Preparation, characterization, and measurement of lipoprotein lipase

[41]

LIPOPROTEIN LIPASE PURIFICATION AND ASSAY

691

[41] P r e p a r a t i o n , C h a r a c t e r i z a t i o n , a n d M e a s u r e m e n t o f Lipoprotein Lipase

By PER-HENRIK IVERIUS and ANN-MARGARETOSTLUND-LINDQVIST Introduction Lipoprotein lipase (EC 3.1.1.3) is secreted by a variety of cell types and plays a key role in the metabolism of the triglyceride-rich lipoproteins, namely, chylomicrons and very-low-density lipoproteins. At its extracellular location on the capillary endothelium in various tissues, the enzyme hydrolyzes the triglyceride component of these particles to glycerol and free fatty acids. The latter are thereby made available for uptake into cells where they are either used as fuel or stored.l The enzyme has attracted considerable clinical interest since various causes of hypertriglyceridemia have been associated with impairment of its function. 2 More recently, lipoprotein lipase (LPL) in adipose tissue has been implicated as a major regulator of fat cell size. 3 After the introduction of affinity chromatography on heparinagarose, 4 several purification procedures adopting that technique have appeared and made it possible to purify the enzyme to homogeneity as well as to investigate its chemical and functional properties in more detail. 5-~° This chapter describes protocols for purification of the enzyme from bovine milk 6 and human postheparin plasma. 8 It also presents a versatile assay procedure that can be adopted for tissue extracts, column effluents, cell cultures, postheparin plasma samples, and adipose tissue biopsy specimens.

1 p. Nilsson-Ehle, A. S. Garfinkel, and M. C. Sclhotz, Annu. Rev. Biochem. 49, 667 (1980). 2 j. D. Brunzell, A. Chait, and E. L. Bierman, Metabolism 27, 1109 (1978). 3 j. D. Brunzell, and M. R. C. Greenwood, in "Biochemical Pharmacology of Metabolic Disease, Vol. I O b e s i t y " (P. B. Curtis-Prior, ed.), p. 175. Elsevier, Amsterdam, 1983. 4 T. Olivecrona, T. Egelrud, P.-H. Iverius, and U. Lindahl, Biochem. Biophys. Res. Commun. 43, 524 (1971). 5 T. Egelrud and T. Olivecrona, J. Biol. Chem. 247, 6212 (1972). 6 p. H. Iverius and A.-M. Ostlund-Lindqvist, J. Biol. Chem. 251, 7791 (1976). 7 p. K. J. Kinnunen, Med. Biol. 55, 187 (1977). s A.-M. Ostlund-Lindqvist, Biochem. J. 179, 555 (1979). 9 L. Wallinder, G. Bengtsson, and T. Olivecrona, Biochim. Biophys. Acta 711, 107 (1982). i0 S. M. Parkin, B. K. Speake, and D. S. Robinson, Biochem. J. 207, 485 (1982).

METHODS IN ENZYMOLOGY. VOL. 129

Copyright © 1986by AcademicPress, Inc. All rights of reproductionin any form reserved.

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METABOLISM OF PLASMA LIPOPROTEINS

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Assay L P L requires the presence of a specific protein cofactor, also called activator, to hydrolyze a triglyceride emulsion at an optimal rate. This factor, apolipoprotein C-II, ll,n is a normal constituent of the natural substrates (chylomicrons and very low-density lipoproteins). Most published assays for the enzyme consist of a triglyceride emulsion, serum as a source of apolipoprotein C-II, and albumin as an acceptor for free fatty acids.13 Assays mainly differ with regard to the type of emulsifier used, the relative concentrations of various assay components, and the method used to quantitate the reaction products. Methods using commercial triglyceride emulsions, e.g., Intralipid (Vitrum AB, Sweden), are highly reproducible but less sensitive than those employing radioactive substrates. Larger variability of the radioactive methods is introduced when a new emulsion is prepared for each assay. Maximal enzyme activity requires a sufficient concentration of triglyceride substrate as well as an optimal concentration of activator. ~4The assay described herein 15 is designed by that principle using a serum activator and radioactive triolein emulsified with lecithin.

