinetis with respet to 1,2dioleoylglyerol onentration. The kineti equations desribing two firstorder reations in sequene are Eqns (24). Fitting the experimental values of the relative onentration of trioleoylglyerol during hydrolysis by RPL to Eqn (2), we determined k, to be.276 2.4 min' (estimate 2 standard error throughout). Using this value and fitting the experimental values of the relative onentration of 1,2dioleoylglyerol to Eqn (3) we found k,to be.243 2.246 min. The urves of Fig. 2A show the time ourses produed from Eqns (24) when k, and k, are replaed by the values determined above. The oeffiients of variation between the experimental and theoretial values were 1.7% for (ayl),gro, 22.% for (ayl),gro, and 4.% for ayl Gro. The ratio of k, to k, is 8.8 and equals the ratio of the respetive reation rates if the onentrations of trioleoylglyerol and 1,2dioleoylglyerol are equal. Thus, under the partiular experimental onditions, RPL hydrolyzes dioleoylglyerol approximately ninefold faster than trioleoylglyerol. The halflives of trioleoylglyerol and dioleoylglyerol derived from the above rate onstants (ln2/k) are 25.1 min and 2.9 min, respetively. Following the above steps in the ase of HPL, we obtained values of k, =.13 +.2 min' and k, =.36+.22 minp indiating that HPL hydrolyzes 1,2dioleoylglyerol 2.8 fold faster than trioleoylglyerol. n agreement with Fig. 2A, the urves of Fig. 2B represent the theoretial time ourses. The oeffiients of variation between the experimental and theoretial values were 1.2% for (ayl),gro, 8.7% for (ayl),gro and 15.3% for aylgro. The halflives of trioleoylglyerol and 1,2 dioleoylglyerol were 53.3 min and 19.3 min, respetively. k, an also be alulated from Eqn (4). The values determined in this way for both RPL and HPL were similar to the ones derived from Eqn (3), differing by 4.5% and 3.3%, respetively. 5 1.oo.75._ + 2 C al.5 al. > m 2.25. :? &. %J A /'. \ '.,., 2 4 6 Time (min) Fig. 2. Time ourse of hydrolysis of trioleoylglyerol (2 mm) by panreati lipases at ph 9. and 37"C, in the presene of 35 mm sodium deoxyholate and.3 mgh olipase. (A) Rabbit panreati lipase, 2 mg/l. (B) Human panreati lipase,.4 mga. Data are representative of two independent experiments. Symbols indiate the experimentally determined relative onentrations of trioleoylglyerol (M), 1,2dioleoylglyerol (O), and monooleoylglyerol (A), whereas urves orrespond to onentrations alulated from Eqns (24) after determination of the rate onstants by fitting the experimental data.
h.2 7. C E v. t m v) C.1 al 2 " * Lykidis et al. (EUK J. Biohem. 23) 895. * / 1 2 3 4 [Enzyme] (nm) Fig. 3. Effet of rabbit panreati lipase onentration on k, and k,. Reations were performed with an initial onentration of 2 mm trioleoylglyerol at ph 9. and 37"C, in the presene of 35 mm sodium deoxyholate and.3 mgll olipase. k drolysis on enzyme onentration. The slopes of the lines of Fig. 3 are the seondorder rate onstants, kayl)3gro and k(uyl)2gro : d [(ayl),gro dt k(ayl)jcro [El [(ayl),gro k~ayl)2(jro [El [(ayl),grol. (6) The values of these onstants were k(ayl)3ro = 6.1 X lo5 M'. min' and kayl)2gro = 5.6X16M'. min'. Having determined the rate onstant of dioleoylglyerol hydrolysis ourring sequentially to trioleoylglyerol hydrolysis, we determined the rate onstant in the presene of 1,2dioleoylglyerol alone. For this purpose, using RPL, we substituted 1,2 dioleoylglyerol for trioleoylglyerol in the reation mixture. Again, hydrolysis was first order with respet to 1,2dioleoylglyerol. When we fitted the data to the following equation : [(ayl),gro = [(ayl),grol, kir, (7) we found k$ to be 83% of k, determined under idential onditions. Although k2 is higher than kl with both lipases studied, the rate of trioleoylglyerol hydrolysis is higher than that of dioleoylglyerol hydrolysis throughout most of the reation period beause the onentration of trioleoylglyerol is far greater than that of dioleoylglyerol. This relationship between the rates is reversed when