Interactions of serum albumin and other proteins with porcine pancreatic lipase

00166086/78/7603-0382$02.00/O GASTEO~NTE~OLQGY 75:382-386, 1978 Copyright Q 1978 by the American Gadmenterological

Vol. 75, No. 3

Association

Printed in U.SA.

INTERACTIONS OF SERUM ALBUMIN AND OTHER PROTEINS WITH PORCINE PANCREATIC LIPASE BENGTBORGSTR~M, M.D., ANDCHARLOTTE ERLANSON, M.D. Department of Physiological Chemistry, University of Lund, Lund, Sweden

Pancreatic lipase is an interfacial enzyme that binds to the oil-water interface of its emulsified triglyceride substrate. Its catalytic activity is inhibited by “hydrophobic” proteins, such as serum albumin and p-lactoglobulin. The inhibition is an effect of a competition for the interface, and lipase is shown to have a -5O- to loo-fold higher affinity for the interface than these proteins, although lipase does not show hydrophobic interactions by other criteria. This indicates that the binding of lipase to its substrate may not only be based on hydrophobic interactions, but also on polar interactions which give specificity and additional strength to the binding. Bile salts compete with proteins including lipase for the substrate interface and in concentrations above the critical micellar concentration clear the interface of proteins. A specific reunion of lipase with the substrate and a restoration of catalytic activity in the presence of bile salts is affected by colipase even in the presence of high concentrations of serum albumin and p-lactoglobulin. It is postulated that an important function of bile salts in the intestinal lumen is to clear the interface of the dietary fat from proteins of exo- and endogeneous sources thus making it available for pancreatic lipolysis. Serum albumin has been shown to interact with pancreatic lipase on emulsified substrates in two different ways. At low concentrations (lo-’ M) it protects lipase from irreversible inactivation; at higher concentrations (>lO* M) it inhibits its catalytic activity on the substrate.‘, 2 These effects have been ascribed to the tendency of albumin to adsorb to the substrate interface; at low concentration lipase is protected from unfolding at the interface, and at higher concentration the interface is blocked. The inhibition of lipase by albumin, furthermore, was found to be reversed by deoxycholate, an effect that was not explained at that time.’ Bile salts are now known to inhibit purified lipase by displacing it from the substrate interface.3 Colipase, contaminating lipase preparations previously used,‘, * reactivates lipase in the presence of bile salts4 In view of these findings, it seemed of interest to reinvestigate these physiologically important complex interactions at the lipase substrate interface. l Chemicals.

Materials and Methods chemicalswere purchasedas follows: tri-

The

butyrylglycerol (Merck, Germany), n-decane (Fluka AG Chemische Fabrik, Buchs, Switzerland), dextran-500 (Pharmacia, Sweden) polyethyleneglycol (PEG) (Carbowax 6000, Union Carbide Corp., New York, N. Y.). Taurodeoxycholate (TDC) was synthesized in this laboratory and was >98% pure as indicated by thin layer chromatography.5 The palmitic acid

ester of PEG (P-PEG) was kindly supplied by Dr. P. A. Albertsson, Department of Biochemistry, Chemical Center, Lund, Sweden.

Proteins. Porcine pancreatic lipase B (MW 52000) was purified as describedby Donn6r.6Colipase (MW 11000) was

purified as previously described.’ Bovine serum albumin (MW 66000) was freed of fatty acids as described.8 p-Lactoglobulin (MW 360009)and ribonuclease A-l from bovine pancreas (MW 13600)were products of Sigma Chemical Company, St. Louis, Missouri. *251-labelingof bovine serum albumin was carried out using solid state lact.operoxidase.10 Lipase activity was determined titrimetrically using tributyrylglycerol as substrate. I1Colipase was determined in 4 mM TDC with an excess of lipase.” In cases where interference by proteins was to be expected, lipase activity was determined in 4 mM TDC with colipase in excess.” The partition of lipase between the aqueous and oil phases of tributyrylglycerol or decane was performed as previously described.‘* The enzyme was added to 15 ml of the aqueous phase, and the substrate was emulsified for 1 min using a glass-coated stirring rod at a speed such that the vortex of the solution reached the bottom of the flask. The aqueous phase was then sampled by siphonage through a nylon filter with 20 by 20 pm pores. The binding of lipase to siliconized glass beads and the effect thereupon of serum albumin were determined as follows. The lipase and serum albumin dissolved in buffer (1 ml) were added to test tubes. Two hundred milligrams of siliconized glass beads (a gift from Dr. Howard Brockman,13 the Hormel Institute, Austin, Minn.) were added. The tube was mixed for 30 set at maximum speed with a Super-Mixer (LabLine Instruments, Inc., Melrose Park, 111.).The glass beads were allowed to sediment and the aqueous phase was sampled for determination of lipase activity. Blanks were run in the absence of glass beads. The hydrophobic interactions of lipase and serum albumin were compared using partition in an aqueous two-pha.se system containing P-PEG.14All reactions were performed using a