Stock Reagents Phosphatidylcholine (egg), 50 mg/ml: Supplied as a chloroform solution by Serdary Research Lab., London, Ontario (Cat. No. A-31), and stored at - 2 0 °. Triolein, 200 mg/ml: Supplied by Sigma Chemical Co., St. Louis, MO (Cat. No. T7502), dissolved in benzene and stored at - 2 0 °. Radioactive triolein, 1.25 I~Ci/ml: Supplied by Amersham, Arlington Heights, IL, as glycerol-tri[1-14C]oleate, 100/~Ci/ml (30-60 mCi/mmol), in toluene. Aliquots of 125/~Ci are evaporated under a stream of nitrogen, redissolved in heptane (3 ml), and further purified by extraction with 7 ml of 0.05 M NaOH in 50% (v/v) ethanol. The purified material is finally evaporated under nitrogen, dissolved in 100 ml of heptane and stored at - 2 0 °" Radioactive oleic acid, 0.5 ~Ci/ml: Supplied by Amersham (see above) as [1-14C]oleic acid, 100/~Ci/ml (>50 mCi/mmol) in toluene. An 11 j. C. LaRosa, R. I. Levy, R. Herbert, S. E. Lux, and D. S. Fredrickson, Biochem. Biophys. Res. Commun. 41, 57 (1970). 12 R. J. Havel, C. J. Fielding, T. Olivecrona, V. G. Shore, P. E. Fielding, and T. Egelrud, Biochemistry 12, 1828 (1973). 13 S. E. Riley and D. S. Robinson, Biochim. Biophys. Acta 369, 371 (1974). 14 A. M. Ostlund-Lindqvist and P. H. Iverius, Biochem. Biophys. Res. Commun. 65, 1447

(1975). 15P.-H. Iverius and J. D. Brunzell, Am. J. Physiol. 249, El07 (1985).

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693

aliquot (50/zCi) is evaporated under nitrogen with 50/zmol (14.124 mg) of cold oleic acid, diluted to 100 ml with heptane and stored at - 2 0 °. 0.223 M Tris-HCl buffer, p H 8.5:Trizma-8.5 (29.289 g), supplied by Sigma Chemical Co., St. Louis, MO, is diluted with water to 1 liter and stored at 4 °. At 37 ° the pH is 8.2. O.78 M NaC1 in Tris buffer (salt correction): 4.558 g NaCI is diluted to 100 ml with Tris buffer (see above) and stored at 4 °. Methanol-chloroform-heptane, 1.41 : 1.25 : I (v/v/v): Methanol (141 ml) is mixed with 125 ml of chloroform, and 100 ml of heptane. 0.05 M Carbonate-borate buffer, p H 10.5: Anhydrous K2CO3 (6.91 g) and boric acid (3.092 g) are dissolved in water, adjusted to pH 10.5 with 5 M KOH, and diluted to 1 liter. Fresh solution is made weekly and stored at 4° . Albumin, 183.3 mg/ml in Tris buffer: Bovine serum albumin (45.825 g) supplied by Sigma (Cat. No. A4503) is dissolved in Tris buffer (see above), diluted to 250 ml, and stored at - 2 0 ° in 10-ml aliquots. Heparin, 1 mg/ml, in Tris or Krebs-Ringer phosphate (KRP) buffer: Sodium heparin (50 mg) supplied by Sigma (Cat. No. H3125) is dissolved in either Tris buffer (see above) or KRP buffer (see below) to a final volume of 50 ml and stored at - 2 0 ° in 2-ml aliquots. Pooled human serum. Blood (400 ml per donor) is drawn in 50-ml plastic syringes with 2.5 ml of disodium EDTA (20 mg/ml). The blood from each donor is centrifuged in separate containers, the plasma collected and centrifuged again before pooling. Solid CaCI2 is added to 0.1% (w/v) followed by bovine thrombin to 1 unit/ml (Topical Thrombin, Parke-Davis, Morris Plains, N J), and the plasma is incubated at 37° for 30 min. After the serum has been recovered from the clot by centrifugation, it is dialyzed against 0.15 M NaCI and heat inactivated at 56° for 30 min in 100-ml glass bottles. Finally, the serum is dialyzed against KRP buffer (see below) and stored in 10-ml aliquots at - 2 0 °. Detergent solution, 2 mg/ml sodium deoxycholate-O.08 mg/ml Nonidet P40-0.05 mg/ml heparin-lO mg/ml BSA-0.25 M sucrose in Tris buffer: Sodium deoxycholate (500 mg) and 21.394 g of sucrose are added to 2 ml of Nonidet P40 (1 g/100 ml), 12.5 ml of heparin (1 mg/ml), and 13.7 ml of albumin. Tris buffer (see above) is used for all the component solutions as well as for dilution to 250 ml after warming to 37 °. The solution is stored in 5-ml aliquots at - 2 0 °. Skim milk standard. Unpasteurized bovine milk is obtained fresh from a diary farm and the cream removed after centrifugation at 4 °. The skim milk (500 ml) is stirred for 30 min at 4 c with solid trisodium citrate dihydrate (14.7 g) added to 0.1 M, and dialyzed against 0.15 M NaC1-5 mM sodium phosphate buffer (pH 7.4) for 3 x 6 hr with a 10-fold excess of