k,[(ayl),gro] = k,[(ayl),gro]. By replaing [(ayl),gro] and [(ayl),gro] with the mathematial expressions of Eqn (2) and Eqn (3) one an alulate that the reversal takes plae at 1.1 min for RPL and 44.3 min for HPL. To establish that the defined parameters, k, and k,, are reliable markers of lipase ativity, we heked their dependene on enzyme onentration. Fig. 3 shows the values of k, and k, in the presene of different RPL onentrations. Both rate onstants exhibited a linear inrease with inreasing enzyme onentrations (r =.99); therefore these rate onstants reflet enzymi atalysis. Moreover, the linearity indiates a firstorder dependene of the rates of trioleoylglyerol and dioleoylglyerol hy Effet of deoxyholate on k, and k,. Fig. 4A shows how the two rate onstants hanged when the deoxyholate onentration in the reation mixture was dereased from 35 mm to 5 mm. Both k, and kz dereased, but not to the same extent; while k, dereased nearly 11 fold, kz underwent a 33fold derease. Consequently, the ratio, k,/kl, dereased from 9, with 35 mm deoxyholate, to 3, in the presene of 5 mm deoxyholate (Fig. 4B). These data suggest that deoxyholate enhanes hydrolysis of dioleoylglyerol preferentially ompared to the hydrolysis of trioleoylglyerol. Effet of olipase on k, and k,. Fig. 5 depits the effet of olipase onentration on k, and k, at two deoxyholate onentrations, 35 mm and 5 mm. Both k, and k2 dereased when we dereased the olipase onentration from.3 mg/l to.6 mg/l; in the presene of 35 mm deoxyholate, k, and k, dereased 4.3 fold and 4.7fold, respetively, whereas at 5 mm deoxyholate 1 h. C E C m v).2 8.1 6 4. 1 1 2 [Sodium deoxyholate] (rnm) Fig. 4. Effet of deoxyholate onentration on k, and k, (A), and kjk, (B) (semilogarithmi plots). Reations were performed with an initial onentration of 2 mm trioleoylglyerol at ph 9. and 37 C in the presene of.3 mg/l olipase and 2 mg/l rabbit panreati lipase.
896 Lykidis et al. (Eu,: J. Biohem. 23) / 3.2 A B '" (u.1 2 2. =, v 1.1 Colipase (mgll).1.1 Colipase (mg/l). 1. 1.1 Colipase (rng/l) Fig. 5. Effet of olipase onentration on k, and k2 (semilogarithmi plots). Reations were performed with an initial onentration of 2 mm trioleoylglyerol at ph 9. and 37 C in the presene of 2 mgfl rabbit panreati lipase and (A) 35 mm sodium deoxyholate or (B) 5 mm deoxyholate. C, ratio of k2 and k, at 35 mm (A) and 5 mm deoxyholate (A). the orresponding values were 3. and 2.. The ratios of k2 to k, (Fig. 5C) remained relatively unhanged with 35 mm deoxyholate, with values in the range 8.29, suggesting a similar effet of olipase on trioleoylglyerol and dioleoylglyerol hydrolysis. At 5 mm deoxyholate, kjk, inreased from 3.3 to 4.9 as the olipase onentration was dereased, indiating a slight seletive stimulation of k, by olipase at low deoxyholate onentrations. Effet of temperature on k, and k,. Fig. 6 shows the dependene of k, and k, on temperature. There was a ontinuous inrease of both rate onstants between 25 C and 37 C. The plots of log(rate onstant) versus 1/T were linear (r>.99 for both k, and k2) at 2533 C. When we also onsidered the values at 37"C, linearity was less satisfatory ( =.97 for k, and.96 for k2). The linearity of log(rate onstant) versus 1T onforms with the Arrhenius law. From the slopes of the lines (based on the data at 2533"C), we alulated the apparent ativation energy for trioleoylglyerol hydrolysis to be 59.8 kj. mol' and for dioleoylglyerol hydrolysis, 53.5 W. mol'. Effet of enzyme ageing on k, and k,. During repetitive determinations of k, and k, we made a startling observation. As expeted, upon ageing, our enzyme preparations underwent partial inativation manifested by a derease in the rate onstants. Quite unexpetedly, though, the relative dereases of k, and k2 were different. Four months after its purifiation, RPL had lost 6% 1.6.'"'''''. 3.2 3 24 3.28 ooo/t (K1) Fig. 6. Effet of temperature on k, and k,. Reations were performed with an initial onentration of 2 mm trioleoylglyerol at ph 9. in the presene of 35 mm sodium deoxyholate,.3 mg/l olipase, and 2 mg/l rabbit panreati lipase. of its ativity toward trioleoylglyerol and 74% of its ativity toward dioleoylglyerol. As a result, the ratio of k2 to k, dereased from 8.8 to 5.6. n the ase of HPL, the effet was even more pronouned. Three months of ageing resulted in a loss of 28 % of its ativity toward trioleoylglyerol and 78 % of its ativ 3 32 3.36