ReceivedDecember13,1977. AcceptedFebruary 16, 1978. Address requests for reprints to: B. Borgstri5m,M.D., Department of Physiological Chemistry, P.O. Box 750, S-220 07 Lund, Sweden. This study was supported by grant B78-03X-00071-14A from the Swedish Medical Research Council. 382

September 1978

PORCINE PANCREATIC LIPOLYSIS, EFFECT OF PROTEINS

bufferpH 7.0, 150 mM in NaCl, 1 mM in CaCl,, 2 mM in Trismaleate, and 0.02% in sodium azide at 25°C.

Results Inhibition of pancreatic lipase by proteins and effect of colipase and bile salts. In these experiments tributyr-

ylglycerol (500 ~1) was preemulsified with the buffer solution (15 ml) containing varying concentrations of serum albumin and/or other additions in the titration vessel for 5 min using magnetic stirring. The lipase was then added in a lo-l,~lsample and the lipase activity was recorded by continuous titration. Figure IA shows the inactivation curve of lipase; 50% inactivation occurred at 1110~~M serum albumin when the lipase concentration calculated in the bulk phase was 1O-gM. The simultaneous presence of colipase at a concentration of 1Om8 M reduced to some extent the inhibition caused by serum albumin. The significance of this effect is, however, not clear as colipase also stimulates lipolysis in the absence of albumin.3 Addition of bile salt (4 mM TDC) to the system lipase plus serum albumin resulted in a complete inhibition of lipase activity at all levels. In the simul-

383

taneous presence of colipase a complete restoration of lipase activity was obtained at the levels of serum albumin tested. As previously noted’ the order of addition of lipase and serum albumin to the substrate was important. When albumin and lipase were mixed before adding to the substrate, the initial rates of hydrolysis were higher compared to when albumin was preemulsified with the substrate. In the former case, however, the rate of the reaction slowed down with time and reached a level comparable to that obtained when lipase was added to the preemulsified substrate. Very similar results were obtained for@-la&globulin (fig. 1B). With this protein 50% inhibition was also seen at a concentration of =W7 M. The simultaneous presence of bile salts and colipase also in this case leads to full activity at all levels of p-la&globulin used. Pancreatic ribonuclease on the other hand had much less effect on lipase activity than either serum albumin or p-la&globulin with 50% lipase inhibition occurring at =1.5*10-’ M protein (lOmgM lipase) (fig. 1C). The simultaneous presence of colipase restored the activity almost completely and colipase in the presence of bile salt completely (not shown). A Lineweaver-Burke plot of lipase activity alone and in the presence of two different concentrations of serum albumin indicates that serum albumin might function as a competitive inhibitor of lipase (fig. 2) in the sense that the two proteins compete for a position at the interface. The lower V,,, obtained for lipase alone is

1.

V

1.5

-

1.0 -

0.5

-

1

I

0.01 FIG. 1. Effect of bovine serum albumin (A), la&globulin (B), and pancreatic ribonuclease (C) on the activity of 10V8M porcine pancreatic lipaee alone (+O), and in the presence of lOWaM colipase (0-O) and lo-* M colipaee in 4. 10msM taurodeoxycholate (o--o).

0.02

g

FIG. 2. Lineweaver-Burke plot of the effect of two different concentrations of bovine serum albumin, 2. 10m7M (A-A) and 4. lo-’ M (V-V) on the activity of porcine pancreatic lipase using tributyrylglycerol as substrate (+O, no serum albumin). Con.~ centration of lipase in the bulk phase was 5. lo-lo M.

384

BORGSTRdMAND

probably explained by a denaturation of lipase at the interface which serum albumin tends to prevent. Effect of serum albumin on the binding of lipase to the oil-water interface and to the interface of siliconized glass beads. The effect of serum albumin on the binding of porcine pancreatic lipase to emulsified tributyrin is seen in figure 3. The tributyrin was preemulsified with serum albumin, lipase was added, and 1 min later the aqueous phase was sampled and lipase concentration was determined. It is seen that with an increase in serum albumin an increasing fraction of lipase will be found in the aqueous phase; i.e., the presence of serum albumin will prevent lipase from binding to the interface of the substrate. These results thus strengthen the kinetic studies which show that lipase and serum albumin compete for the interface of the substrate and that the inhibition is an effect of the physical separation of lipase from the interface. The binding of lipase to a hydrocarbon interface measured in the same way was less complete than to tributyrin, and a lower concentration of serum albumin was needed to displace lipase into the aqueous phase. Lipase binds to siliconized glass beads as previously reported15 and is displaced from such binding by the simultaneous presence of serum albumin. Approximately a Xl-fold higher concentration of serum albumin is needed to displace lipase (to 50%) from a triglyceride surface than from a siliconized glass bead surface (fig. 4). Effect of bile salt on the binding of serum albumin to the tributyrin-water interface. In these experiments 500 ~1 of tributyrin were sonicated for 30 set in 3 ml of lop6 M 1251-serum albumin containing from 0.4 to 24 mM taurodeoxycholate. Emulsions were formed in all tubes which slowly settled to give a clear aqueous subphase that could be sampled for radioactivity determination. With no or 0.4 mM bile salt an apparently stable emulsion separated, at higher bile salt concentrations the separated oil phase contained increasingly larger