694

METABOLISM OF PLASMA LIPOPROTEINS

[41]

buffer. After the dialysis, glycerol is added to 30% (v/v) and the standard is frozen in 4-ml aliquots with ethanol-dry ice and stored at - 7 0 °.

Fresh Reagents Krebs-Ringer phosphate (KRP) buffer, pH 7.4: NaCI (45.0 g/liter), 100 ml KCI (57.4 g/liter), 5 ml CaCI2" 2H20 (81.4 g/liter), 1.5 ml MgSO4" 7HzO (191.0 g/liter), 1.0 ml Distilled water, 432 ml NaHzPO4" H20 (13.80 g/liter), 15 ml Na2HPO4" anhydr (14.196 g/liter), 85 ml

Elution buffer, 25% (v/v) serum-O.05 mg/ml heparin: Serum, 5 vol Heparin (1 mg/ml in KRP buffer), 1 vol KRP buffer, 14 vol

Diluted serum, 25% (v/v): Serum, 1 vol KRP buffer, 3 vol

Triglyceride emulsion. Aliquots of phosphatidylcholine (0.12 ml; 6 rag), triolein (0.25 ml; 50 mg), and radioactive triolein (2 ml; 2.5/zCi) are transferred to a 2.2 x 7 cm flat-bottomed glass vial and the solvents evaporated under a stream of nitrogen. After the addition of Tris buffer (1.95 ml), salt correction (1 ml), and albumin (3 ml), the mixture is cooled on ice and sonicated at 50 W with 10 repeated 10-sec bursts interrupted by 10-sec pauses using a ½-in. diameter probe with a flat tip. The emulsion is kept at 0° and used within 2 hr. Each batch of 6 ml is enough for l0 assay incubations. When several batches are required, they are pooled before use.

Sample Preparation Elution of Adipose Tissue. Weighed adipose tissue ( - 5 0 mg), obtained by needle aspiration and cut into pieces of 5-7 mg size, is transferred to 12 x 75 mm glass tubes containing 0.3 ml of elution buffer. The tubes are covered with Parafilm and incubated in a shaking water bath (80 cycles/ min) at 37° for exactly 30 min. At the end of the incubation, an aliquot (0.2 ml) of the buffer is removed and assayed for more enzyme activity (see below). Detergent Extraction of Adipose Tissue. Stock detergent solution is thawed at 37° to ensure complete dissolution of its components. Approximately 50 mg of weighed tissue is homogenized at room temperature with

[41]