Lykidis et al. (Eul: J. Biohem. 23) 897 ity toward dioleoylglyerol, and kdk, dereased from 2.8 to.9. This finding neessitated the use of fresh (up to onemonth old) enzyme preparations. DSCUSSON Panreati lipase has already been purified and haraterized from various speies. The gene sequene of RPL was revealed reently (AlemanGomez et al., 1992), although no purifiation of the enzyme was reported. Following the proedure desribed in the Materials and Methods setion we purified this enzyme to apparent homogeneity and studied some of its physial properties. The moleular mass determined is onsistent with the sequene of the gene. The degree of purifiation and the final yield are omparable to those of panreati lipases from other soures. We also purified HPL aording to an established proedure for omparison to RPL. We have studied the ation of both lipases against emulsified triaylglyerol. The time ourses obtained show similarities with the previously studied porine panreati lipase, i.e. exponential deay of triaylglyerol and transient aumulation of 1,Zdiaylglyerol as well as no prodution of 1,3diaylglyerol or glyerol. Having determined the hanges in the relative onentrations of the omponents of lipolysis with time and by applying the alulus of sequential reations to the data of Fig. 2, we determined the firstorder rate onstants for the hydrolysis of triaylglyerol and diaylglyerol. Our results indiate that the kineti equations of sequential reations provide a satisfatory desription of the whole proess of lipolysis. The approah of sequentialreation kinetis has also been followed in the ase of other lipolyti enzymes. Wang et al. (1988) used the kineti equations for sequential reations to investigate the effet of tauroholate on human milk bilesaltativated lipase. Muderhwa et al. (1992) studied the exhange of " between water and fatty aids atalyzed by arboxylester lipase. We therefore believe that kineti equations for sequential reations are useful tools in studying lipolyti reations. We have alulated the rate onstant of dioleoylglyerol hydrolysis (k2) to be higher than that of trioleoylglyerol hydrolysis (k,) with both lipases tested. Additionally, when dioleoylglyerol alone was the substrate for panreati lipase the rate onstant determined was similar to k2, remaining higher than k,. This finding ontrasts with the report of Lagoki et al. (1973) on the faster hydrolysis of triotanoylglyerol ompared to diotanoylglyerol by porine panreati lipase. That study employed the monolayer tehnique in the absene of bile salts, whih we show to favor the hydrolysis of diaylglyerol in the present study. The nature of the ayl groups attahed to glyerol, ph, ioni strength, et. ould also modulate the ratio of k2 to k,. Further studies are therefore needed to determine the fators regulating this ratio. Lipolysis an be divided into three events. These inlude substrate partitioning to the lipid/water interfae, enzyme partitioning, and atalysis at the interfae (Brokman, 1984). Therefore, the seondorder rate onstants k~ayl),(jro and k(ay,)2(iro that we determined should be onsidered as apparent rate onstants of the proess. The differene between k(ayl)goro and k(ryl)2gro will orrespond to differenes in the interation between lipase and substrate if triaylglyerol and diaylglyerol are topologially equivalent in the heterogeneous reation mixture, meaning that, after being produed, diaylglyerols do not leave the interfae but are arranged on it together with the remaining triaylglyer 1s. This has been verified by other researhers. Patton and Carey (1979) showed that diaylglyerol formed