ERLANSON

Vol.75.No.3

tributyrin droplets in spite of the presence of a high concentration of bile salt. With the highest concentration of bile salt the tributyrin separated out in almost one single drop. The radioactivity of the aqueous phase indicated that above the critical micellar concentration of TDC (-0.8 mM) the serum albumin had been displaced from the oil-water interface into the aqueous phase. Hydrophobic interactions of lipase. The hydrophobic interactions of porcine pancreatic lipase determined in an aqueous two-phase system containing P-PEG are given in figure 5 and compared to serum albumin. In

-8

-7

-6

-5

-4

LOG. CONC. SERUM ALBUMIN M

4. Effect of bovine eerum albumin on the binding of lipaee to siliconized glass beads. One milliliter of lo-’ M lipaee containing different concentrations of eerum albumin wae added with 200 mg of silicon&d glass beads, mixed, and the aqueous phase was sampled for lipaee assay. FIG.

ALOGK

l*"P-0.8 II I

20 96P -PEG

-8

-7

-8

-8

FIG. 5. Change in A log K for bovine serum albumin and pancreatic lipaee ae a function of the emount of polyethyleneglycol FIG. 3.Effect of bovine serum albumin on the binding of pan- palmitate in the eyetern (expressed as the fraction (in percent) of the creatic lipaee to the aqueous interface of emulsified tributyrylgly- total amount of polyethyleneglycol that carries a palm&ate group). cerol (A-A) and ndecane W-W. Oil 600 ~1) wae preemulei- Phaee system: 7% dextran 500, 4.4% polyethyleneglycol including tied with 15 ml of the albumin solution for 5 min wing a glaee-coated varying amounta of ita palm&ate, 150 mu NaCl, and 6 mu Triemagnetic stirrer. Concentration of lipaee in the bulk phaee wae 1O-8 maleate buffer pH 7.0. Symbols: bovine eerum albumin (M), M. pancreatic lipaee (O---O). LOQ.

CONC. SEWN

ALSUYIN

M

September 1978

PORCINE PANCREATIC

LIPOLYSIS,

these experiments the concentration of lipase or serum albumin was determined in the two phases and the partition coefficient K (upper-lower) calculated. In accordance with an earlier report14serum albumin has a high affinity for the P-PEG phase with a positive value of A log K indicative of a high degree of hydrophobic interactions. Lipase in contrast has a low affinity for the P-PEG phase at low concentrations of P-PEG and is repelled into the lower phase at higher concentrations with a strongly negative value of A log K. The recovery of lipase from the two phases after equilibration summed up to =lOO% and thus no denaturation of the protein occurred. Pancreatic lipase in this system thus shows predominating hydrophilic interactions. l4 Discussion The results obtained in the present report indicate that proteins inhibit the activity of pancreatic lipase on its water-insoluble triglyceride substrate by competing for the interface. Most active in this respect are hydrophobic proteins, exemplified by serum albumin and @lactoglobulin.*4 The inhibitory effect of serum albumin was already documented in the paper by Frazer and Nicol’ and later by Brockerhoff,2 but the molar relationships were not considered. Our results indicate that lipase has 50- to lOO-foldhigher affinity for the substrate than serum albumin. The reversal of lipase inhibition by bile salts in the previous experiments’ indicates that the lipase preparation used contained colipase. When pure lipase is used as in the present investigation, bile salts do not activate lipase activity which has been inhibited by high concentrations of protein. Bile salts, however, clear the triglyceride interface from proteins including lipase and thus separate the enzyme physically from the substrate. Such an effect of bile salts would be a general detergent effect earlier described for sodium dodecyl laurate on a serum albumin-stabilized decane emulsion.‘” The simultaneous presence of colipase, a polypeptide cofactor of pancreatic juice that has the property to bind to the lipase substrate even in the presence of bile salts, restores the catalytic activity of lipase.“, ~3” An important function of bile salts in the intestinal content, therefore, may be to clear the triglyceride interface from proteins which by themselves would block the binding of lipase. Because of the specificity of colipase for binding to the lipase substrate in bile salt solution, lipase can be anchored to the substrate and a high specificity can be achieved. Such a situation prevents the nonspecific binding of lipase to hydrophobic interfaces which causes the irreversible denaturation of the enzyme.’ A recent study I8 has shown that emulsification of triglyceride with protein facilitated lipolysis. The protein utilized was a 1:l mixture of bovine hemoglobin and ovalbumin (or a peptic digest thereof) which are of low hydrophobicity,14 and the results, therefore, are in line with the present investigation. The high affinity binding of lipase for its substrate interface compared to other proteins indicates special qualities of the interaction the nature of which is not known at the present

