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Comparative studies of human and porcine pancreatic lipases : N-terminal sequences, sulfhydryl groups and interfacial activity
BIOCHIMIE, 1981, 63, 799-801.
Comparative studies of human and porcine pancreatic lipases : N-terminal sequences, sulfhydryl groups and intcrfacial activity. Alain D E CARO *, Jacques BONICEL **, G6rard P I E R O N I ** and Odette GUY * <>. (Revue le 24-5-1981, acceptde aprds rgvision le 9-9-1981).
* Unitd de Recherches de Patho!ogie Digestive, I N S E R M U 31 - 46 boulevard de !a Gaye, 13009 Marseille. ** Centre de Biochimie et de Biologic Moldculaire, C N R S , 3 l Chemin Joseph Aiguier, 13009 Marseille.
Mots-cl~s : lipase / pancreas.
Key-words : lipase / pancreas.
Human pancreatic lipase consists of a single polypeptide chain of 48000 daltons [1]. A first comparison with other mammalian lipases such as porcine [2], bovine [3] and ovine [4] has shown a great similarity between these enzymes: same molecular weight, similar amino-acid composition and common antigenic determinants. However, in contrast with bovine and ovine, human and porcine lipases are glycosylated. Immunological crossreactions between these two lipases indicate only a partial identity between the two proteins [1]. This paper reports a comparison between human and porcine lipases concerning the N-terminal sequences, sulfhydryl groups reactivity, and enzymatic activity using substrate monolayers.
minal sequence was performed by the automatic sequential degradation of 0.2 ~mole of reduced and S-carboxy-methylated lipase. One degradation was carried out with a Socosi PS 100 sequencer using dimethyl benzylamine buffer [6]. A second experiment was performed with a Beckman 890 C sequencer using 0.l M quadrol buffer [7]. P T H ammo acids were identified by gas chromatography, thin-layer chromatography and, when necessary, by amino-acid analysis after regeneration of the parent amino-acid. In one experiment the identification was made by H P L C using Waters apparatus according to the conditions described [8]. Repetitive yields were 93 per cent and initial yield was 30 per cent. The titration of sulfhydryl groups was performed with DTNB at 25°C, according to Ellman [9], and the absorbance change was measured at 412 nm using 14 150 as molar extinction coefficient [10]. Reaction was made on 2530 nanomoles of lipase, using a 15-fold molar excess of reagent over enzyme. Titrations were carried out on native and denaturated enzyme, in the presence of 0.25 per cent sodium dodecyl sulfate. Lipase activity on monomolecular films was measured in a special zero order trough as described by Verger and De Haas [11], using the barostat method and 1,3 dicaprin as substrate [12].
Human pancreatic lipase was purified from pancreatic juicz, as previously described [1], except for the second step of purification which was a chromatography on CM-Sepharose instead of CMSephadex. Porcine colipase I + II was a generous gift from M Charles (C.N.R.S. Marsei!le). DTNB, 5-5'-dithiobis-(2-nitrobenzoic acid) was a product from Aldrich. 1,3 dicaprin was synthetized by M. Derbesy (Ecole Sup6rieure de Chimie de Marseille). Protein was reduced with mercaptoethanol in 8 M urea and subsequently alkylated with iodoacetate according to Crestfield et al. [5]. In one experiment 14C-labeled iodoacetate (105 d p m / umole) was used to facilitate the characterization of half-cystine residues. Carboxy-methylated protein was purified by filtration on a Sephadex G-200 column (H = 180 cm) equilibrated in a 0.5 per cent ammonium bicarbonate solution. The N-ter-
To whom all correspondence should be addressed.
Figure 1 shows the first 29 N-terminal amino acids of human lipase with 4 unidentified residues in position 15, 18, 23 and 26. Compared to the N-terminal sequence of porcine lipase [13], three substitutions are located near the N-terminal end of the polypeptide chain at positions 1, 5 and 6, and a conservative substitution is located at residue 21. These results show an extensive homology between the N-terminal regions of the two proteins. Particularly, the cysteinyl residues are at
800
A. de Caro and coll.
these results suggest that human lipase is more sensitive to interfacial denaturation than porcine lipaso. This sensitivity can explain the absence of
the same position, suggesting that the first disulfide bridge 4-10 (M. Rovery et al., unpublished) is conserved.
