Binding of deoxycholate to lipoprotein lipase

471 Biochimica et Biophysics Acta, 575 (1979) 471-474 0 ElsevierlNorth-Holland Biomedical Press

BINDING

GUNILLA

OF DEOXYCHOLATE

BENGTSSON

and THOMAS

TO LIPOPROTEIN

LIPASE

OLIVECRONA

Department of Chemistry, Section on Physiological Chemistry, University of Umed, S-901 87 Ume8 (Sweden) (Received

August

lOth, 1979)

Key words: Deoxycholate

binding; Lipoprotein

lipase; (Bovine milk)

Summary Binding of deoxycholate to lipoprotein lipase from bovine milk was demonstrated by equilibrium dialysis and by charge shift electrophoresis. The detergent increased the solubility and stability of the enzyme in aqueous buffers, but did not change its elution position on gel filtration. It is concluded that each molecule of enzyme binds several molecules of deoxycholate without denaturation. This signifies the presence of a lipid-binding region in the native structure of the enzyme.

Helenius and Simons [l] demonstrated that deoxycholate binds to membrane proteins but not to most water soluble proteins and have suggested that binding of this detergent signifies the presence of a lipid binding region in the native structure of the protein. In further support of this, deoxycholate was shown to bind to several apolipoproteins [2]. The action of lipoprotein lipase involves binding of the enzyme to the lipoprotein substrate [3] suggesting that this enzyme has a lipid binding region. Deoxycholate, long chain fatty acids and some detergents stabilize lipoprotein lipase [4,5]. Deoxycholate also desorbs the enzyme from emulsions of long [ 61 and short [ 71 chain triacylglycerols and decreases its binding to heparinSepharose [8] as well as to unsubstituted Sepharose [9]. These observations provide indirect evidence for binding of this detergent to the lipase. More recently we have observed that under some conditions deoxycholate as well as fatty acids interfere with the lipolysis-promoting effects of apolipoprotein CII and other proteins with cofactor activity for lipoprotein lipase. The aim of the present study was to obtain direct evidence for, or against, binding of deoxycholate to this enzyme. Lipoprotein lipase was purified as described [ 91. Stock solutions of 0.2-l mg/ml were stored frozen in 1.2 M NaCl, pH 7.4. Enzyme activity

472

was determined using a gum arabic emulsion of [3H] oleic acid labeled triolein [lo] . Deoxycholate and cetyltrimetylammonium bromide were from Merck, Darmstadt, F.R.G. Oleic acid, bovine serum albumin, a-chymotrypsinogen and transferrin were from Sigma, St. Louis, MO, U.S.A. Human antithrombin 3 was a kind gift from Lam-Olof Andersson, Kabi Research Laboratories, Stockholm. Lipase from Pseudomonas fluorescens [ 111 was a kind gift from Amano Pharmaceutical Co. Nagoya, Japan. [‘“Cl Deoxycholate was from New England Nuclear, Dreieich, F.R.G. Triton X-100 was from Packard Instrument, Downers Groove, IL, U.S.A. Agarose was from Miles Laboratories, Stoke Poges, U.K. On equilibrium dialysis in 2 mM deoxycholate lipoprotein lipase bound 17-20 mol deoxycholate per mol of enzyme (Table I). For this calculation TABLE

I

BINDING

OF DEOXYCHOLATE

TO LIPOPROTEIN

LIPASE

DURING

EQUILIBRIUM

DIALYSIS

2 dialysis bags (0.8 ml each) containing lipoprotein lipase at a concentration of 1 mg/ml in 5 mM unlabeled deoxycholate. 10 mM Tris-HCl. pH 8.5 were immersed in 1 1 of the same buffer with 2 mM deoxycholate - 10 NCi [“Cl deoxycholate. Bags were removed after 5 and 7 days of dialysis at 4% with stirring. Radioactivity per volume was determined in the fluids inside and outside the bag. Deoxucholate excess is the ratio between these radioactivities. Protein content in the dialysis bags was determined by quantitative amino acid analysis. As controls dialysis bags containing 1 mg/ml ovalbumin, or human antithrombin 3 or llpase from Pseudomonas fluorescens, or containing buffer only were treated in the same manner. There was no significant deoxycholate excess inside any of these bags. The molar ratio of deoxycholate excess to enzyme (DOC/LPL) was calculated assuming a molecular weight of 100 000 for the enzyme 1121.