LIPOPROTEIN LIPASE PURIFICATION AND ASSAY

695

0.2 ml of detergent solution using an all-glass tissue grinder (Duall, size 20, Kontes, Berkeley, CA). After the addition of another 0.3 ml of detergent solution followed by brief mixing, the homogenate is immediately transferred to centrifuge tubes and spun at 4° and 12,000 g for 15 min. An aliquot (0.2 ml) of the infranatant is assayed for enzyme activity (see below). Postheparin Plasma. Postheparin blood is collected in Li-heparinate Vacutainer tubes (Becton-Dickinson, Rutherford, N J), the plasma recovered by centrifugation, frozen with dry ice-ethanol, and stored at - 7 0 °. Before analysis, the plasma is thawed rapidly in cold water and an aliquot (0.05 ml) diluted with 0.2 ml of pooled serum, 0.05 ml of heparin (1 mg/ml in KRP buffer), and 0.2 ml of KRP buffer. An aliquot (0.15 ml) of the above dilution is added to an equal volume of inhibiting antiserum or antibody to LPL, appropriately diluted with KRP buffer. A control sample is similarly prepared using diluted nonimmune serum or IgG. The mixtures are incubated at 4° for 2 hr before an aliquot (0.2 ml) is assayed for lipase activity. Cell Culture Dishes. 16Culture medium is assayed without further dilution. The cell layer of a 35-mm culture dish is rinsed twice with 1 ml of saline. After careful removal of the second rinse, 0.5 ml of detergent solution is added and the cells are lysed by scraping with a Teflon policeman. The extract is then homogenized and centrifuged as described above for adipose tissue. Skim Milk Standard. Frozen standard is rapidly thawed in cold water and an aliquot (0.1 ml) gently added to the bottom of a test tube with 2 ml of ice-cold Tris buffer. Immediately before taking an aliquot (0.2 ml) for assay, the standard is mixed with the buffer. Miscellaneous Enzyme Sources. When needed, a sample is appropriately diluted with ice-cold Tris buffer either to fit the assay range or to reduce the effect of interfering substances, e.g., salt. Procedure Assay Mixture. The complete assay mixture (pH 8.2 at 37° and ionic strength 0.16) contains 0.178 M Tris-HCl buffer (ionic strength 0.05), 0.11 M NaC1, 55 mg/ml of albumin, 0.01 mg/ml of heparin, 5% (v/v) of serum, and 5 mg/ml of glycerol-tri[1-~aC]oleate (0.05/zCi/mg) emulsified with 0.6 mg/ml of lecithin. Blank incubations consist of 0.6 ml of triglyceride emulsion, 0.2 ml of Tris buffer, and 0.2 ml of elution buffer. ~6 A. Chait, P.-H. Iverius, and J. D. Brunzell, .I. Clin. Invest. 69, 490 (1982).

696

METABOLISM OF PLASMA LIPOPROTEINS

[41]

Aliquots (0.2 ml) of adipose tissue eluates or diluted postheparin plasma samples (see above) are mixed with 0.6 ml of triglyceride emulsion and 0.2 ml of Tris buffer. Aliquots (0.2 ml) of samples in Tris buffer, i.e., detergent extracts, skim milk standard, or miscellaneous samples are mixed with 0.6 ml of triglyceride emulsion and 0.2 ml of diluted serum. Incubation. Test tubes containing incomplete assay mixture (0.8 ml) are kept on ice until enzyme (0.2 ml) is added, then covered with Parafilm, and incubated in a 37° shaking water bath (80 cycles/min) for 60 min. Incubations are terminated by the extraction of free fatty acids for counting of radioactivity. Fatty Acid Extraction. ~7Test tubes (13 x 100 mm) containing 3.25 ml of methanol-chloroform-heptane are prepared in advance. Four aliquots (0.2 ml) from each incubation mixture are transferred to extraction tubes which immediately are agitated vigorously. Carbonate-borate buffer (1.05 ml) is added, the tubes are stoppered and agitated again. Phase separation is accomplished by centrifugation at room temperature for 20 min using a swingout rotor. An aliquot (2 ml) of the aqueous upper phase is then transferred to a scintillation vial containing 4 ml of scintillation liquid (Aquasol, New England Nuclear, Boston, MA) and counted for 10 min in a liquid scintillation spectrometer. Specific Radioactivity of Triolein. Radioactive triolein solution (20 pJ) is transferred in duplicate to empty scintillation vials and the solvent evaporated under a stream of nitrogen. Prior to counting of radioactivity, scintillation liquid (4 ml) and the aqueous phase (2 ml) from a blank extraction are added. Extraction Recovery. Radioactive oleic acid solution (0.2 ml) is evaporated in two test tubes as well as in two scintillation vials under a stream of nitrogen. The test tubes are processed as blank incubations (see above) which are followed by fatty acid extraction and scintillation counting. The scintillation vials receiving tracer are counted after the addition of scintillation liquid (4 ml) and the aqueous phase from a blank extraction (2 ml). Comments. Blank incubations are run in triplicate with one replicate used in the determination of triolein specific radioactivity and extraction recovery. It is essential that hydrolysis products of the radioactive triolein are removed by the procedure described above in order to ensure low background radioactivity of the blank. The procedures used to recover enzyme, e.g., elution of enzyme or detergent extraction of enzyme from adipose tissue, have more variability than the proper assay. Therefore, it is recommended that these proceJ7 p. Belfrage and M. V a u g h a n , J. LipidRes. 10, 341 (1969).