during fat digestion by human panreati lipase, in the presene of bile salts and olipase, remains in the oil phase while monoaylglyerol enters the aqueous phase. Lagoki et al. (1973) studied the hydrolysis of triotanoylglyerol and the partition of 1,2diotanoylglyerol between the lipid and the aqueous phase. They onluded that the diester remains on the surfae, while both 2 monotanoylglyerol and otanoi aid enter the aqueous phase. Finally, Sow et al. (1979) indiated that diaylglyerol formed by the ation of lipoprotein lipase remains and spreads at the interfae. Consequently, the assumption of topologial equivalene between triaylglyerol and diaylglyerol seems to hold and the relationship between k~ayl)3m,ro and k(ayl)2ro may reflet the relationship between the true rate onstants of the reations. Bile salts and olipase are main ofators of panreati lipases. Their presene is known to inrease rates of lipolysis with no distintion made between the effets on eah of the two reations. After validation of the kineti sheme desribed by the sequentialreation model, investigation on the effet of these ofators on eah reation was feasible. We found deoxyholate and olipase to enhane both trioleoylglyerol and dioleoylglyerol hydrolysis, as manifested by the inrease in k, and k2 (Fig. 4A, Fig. 5A and B). t was, therefore, of interest to investigate whether deoxyholate and olipase exerted any seletive influene on trioleoylglyerol or dioleoylglyerol hydrolysis. ndeed, deoxyholate enhanes dioleoylglyerol hydrolysis seletively resulting in an inrease of kjk, (Fig. 4B). n ontrast, olipase (Fig. 5C) does not alter k,lk, onsiderably at high onentrations of bile salt. At low onentrations of bile salt, however, olipase seems to favor trioleoylglyerol hydrolysis. Several mehanisms for the influene of bile salts on panreati lipase ativity have been proposed. These inlude stabilization of the enzyme moleule (Momsen and Brokman, 1976), removal of the fatty aids produed from the interfae (Borgstrom, 1964), modulation of the adsorption of lipase to the lipid/ water interfae (Lairon et al., 1978, 198), regulation of the onformational status of substrate at the interfae (Momsen et al., 1979). Bile salts ould also regulate the partitioning of the substrate between the lipid bulk phase and the surfae phase and the onformation of lipase at the interfae. The effet of bile salts on stabilization of the enzyme, removal of the produts, and adsorption of the enzyme is not expeted to alter k, or k2 seletively. Therefore, the differential effet of deoxyholate on the rate onstants should be attributed to seletive alteration of the interation between enzyme and trioleoylglyerol or dioleoylglyerol or of the partitioning of the two substrates. At present we are unable to distinguish between these possibilities. Colipase, however, is believed to anhor panreati lipase (Verger et al., 1977) to the lipid/water interfae. t also protets lipase from interfaial denaturation (Momsen and Brokman, 1976). Aording to these findings, olipase would be expeted to alter k, and k, uniformly. This is what we found at high onentration of deoxyholate. There is no obvious explanation for the preferential effet of olipase on k, at low deoxyholate onentration. From the variation of k, and k, with respet to temperature (Fig. 6), we alulated the apparent ativation energies for trioleoylglyerol and dioleoylglyerol hydrolysis aording to the Arrhenius law. These energies inlude the true ativation energy of eah reation, the energy of adsorption of lipase to the interfae and the energy of transloation of the substrate from the bulk lipid phase to the interfae. Considering the topologial equivalene of triaylglyerol and diaylglyerol, we may assume that the seond and third of the above fators ontribute equally to the apparent ativation energies. Consequently, the differene in the experimentally determined values may reflet the differene in the true ativation energies of triaylglyerol and diaylglyerol hydrolysis.