___-__

Time Deoxycholate excess days in dialysis bag l__l_-.~_~_

Enzyme activity % of initial

Protein (mglml)

DOCILPL (mol/mol)

5 7

96 91

0.95 0.90

17 20

1.08 1.09

___

we assumed a molecular weight of 100 000 corresponding to a dimer. This is based on the ultracentrifugal studies by Iverius and ‘bstlund-Lindqvist [ 121. On gel filtration in 5 mM deoxycholate at pH 8.5 the enzyme eluted in the same position as it does in 1.5 M NaCl at pH 7.4 [ 131. This position corresponds to that of a globular protein with a molecular weight of 115 000. Thus, deoxycholate did not dissociate the enzyme to a monomeric state. Immunological reactivity (double diffusion) and enzyme activity (Table I) was retained after the dialysis. Thus, binding of deoxycholate must have occurred without any irreversible changes in the structure of the enzyme. Binding of deoxycholate was also demonstrated by charge shift electrophoresis (Fig. 1) as introduced by Helenius and Simons [l].Lipoprotein lipase did not move from the origin during agarose gel electrophoresis at pH 8.6. This may be due to its limited solubility under these conditions (Table II) but is also in accord with its isoelectric point of about 9 [14]. In contrast, the enzyme moved rapidly toward8 the anode when 5 mM deoxycholate was included in the buffer. Deoxycholate increased the solubility of the enzyme. When the salt concentration of a stock solution of enzyme was reduced below 0.5 M NaCl a precipitate formed. This precipitate dissolved in deoxycholate and all the

473

E 8 20000

z 3 i=

10000 -1.

2 0 ii I In N F

Fig. 1. Charge shift electrophoresis. Conditions: 1% agarose. 0.12 M sodium barbital buffer pH 8.6. 5 mM deoxycholate. 15°C. ‘*%-labeled lipoprotein lipase was dialyzed against this buffer for 2 days, essentially as In Table II. The plate was prerun for 10 min at 4 V/cm before 5 kg lipoprotein lipase (in 5 ~1) was applied. Electiophoresls was for 2 h at 4 V/cm. One strip was stained with Coomassie Brilliant Blue. Another parallel strip was cut into 5 mm sections which were soaked in 0.2 ml of electrophoresis buffer. Lipoprotein lipase (LPL) activity was determined ln aliquots of these extracts. The overall recovery of enzyme activity was about 25%. Yet another parallel strip was cut into sec0, enzyme activity; 0, ‘?-radioactivity. The arrows intions which were assayed for “‘I-radioactivity. dicate the position of (a) wchymotrypsinogen, (b) transferrin and (c) bovine serum albumin when electrophoresed under the same conditions. When deoxycholate was omitted from the electrophoresis buffer the standard proteins migrated at the same rates, but lipoprotein lipase remained at the origin No enzyme activity was recovered after as evidenced by Coomassle staining and ‘ZSI-radioactivity. this electrophoresis. On electrophoresis in 0.25 mM cetyltrimethyl ammonium bromide-l% Triton enzyme protein moved towards the cathode but the enzyme activity was lost. Thus, it formed a complex with this positively charged detergent, but was denaturated.

enzyme activity was recovered in a clear solution (Table II). In contrast, the precipitated enzyme did not dissolve in Triton X-100. Baginsky and Brown [5] and Kinnunen et al. [8] found that transfer of lipoprotein lipase into deoxycholate containing buffers resulted in enhanced enzyme activity suggesting that some of their enzyme was in an aggregated state. Borensztajn et al. [15] have observed that deoxycholate increases the amount of lipoprotein lipase which can be brought into solution from homogenates of rat hearts. As has previously been reported for rat [ 41 and human [ 51 lipoprotein lipases, deoxycholate and oleate stabilized the bovine lipoprotein lipase. When the enzyme was diluted into 0.1 M NaCl pH 8.5 and incubated at 37°C the enzyme activity rapidly declined; less than 10% remained after 5 min. When the buffer contained 2 mM deoxycholate there was an initial rapid loss of lo-20% of the activity within the first 10 min, but the activity