[41]

LIPOPROTEIN LIPASE PURIFICATION AND ASSAY

697

dures are carried out in triplicate to allow computation of the standard error. Computations Blank Correction. The gross radioactivity (cpm) of blanks, standards, and unknown samples is computed as the mean of all extractions from a particular sample. Net radioactivity, which is obtained by subtracting the mean of the blanks, is used in all further computations.

Extraction recovery (R):

R = 5E/F

where R is the fraction of oleic acid that distributes to 2 ml of the aqueous phase in the extraction tube. F represents the total oleic acid radioactivity (cpm) added to a separate scintillation vial or to a blank incubation (1 ml) and E is the part of that radioactivity extracted from a 0.2-ml aliquot into 2 ml of aqueous phase. T × 10 x 885.43 S = (5 x 10-3)(3 x 109) = 5.903 x 10-4T

Specific radioactivity (S):

T is the radioactivity of triolein that was directly added to a scintillation vial and T x 10 represents the radioactivity in I ml of complete assay mixture. The remaining figures convert the triolein (Mr 885.43), present at 5 mg/ml, into fatty acid equivalents (nanomoles). Thus the unit for S is cpm/nmol. Enzyme activity (A):

A =

C R x S :× 0.2 x 0.2 x 6 0 - R

C x S x 2.4

The enzyme activity (A), expressed as, nanomoles of fatty acid released per minute and per milliliter sample, assumes that the latter was added to the assay mixture as a 0.2-ml aliquot. The parameters R and S are defined above. C is the mean net radioactivity for a particular sample. Interassay variation is controlled for by multiplying apparent enzyme activity by a correction factor (f): f = Ao/Al

AI is the apparent activity of the skim milk standard on a particular assay occasion and A0 an arbitrarily chosen reference value for the standard. Postheparin plasma lipolytic activity is obtained by multiplying the apparent activity by 20 (dilution factor). Activity remaining after the incubation with inhibiting antibody to LPL represents hepatic lipase) 8 The 18 j. K. Huttunen, C. Ehnholm, P. K. J. Kinnunen, and E. A. Nikkil/i, Clin. Chim. Acta 63, 335 (1975).

698

METABOLISM OF PLASMA LIPOPROTEINS

[41]

LPL activity is then obtained by subtraction. Calculation of lipase activity in adipose tissue and other sources needs no further explanation.

General Comments The assay is linear for sample enzyme activities up to 200 nmol/ min. ml. Using a skim milk standard, the interassay coefficient of variation is 5.2% whereas the intraassay variation is only 1.2%. Detergent extraction of adipose tissue reflects total tissue enzyme and yields higher activity than any other method to recover the enzyme. Therefore this procedure should be particularly useful when it is important to distinguish a low activity from zero activity. Elution of adipose tissue with serum and heparin at 37 ° recovers less than 50% of the activity in a detergent extract. However, the eluted activity is more sensitive to physiological perturbations and is probably also a better indicator of the physiologically active enzyme fraction. Some applications of this assay to human adipose tissue and postheparin plasma will appear elsewhere.15 Purification Procedures