Human
NIl 2
GLu-Val-Cy
Ty
G1
Arg-Leu-Gly-Cys-Phe-Ser-Asp-Asp-
X -Pro-
Porcine
N H ~l-Ser~tC, l u - V a l - C v s4P h e 4 P roqA r g - L e u - G l y - C y s - P h e - S e r - A s p - A s p - A l a - P r o ZL___J ~ / / I I i 2 3 /4 5 6 7 8 9 i0 ii 12 13 i~ 15 16
tluman
Trp- X -Gly-IleIlleJGln- X -Pro-Leu- X -lle-Leu-Pro-
Porcine
Trp-Al a-G1y- Ile-[~J~G1n-Arg-P ro-Leu-Ly s- 1le-Leu-P to-
f"---3
I
17
18
19 20
I
21 22
23
2~
25
26
27
28
29
FIG. 1. - - N-terminal sequences of human and porcine lipases. Sequence of porcine lipase from [13].
0.57 and 1.73 SH groups have been titrated on the native and denatured human lipases, respectively. Similar values were obtained with porcine lipase, suggesting the presence of two SH groups, one of them buried inside the protein [14] The same conclusion is probably true for human lipase. As shown in figure 2 human lipase does not significantly hydrolyze dicaprin at pressures below 18 dynes/cm. Above this value, the activity increases rapidly and an 11-fold increase in activity can be noticed between 20 and 30 dynes/cm. This increase of activity with surface pressure is even larger when human lipase is assayed in the presence of porcine colipase. With colipase, the enzyme activity is detected at lower surface pressures (13 dynes/cm) and increases continuously up to 30 dynes/cm. The behaviours observed with the human and porcine lipases as a function of surface pressure are different. Porcine lipase without colipase exhibits enzymatic activity at surface pressures as low as 1 dyne/cm [15]. At this surface pressure, the activation factor, as defined by the ratio of the velocities with and without colipase, is as high as 25 (figure 3). Under the same experimental conditions, human lipase never reaches such a high activation factor but the human enzyme which is inactive at low surface pressures is stimulated 10fold by colipase at 16 dynes/cm. At this surface pressure there is only an activation factor of 2 of porcine lipase [15]. Since the difference observed between human and porcine lipases are well outside the range of between-experiments variability, BIOCHIMIE, 1981, 63, n ° 10.
activity at low surface pressure (high interfacial energy) (figure 2). The appreciable stimulation by colipase, even at high surface pressures (figure 3), can also be attributed to a protective effect against surface denaturation [15]. X 10 -zo
~,o 7
t/
/
/
E E
~, * o
e/
m ,.i
l
g
t
./ / ¢
S w
20
"
:
0
d"
/
./ ~.
w
/
/ o/f~
/
10
el
A--~---~? 10
.4"
. . . . . . . o15
1"
I
o~'/ I
20 25 SURFACE PRESSURE ( d y n e s / ¢ m )
I 3O
Fro. 2. - - Variation o] human lipase activity with surface pressure, using 1,3 dicaprin film as substrate at 25°C. The diagram represents the activity of 3 tag of lipase measured in the absence (© . . . . . ©) and in the presence (O . . . . . e ) of 36 lag of porcine colipase added 5 min before the injection of lipase.
Human pancreatic lipase. In conclusion, although human and porcine lipases present some structural homologies, some differences may exist in the binding site of the en-
801
Acknowledgements. The authors are glad to thank Dr. C. Figarella /o1" stimulating discussion. It is a pleasure to acknowledge Pro]. H. Sarles for his interest throughout this work and P. Couchoud /or her technical assistance. This research was supported by grant n ° 80.70.14 ]rom Ins'titut National de la Santd et de la Recherche M~dicale.
VL + COL VL
REFERENCES. 20
15
10
5
0
| 5
I 10
I 115 210 15 30 SURFACE PRESSURE (dynes/crn)
FIG. 3. - - Pressure dependence of the activation factor (Vx,4_cor,/Vr,) on the hydrolysis o / 1 , 3 dicaprin films by human (© ©) and porcine ( e e) lipases. The reaction rates (VL in the absence of colipase and VL4_co~, in the presence of cofactor) were derived from the maximal slope of the recorded curves. ( e e) data taken from reL [15].
zymes to lipid interfaces. This could explain the peculiar activity of human lipase at high surface pressures.