EFFECT

OF PROTEINS

385

time. They may be both hydrophobic and electrostatic.‘s In the two-phase aqueous partition system containing P-PEG, lipase does not show indications of hydrophobic interactions, but rather is forced out of the hydrophobic phase behaving as a highly polar protein. The reason for these discrepancies is not clear; they may be caused by a specific binding of ionic character to the substrate interface or by a specific binding of a localized hydrophobic site on the protein that does not give the lipase an over-all hydrophobic character as measured by the PPEG phase partition test. Another possibility is that the lipase conformation is changed at the substrate interface displaying a specific binding site, an interfacial activation of the enzyme in the sense previously suggested. *OA practical consequence of the present results is that pancreatic lipase, in the presence of hydrophobic proteins such as serum albumin, has to be assayed with bile salts and colipase present. ’* REFERENCES

4. 5.

6. I.

8. 9. 10.

11.

12.

13.

14.

15.

16.

17.

Fraser GP, Nicol AD: Studies in human pancreatic lipase. Clin Chim Acta 13:552-562, 1966 Brockerhoff H: On the function of bile salts and proteins as cofactors of lipase. J Biol Chem 246:5828-5831, 1971 Borgstrom B, Erlanson C: Pancreatic lipase and colipase. Interactions and effects of bile salts and other detergents. Eur J Biochem 37:60-68,1973 Borgstrom B, Erlanson C: Pancreatic juice colipase: physiological importance. Biochim Biophys Acta 242:509-513,197l Hofmann AF: The preparation of chenodeoxycholic acid and its glycine and taurine conjugates. Acta Chem Stand 17:173-186, 1963 Donner J: Preparation of porcine pancreatic lipase free of colipase activity. Acta Chem Stand B30:430-434, 1976 Erlanson C, Fernlund P, Borgstrom B: Purification and characterization of two proteins with colipase activity from porcine pancreas. Biochim Biophys Acta 310~437-445, 1973 Chen RF: Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem 242:173-181,1968 Piez KA, Davie EW, Folk JE: p-lactoglobulin A and B. J Biol Chem 236:2912-2916,196l David GS: Solid state lactoperoxidase: a highly stable enzyme for simple, gentle iodination of proteins. Biochem Biophys Res Commun 48:464-471, 1972 Borgstrom B, Hildebrand H: Lipase and colipase activities of human small intestinal contents after a liquid test meal. Stand J Gastroenterol 10:585-591, 1975 Borgstrom B: On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts. J Lipid Res 16:411-417, 1975 Momsen WE, Brockman HL: Effect of colipase and taurodeoxycholate on the catalytic and physical properties of pancreatic lipase B at an oil-water interface. J Biol Chem 251:378-383,1976 Shanbhag VP, Axelsson CG: Hydrophobic interaction determined by partition in aqueous two-phase systems. Eur J Biothem 60:7-22, 1975 Chapus C, Sari H, Semeriva M, et al: Role of colipase in the interfacial adsorption of pancreatic lipase at hydrophilic interfaces. FEBS Lett 58~155-158, 1975 Cockbain EG: The adsorption of serum albumin and sodium dodecylsulfate at emulsion interfaces. J Colloid Sci 11575-584, 1956 Vandermeers A, Vandermeers-Piret MC, Rathe J, et al: Effect of colipase on adsorption and activity of rat pancreatic lipase on emulsified tributyrin in the presence of bile salt. FEBS I&t

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49:334-331,1975 18. Meyer JH, Stevenson EA, Watts BS: The potential role of protein in the absorption of fat. Gastroenterology 70:232-239, 1976 19. Borgstrom B, Donner J: The polar interactions between pan-

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creatic lipase, colipase and the triglyceride substrate. FEBS Lett 83:23-26, 1977 20. Chapus C, Semeriva M, Bouter-Lapierre C, et al: Mechanism of pancreatic lipase action. I. Interfacial activation of pancreatic lipase. Biochemistry 15:4960-4987, 1976