Reagents Heparin-agarose: Heparin is supplied by Sigma (see above). Heparin with low affinity for anti-thrombin is fractionated on anti-thrombinSepharose as described by Nordenman and Bj6rk. 19Conventional heparin and low-affinity heparin are covalently linked to Sepharose CL-6B as described. 6,2°,21 After use, the gels are regenerated by alternating washes with 10 bed volumes of 0.1% Triton X-100 in 2 M NaCI and 10 vol of water C~-aluminum hydroxide gel: Supplied by Sigma (Cat. No. A8628) Bovine milk LPL 6 Skim milk: Bovine milk is obtained fresh from a cow that has been screened beforehand for high enzyme activity. Within 1 hr the milk is chilled to 0 ° and then centrifuged. The fat cake is pierced with Tygon tubing and the skim milk siphoned off. After another centrifugation to remove traces of fat, the milk (3 liters) is adjusted to 0.1 M with solid trisodium citrate dihydrate (88.2 g) Heparin-Agarose Chromatography. Conventional heparin-agarose gel (400 ml) is added to the citrate-treated skim milk (3 liters) and kept at 19 B. Nordenman and I. Bj6rk, Biochemistry 17, 3339 (1978). 2o P.-H. Iverius, Biochem. J. 124, 677 (1971). 21 A.-M. Ostlund-Lindqvist and J. Boberg, FEBS Lett. 83, 231 (1977).

[41]

LIPOPROTEIN LIPASE PURIFICATION AND ASSAY

699

0° for 30 min under frequent stirring. The gel is separated from the skim milk on a sintered glass filter, washed three times with 400 ml of ice-cold 0.5 M NaC1-30% (v/v) glycerol-0.01 M phosphate (pH 7.5), resuspended in the buffer, and packed into a column (7 × 10.5 cm). The column, kept at 4 ° and run at 174 ml/hr, is washed with one bed volume of buffer and then eluted with a 3-liter linear gradient of 0.5 to 1.5 M NaCI in 30% (v/v) glycerol-0.01 M phosphate, pH 7.5 (Fig. 1). Adsorption to C~ Gel. The pooled enzyme fractions (Fig. 1) are diluted with an equal volume of 30% (v/v) glycerol-0.01 M phosphate (pH 7.5) and 20 ml of sedimented Cr gel in water are added. The suspension is stirred at 4 ° for 30 rain, after which the gel is collected by centrifugation. The gel is washed twice by suspending in 250 ml of 0.5 M NaCI-30% (v/v) glycerol-0.01 M phosphate (pH 7.5) and then centrifuging. The suspension should be accomplished gently with a glass rod or spatula, since more efficient tools like a tissue homogenizer may destroy the enzyme activity. The enzyme is finally eluted from the gel by suspending and centrifuging four times in 50 ml of 1.2 M NaCI-30% (v/v) glycerol-0.01 M phosphate (pH 7.5). The combined eluates (200 ml) are spun at 4 ° and 12,000 g for 20 min to remove traces of the gel, and thereafter dialyzed for 20 hr at 4 ° against 3.6 M ammonium sulfate-0.01 M phosphate (pH 6.5). After centrifugation at 4 ° and 30,000 g for 15 min using one centrifuge tube repeatedly, the white precipitate is dissolved in the buffer for the final fractionation step (see below). Intervent Dilution Chromatography. The enzyme obtained above after ammonium sulfate precipitation, is dissolved in 3 ml of 0.15 M NaCI-15% 20~- "r~ ~_. ~-

~o !

~

2.0 -~

0.4

L5 x_x-=X~ 1,0 (b

,--,-,-,-,-,-s I0 FRACTION

20

30

4o

NUMBER

FIG, ]. £1ution pattern of bovine milk LPL from conventional heparin-agarose. The fractions (58 ml) were analyzed for enzyme activity (0) by a previously described assay, absorbance at 280 nm (©), and sodium chloride concentration ( x ) by measuring conductivity. The 10 fractions between the arrows were pooled. The graph is taken from P.-H. lverius and A.-M. 0stlund-Lindqvist, J. Biol. Chem. 251, 7791 (1976).

700

[41]

METABOLISM OF PLASMA LIPOPROTEINS

250 -

.3.0A280

~ ~ 2ooE

-2.0

'~ '~ 150-

1oo

-~ 5

1o

0

20

20

4,0

60

80

FRACTION NUMBER

FIG. 2. Intervent dilution chromatography of partially purified bovine milk LPL on conventional heparin-agarose. The fractions (3.5 ml) were analyzed for enzyme activity (O), absorbance at 280 nm (O), and conductivity (×). The three fractions containing the highest enzyme activities were pooled. The graph is taken from P.-H. Iverius and A.-M. Ostlund-Lindqvist, J. Biol. C h e m . 251, 7791 (1976).