BIOCHIMIE, 1981, 63, n ° 10.
I. De Caro, A., Figarella, C., Amic, J., Michel, R. & Guy, O. (1977) Biochim. Biophys. Acta, 499, 411-419. 2. Semeriva, M. 8, Desnuelle, P. (1978) Adv. Enzymol., 48, 319-370. 3. Julien, R., Rathelot, J., Canioni, P., Sarda, L. 8, Plummer, T. H. (1975) Biochim. Biophys. Acta, 379, 157-163. 4. Canioni, P., Benajiba, A., Julien, R., Rathelot, J., Benabdeljlil, A. & Sarda, L. (1975) Biochimie, 57, 35-41. 5. Crestfield, A. M., Moore, S. & Stein, W. H. (1963) J. Biol. Chem., 238, 622-627. 6. Hermodson, M. A., Ericson, L. H., Titani, K., Neurath, H. 8, Walsch, K. A. (1972) Biochemistry, 11, 4493-4502. 7. Brauer, A. W., Margolies, M. N. & Haber, E. (1975) Biochemistry, 14, 3029-3035. 8. Bonicel, J., Couchoud, P., Foglizzo, E., Desnuelle, P. & Chapus, C. (1981) Biochim. Biophys. Acta, 669, 39-45. 9. Ellman, G. L. (1959) Arch. Bioctlem. Biophys., 82, 70-77. 10. Riddles, P. W., Blakeley, R. L. & Zerner, B. (1979) Anal. Biochem., 94, 75-81. 11. Verger, R. & De Haas, G. H. (1973) Chem. Phys. Lipids, 10, 127-136. 12. Rietsch, J., Pattus, F., Desnuelle, P. & Verger, R. (1977) J. Biol. Chem., 252, 4313-4318. 13. Bianchetta, J. D., Bidaud, J., Guidoni, A. A., Bonice[, J. J. ~ Rovery, M. (1979) Eur. J. Bioehem., 97, 395-405. 14. Verger, 1~., Sarda, L. & Desnuelle, P. (1971) Biochim. Bic)phys. Acta, 242, 580-592. 15. Verger, R., Rietsch, J. a Desnuelle, P. (1977) J. Biol. Chem, 252, 4319-4325.
Comparative studies of human and porcine pancreatic lipases : N-terminal sequences, sulfhydryl groups and intcrfacial activity. Alain D E CARO *, Jacques BONICEL **, G6rard P I E R O N I ** and Odette GUY * <>. (Revue le 24-5-1981, acceptde aprds rgvision le 9-9-1981).
* Unitd de Recherches de Patho!ogie Digestive, I N S E R M U 31 - 46 boulevard de !a Gaye, 13009 Marseille. ** Centre de Biochimie et de Biologic Moldculaire, C N R S , 3 l Chemin Joseph Aiguier, 13009 Marseille.
Mots-cl~s : lipase / pancreas.
Key-words : lipase / pancreas.
Human pancreatic lipase consists of a single polypeptide chain of 48000 daltons [1]. A first comparison with other mammalian lipases such as porcine [2], bovine [3] and ovine [4] has shown a great similarity between these enzymes: same molecular weight, similar amino-acid composition and common antigenic determinants. However, in contrast with bovine and ovine, human and porcine lipases are glycosylated. Immunological crossreactions between these two lipases indicate only a partial identity between the two proteins [1]. This paper reports a comparison between human and porcine lipases concerning the N-terminal sequences, sulfhydryl groups reactivity, and enzymatic activity using substrate monolayers.
minal sequence was performed by the automatic sequential degradation of 0.2 ~mole of reduced and S-carboxy-methylated lipase. One degradation was carried out with a Socosi PS 100 sequencer using dimethyl benzylamine buffer [6]. A second experiment was performed with a Beckman 890 C sequencer using 0.l M quadrol buffer [7]. P T H ammo acids were identified by gas chromatography, thin-layer chromatography and, when necessary, by amino-acid analysis after regeneration of the parent amino-acid. In one experiment the identification was made by H P L C using Waters apparatus according to the conditions described [8]. Repetitive yields were 93 per cent and initial yield was 30 per cent. The titration of sulfhydryl groups was performed with DTNB at 25°C, according to Ellman [9], and the absorbance change was measured at 412 nm using 14 150 as molar extinction coefficient [10]. Reaction was made on 2530 nanomoles of lipase, using a 15-fold molar excess of reagent over enzyme. Titrations were carried out on native and denaturated enzyme, in the presence of 0.25 per cent sodium dodecyl sulfate. Lipase activity on monomolecular films was measured in a special zero order trough as described by Verger and De Haas [11], using the barostat method and 1,3 dicaprin as substrate [12].