898 Lykidis et al. (EuP: J. Biohem. 23) The main findings of the present study are summarized as follows : (a) RPL hydrolyzes dioleoylglyerol faster than trioleoylglyerol; (b) deoxyholate enhanes dioleoylglyerol hydrolysis preferentially ; () olipase leads to a similar inrease in the rate of both reations at high deoxyholate onentration in ontrast to a slight seletivity toward trioleoylglyerol at low deoxyholate onentration ; (d) upon storage the enzyme suffers a greater loss of ativity toward dioleoylglyerol as ompared to trioleoylglyerol ; (e) kineti properties and stability patterns appear to be very similar between RPL and HPL. Overall, our results indiate intrinsi differenes in the hydrolysis of triaylglyerol and diaylglyerol by panreati lipases. Further studies are needed to eluidate the exat moleular events responsible for these differenes. This study was funded in part by the Community Bureau of Referene (BCR) of the European Union. REFERENCES AlemanGomez, J. A,, Colwell, N. S., Sasser, T. & Kumar, V. B. (1992) Biohem. Biophys. Res. Commun. 188, 964971. Arzoglou, P. L., Tavridou, A. & Balaska, C. (1989) Anal. Lett. 22, 14591471. Borgstrom, B. (1964) J. Lipid Res. 5, 522531. Bradford, M. M. (1976) Anal. Biohem. 72, 248254. Brokman, H. L. (1984) in Lipuses (Borgstrom, B. & Brokman, H. L., eds) pp. 3 46, Elsevier, Amsterdam. Constantin, M. J., Pasero,. & Desnuelle, P. (196) Biohim. Biophys. Atu 43, 1319. Dole, V. P. (1956) J. Clin. nvest. 35, 15154. Lagoki, J. W., Law, J. H. & Kezdy, F. J. (1973) J. Biol. Chem. 248, 58587. Lairon, D., Nalbone, G., Lafont, H., Leonardi, J., Domingo, N., Hauton, J. C. & Verger, R. (1978) Biohemistry 17, 2528. Lairon, D., Nalbone, G., Lafont, H., Leonardi, J., Vigne, J.L., Chabert, C., Hauton, J. C. & Verger, R. (198) Biohim. Biophys. Atu 618, 119 128. Momsen, W. E. & Brokman, H. L. (1976) J. Biol. Chem. 251, 378 383. Momsen, W. E., Smaby J. M. & Brokman, H. L. (1979) J. Bid. Chem. 254, 8855886. Muderhwa, J. M., Shmid, P. C. & Brokman, H. L. (1992) Biohemistry 31, 141 148. Patton, J. S. & Carey, M. C. (1979) Siene 24, 145148. Roberts, D. V. (1977) in Enzyme kinetis, pp. 1214, Cambridge University Press, Cambridge. Sow, R. O., Desnuelle, P. & Verger, R. (1979) J. Biol. Chenz. 254, 6456 6463. Tavridou, A,, Avranas, A. & Arzoglou, P. (1992) Biohem. Biophys. Res. Commun. 186, 746752. Verger, R., Rieth, J. & Desnuelle, P. (1977) J. Biol. Chem. 252, 4319 4325. Wang, C.S., Hartsuk, J. A. & Downs, D. (1988) Biohemistry 27, 4834488.