(v/v) glycerol-0.01 M phosphate (pH 7.5) and applied to a 1.5 x 85 cm column of conventional heparin-agarose. After washing with one bed volume (150 ml) of the above buffer, the column is eluted with 1.5 M NaCl-15% (v/v) glycerol-0.01 M phosphate (pH 7.5) at a rate of 14 ml/hr. The enzyme, which emerges as a sharp peak at the front of the elution buffer (Fig. 2), is dialyzed against 3.6 M ammonium sulfate-0.01 M phosphate (pH 6.5) for 20 hr and centrifuged at 4 ° and 30,000 g for 15 min. The precipitate, which at this stage has a gelatinous appearance, is dissolved in 1 ml of 50% (v/v) glycerol-0.01 M phosphate (pH 7.5) and stored at - 2 0 °" C o m m e n t s . This procedure produces 1.5-2 mg of enzyme with a recovery of 10-13%. The purity, checked by sodium dodecyl sulfate (SDS)polyacrylamide electrophoresis, may vary between different preparations but is usually better than 95%. Most of the impurities, which almost always have a faster mobility than the enzyme, do react with an affinitypurified antibody to LPL after Western blotting, indicating that they are proteolytic fragments of the enzyme (unpublished results). A recent report confirms this finding and advocates the addition of 1 mM phenylmethanesulfonyl fluoride to the milk in order to inhibit proteolysis, z2 Gel electrophoretic methods other than those employing SDS have been unL. Socorro and R. L. Jackson, J. Biol. Chem. 260, 6324 (1985).

[41]

701

L I P O P R O T E I N L I P A S E P U R I F I C A T I O N A N D ASSAY

successful for testing of purity, since aggregation of the enzyme prevents it from entering the gel. The enzyme produced by this particular procedure has not been exposed to detergents and is obtained in the native form at a high concentration. Storage in 50% glycerol at - 2 0 ° as a liquid solution is extremely convenient, and such preparations have been kept for more than 6 years without substantial loss of activity. Human Postheparin Plasma LPL 8,21 Postheparin Plasma. Volunteers are injected with heparin intravenously (100 U/kg). After 20 min, plasma is obtained either by plasmapheresis or as described for the initial steps to make pooled human serum (see "Assay"). The plasma (1000 ml) is immediately chilled to 0 °, mixed with solid NaCI (23.38 g) to a concentration of 0.4 M, and 120 ml of conventional heparin-agarose. After stirring at 0° for 30 min, the gel is washed six times with 200 ml of 0.4 M NaC1--30% (v/v) glycerol-0.01 M phosphate (pH 7.5) and packed into a column (2.5 × 25 cm). The column, kept at 4 ° and run at 60 ml/hr, is washed with another 3 bed volumes of the buffer and then eluted with a 700-ml linear gradient of 0.4 to 1.5 M NaC1 in 30% (v/v) (glycerol-0.01 M phosphate, pH 7.5 (Fig. 3). The peak fractions of L P L are pooled (200 ml), dialyzed against 3.6 M ammonium sulfate0.01 M phosphate (pH 6.5), and the precipitate is dissolved in 1 ml of 50% (v/v) glycerol-0.01 M phosphate (pH 7.5) before storing at - 2 0 °. A280

I

I

•E 8 .04

.,

0.5-

J

2O0

4O0 EFFLUENT

6OO

80O

VOLUME (rot)

FIG. 3. Elution pattern of postheparin plasma lipolytic enzymes from heparin-agarose. The fractions (5 ml) were analyzed for LPL activity (0) and hepatic lipase activity (&) by previously described assays, absorbance at 280 nm (©), and sodium chloride concentration (x) by measuring conductivity. The graph is taken from A.-M. Ostlund-Lindqvist and J. Boberg, F E B S L e t t . 83, 231 (1977).