Human pancreatic lipase was purified from pancreatic juicz, as previously described [1], except for the second step of purification which was a chromatography on CM-Sepharose instead of CMSephadex. Porcine colipase I + II was a generous gift from M Charles (C.N.R.S. Marsei!le). DTNB, 5-5'-dithiobis-(2-nitrobenzoic acid) was a product from Aldrich. 1,3 dicaprin was synthetized by M. Derbesy (Ecole Sup6rieure de Chimie de Marseille). Protein was reduced with mercaptoethanol in 8 M urea and subsequently alkylated with iodoacetate according to Crestfield et al. [5]. In one experiment 14C-labeled iodoacetate (105 d p m / umole) was used to facilitate the characterization of half-cystine residues. Carboxy-methylated protein was purified by filtration on a Sephadex G-200 column (H = 180 cm) equilibrated in a 0.5 per cent ammonium bicarbonate solution. The N-ter-
To whom all correspondence should be addressed.
Figure 1 shows the first 29 N-terminal amino acids of human lipase with 4 unidentified residues in position 15, 18, 23 and 26. Compared to the N-terminal sequence of porcine lipase [13], three substitutions are located near the N-terminal end of the polypeptide chain at positions 1, 5 and 6, and a conservative substitution is located at residue 21. These results show an extensive homology between the N-terminal regions of the two proteins. Particularly, the cysteinyl residues are at
800
A. de Caro and coll.
these results suggest that human lipase is more sensitive to interfacial denaturation than porcine lipaso. This sensitivity can explain the absence of
the same position, suggesting that the first disulfide bridge 4-10 (M. Rovery et al., unpublished) is conserved.
Human
NIl 2
GLu-Val-Cy
Ty
G1
Arg-Leu-Gly-Cys-Phe-Ser-Asp-Asp-
X -Pro-
Porcine
N H ~l-Ser~tC, l u - V a l - C v s4P h e 4 P roqA r g - L e u - G l y - C y s - P h e - S e r - A s p - A s p - A l a - P r o ZL___J ~ / / I I i 2 3 /4 5 6 7 8 9 i0 ii 12 13 i~ 15 16
tluman
Trp- X -Gly-IleIlleJGln- X -Pro-Leu- X -lle-Leu-Pro-
Porcine
Trp-Al a-G1y- Ile-[~J~G1n-Arg-P ro-Leu-Ly s- 1le-Leu-P to-
f"---3
I
17
18
19 20
I
21 22
23
2~
25
26
27
28
29
FIG. 1. - - N-terminal sequences of human and porcine lipases. Sequence of porcine lipase from [13].
0.57 and 1.73 SH groups have been titrated on the native and denatured human lipases, respectively. Similar values were obtained with porcine lipase, suggesting the presence of two SH groups, one of them buried inside the protein [14] The same conclusion is probably true for human lipase. As shown in figure 2 human lipase does not significantly hydrolyze dicaprin at pressures below 18 dynes/cm. Above this value, the activity increases rapidly and an 11-fold increase in activity can be noticed between 20 and 30 dynes/cm. This increase of activity with surface pressure is even larger when human lipase is assayed in the presence of porcine colipase. With colipase, the enzyme activity is detected at lower surface pressures (13 dynes/cm) and increases continuously up to 30 dynes/cm. The behaviours observed with the human and porcine lipases as a function of surface pressure are different. Porcine lipase without colipase exhibits enzymatic activity at surface pressures as low as 1 dyne/cm [15]. At this surface pressure, the activation factor, as defined by the ratio of the velocities with and without colipase, is as high as 25 (figure 3). Under the same experimental conditions, human lipase never reaches such a high activation factor but the human enzyme which is inactive at low surface pressures is stimulated 10fold by colipase at 16 dynes/cm. At this surface pressure there is only an activation factor of 2 of porcine lipase [15]. Since the difference observed between human and porcine lipases are well outside the range of between-experiments variability, BIOCHIMIE, 1981, 63, n ° 10.