474

TABLE

II

EFFECTS

OF DETERGENTS

ON THE SOLUBILITY

OF LIPOPROTEIN

LIPASE

2 ml lipoprotein lipase (0.4 mg/ml) in 5 mM sodium veronal, 1.2 M N&l, pH 7.4. was dialyzed at 4’C against 10 mM Tris-HCl pH 8.5 with the additions listed in the table. A precipitate formed in all three dialysis bags after a few hours. This precipitate then dissolved again during dialysis against deoxycholate, but not during dialysis against Triton or without detergent. After 24 h the contents of the dialysis bags were transferred to centrifuge tubes. Aliquots were taken for assay of enzyme activity. With the turbid enzyme solutions in Triton X-100 or buffer only, there was a lag phase of several min during which the rate of hydrolysis slowly increased. The values listed represent the linear rates then attained. The tubes were then centrifuged at 25 000 X g, 20 min. The supernatants were decanted and their enzyme activity determined. To the precipitates was added 2 ml 5 mM deoxycholate - 0.5 mM oleate in 10 mM Tris-HCl. pH 8.5. After 24 h of gentle agitation at 4% the precipitates had completely dissolved and aliquots were assayed for enzyme activity. Conditions dialysis

for

5 mM deoxycholate 1% Triton X-100 Buffer only

Lipoprotein

lipase activity

(I.tmol fatty acid released/min

Before centrifugation ____._.

After centrifugation

In redissolved precipitate

200 160 140

200 0 0

-

per ml at 25%.

PH 8.5)

170 170

___.-

then remained essentially unchanged for several hours. This indicate8 that binding of the detergent to the enzyme was not instantaneous, but once the complex had formed the enzyme became stable. Further experiment8 showed that 0.5 mM deoxycholate was enough for marked stabilization. This is well below the critical micellar concentration of deoxycholate [ 161 which suggests that binding occurred by formation of a micelle-like detergent aggregate around some part of the enzyme rather than by binding of a preformed detergent micelle. References 1 2 3 4 5 6 7 8 9 10 11 12 13

Helenius, A. and Simon% K. (1977). Proc. Natl. Acad. Sci. U.S.A. 74. 529-532 Uterman, G. and Beisiegel. U. (1979) FEBS Lett. 97. 245-248 Fielding, C.J. (1976) Biochim. Biophys. Acta 206, 118-124 Fielding. C.J. (1968) Biochim. Biophys. Acta 159, 94-102 Baginsky, M.L. and Brown, W.V. (1977) J. Lipid Res. 18, 423-437 Fielding, C.J. (1969) Biochim. Biophys. Acta 178,499-507 Rapp, D. and Olivecrona. T. (1978) Eur. J. Biochem. 91. 379-385 Kinnunen, P.K.J., Huttunen, J.K. and Ehnholm, C. (1976) Biochim. Biophys. Acta 450, 342-351 Bengtsson, G. and 9livecrona. T. (1977) Biochem. J. 167,109-119 Hernell, 0.. Egelrud. T. and Olivecrona, T. (1975) Biochim. Biophys. Acta 381, 233-241 Sugiura, M., Oikawa, T., Hirano. K. and Inukai. T. (1977) Biochim. Biophys. Acta 488, 353-358 Iverius, P.H. and Ostlund-Lindqvist, A.M. (1976) J. Biol. Chem. 251, 7791-7795 Olivecrona, T., Egelrud, T., Hernell. 0.. Castberg. H. and Solberg, P. (1975) Int. Dairy Fed. Document No. 86, Bruxelles, pp. 61-72 14 Bengtsson, G. and Olivecrona, T. (1977) in Electrofocusing and Isotachophoresis (Radola, B.J. and Graesslin, D., eds.), PP. 189-195. de Gruyter. Berlin 15 Borensztajn. J., Otway, S. and Robinson, D.S. (1970) J. Lipid Res. 11, 102-110 16 Helenius, A., MC Caslln. D.R.. Fries, E. and Tanford, C. (1979) Methods in Enzymology. in the press