activity at low surface pressure (high interfacial energy) (figure 2). The appreciable stimulation by colipase, even at high surface pressures (figure 3), can also be attributed to a protective effect against surface denaturation [15]. X 10 -zo
~,o 7
t/
/
/
E E
~, * o
e/
m ,.i
l
g
t
./ / ¢
S w
20
"
:
0
d"
/
./ ~.
w
/
/ o/f~
/
10
el
A--~---~? 10
.4"
. . . . . . . o15
1"
I
o~'/ I
20 25 SURFACE PRESSURE ( d y n e s / ¢ m )
I 3O
Fro. 2. - - Variation o] human lipase activity with surface pressure, using 1,3 dicaprin film as substrate at 25°C. The diagram represents the activity of 3 tag of lipase measured in the absence (© . . . . . ©) and in the presence (O . . . . . e ) of 36 lag of porcine colipase added 5 min before the injection of lipase.
Human pancreatic lipase. In conclusion, although human and porcine lipases present some structural homologies, some differences may exist in the binding site of the en-
801
Acknowledgements. The authors are glad to thank Dr. C. Figarella /o1" stimulating discussion. It is a pleasure to acknowledge Pro]. H. Sarles for his interest throughout this work and P. Couchoud /or her technical assistance. This research was supported by grant n ° 80.70.14 ]rom Ins'titut National de la Santd et de la Recherche M~dicale.
VL + COL VL
REFERENCES. 20
15
10
5
0
| 5
I 10
I 115 210 15 30 SURFACE PRESSURE (dynes/crn)
FIG. 3. - - Pressure dependence of the activation factor (Vx,4_cor,/Vr,) on the hydrolysis o / 1 , 3 dicaprin films by human (© ©) and porcine ( e e) lipases. The reaction rates (VL in the absence of colipase and VL4_co~, in the presence of cofactor) were derived from the maximal slope of the recorded curves. ( e e) data taken from reL [15].
zymes to lipid interfaces. This could explain the peculiar activity of human lipase at high surface pressures.
BIOCHIMIE, 1981, 63, n ° 10.
I. De Caro, A., Figarella, C., Amic, J., Michel, R. & Guy, O. (1977) Biochim. Biophys. Acta, 499, 411-419. 2. Semeriva, M. 8, Desnuelle, P. (1978) Adv. Enzymol., 48, 319-370. 3. Julien, R., Rathelot, J., Canioni, P., Sarda, L. 8, Plummer, T. H. (1975) Biochim. Biophys. Acta, 379, 157-163. 4. Canioni, P., Benajiba, A., Julien, R., Rathelot, J., Benabdeljlil, A. & Sarda, L. (1975) Biochimie, 57, 35-41. 5. Crestfield, A. M., Moore, S. & Stein, W. H. (1963) J. Biol. Chem., 238, 622-627. 6. Hermodson, M. A., Ericson, L. H., Titani, K., Neurath, H. 8, Walsch, K. A. (1972) Biochemistry, 11, 4493-4502. 7. Brauer, A. W., Margolies, M. N. & Haber, E. (1975) Biochemistry, 14, 3029-3035. 8. Bonicel, J., Couchoud, P., Foglizzo, E., Desnuelle, P. & Chapus, C. (1981) Biochim. Biophys. Acta, 669, 39-45. 9. Ellman, G. L. (1959) Arch. Bioctlem. Biophys., 82, 70-77. 10. Riddles, P. W., Blakeley, R. L. & Zerner, B. (1979) Anal. Biochem., 94, 75-81. 11. Verger, R. & De Haas, G. H. (1973) Chem. Phys. Lipids, 10, 127-136. 12. Rietsch, J., Pattus, F., Desnuelle, P. & Verger, R. (1977) J. Biol. Chem., 252, 4313-4318. 13. Bianchetta, J. D., Bidaud, J., Guidoni, A. A., Bonice[, J. J. ~ Rovery, M. (1979) Eur. J. Bioehem., 97, 395-405. 14. Verger, 1~., Sarda, L. & Desnuelle, P. (1971) Biochim. Bic)phys. Acta, 242, 580-592. 15. Verger, R., Rietsch, J. a Desnuelle, P. (1977) J. Biol. Chem, 252, 4319-4325.