702

[41]

METABOLISM OF PLASMA LIPOPROTEINS

Affinity Chromatography on Heparin-Agarose with Low Affinity for Anti-Thrombin. Four partially purified LPL preparations, obtained as described above by chromatography on conventional heparin-agarose, are pooled, adjusted to 0.5 M NaCI, and applied to a heparin-agarose column (1.6 x 5 cm) with low affinity for anti-thrombin. The column is washed with 160 ml of 0.5 M NaCI-30% (v/v) glycerol-0.01 M phosphate (pH 7.5) and then eluted with 1.2 M NaCI in the same buffer (Fig. 4). Adsorption to C~ Gel. The pooled enzyme fractions (Fig. 4) are diluted with an equal volume of 30% (v/v) glycerol-0.01 M phosphate (pH 7.5) and adsorbed to 2 ml of sedimented C~ gel. Washing of the gel and elution is carried out as described above under "Bovine Milk L P L " except that the procedure is carried out in a 10-fold smaller scale. The enzyme fraction is finally dialyzed against ammonium sulfate, the precipitate dissolved in 1 ml of 50% (v/v) glycerol-0.01 M phosphate (pH 7.5) and stored at - 2 0 °. Comments. The above procedure is designed for the isolation of both hepatic lipase and LPL from postheparin plasma. By contrast to milk, plasma contains significant amounts of anti-thrombin, which binds to heparin and coelutes with LPL. The purification step on heparin-agarose with low affinity for anti-thrombin is therefore added to remove this protein. Although the resulting lipase fraction (0.1 rag) is free from antithrombin, as tested by immunodiffusion, it shows more than one band on SDS electrophoresis and has a specific activity which is approximately half of that of pure bovine enzyme. Nevertheless, this fraction has been

2

12

10~.

E

8~

~

J~u

50 100 150 200 250 EFFLUENT VOLUME (m/)

Fro. 4. Chromatography of partially purified postheparin plasma LPL on fieparinagarose with low affinity for anti-thrombin. The fractions (4 ml) were analyzed for LPL activity (Q) and absorbance at 280 nm (O). The three fractions of highest enzyme activity were pooled. The graph is taken from A.-M. Ostlund-Lindqvist and J. Boberg, F E B S Lett. 113, 231 (1977).

[41]

LIPOPROTEIN LIPASE PURIFICATION AND ASSAY

703

useful for further purification by preparative SDS electrophoresis. The homogeneous protein resulting from this step has been employed for antibody production and amino acid analysis. 8

Properties of Purified LPL There are striking similarities between LPLs from different species. For instance, the human, 2~ the bovine, 4 and the rat enzyme l° all bind to heparin-agarose and elute at a similar salt concentration, are inhibited when assayed in the presence of 1 M NaC1, 23,z4 and are all prone to inactivation in the purified state. Furthermore, the human serum activator (apolipoprotein C-II) can also activate the bovine and the rat enzyme, and antibodies raised against the bovine enzyme may cross-react with human, rat, and mouse enzymes. 25 Although not yet substantiated by amino acid sequences, these data suggest that LPL has been well conserved during mammalian evolution. Bovine LPL, which so far is the best characterized enzyme, may therefore serve as a model for lipases from other species. The bovine lipase is a glycoprotein containing 8.3% carbohydrate including mannose, galactose, glucose, N-acetylglucosamine, and sialic acid. The amino acid composition, which has been published elsewhere, 6 is very similar to that of the human e n z y m e : The extinction coefficient ( L:,1% "~ ~l cmJ is 14.0 at 280 nm. 6 The molecular weight determined by sedimentation equilibrium ultracentrifugation in guanidine under reducing as well as nonreducing conditions is 48,300. 6 Higher values recorded by SDS-polyacrylamide electrophoresis are probably a result of the carbohydrate moiety causing anomalous behavior of the protein in this method. 26 The native enzyme has a sedimentation coefficient (S~0,w) of 5.40 S and a diffusion coefficient (D ~0.w)of 48.8/zm2/S. Using the Svedberg equation, the molecular weight of the native enzyme is 96,900. Thus the native enzyme is a dimer held together by noncovalent interactions: The functional significance of this finding is unclear. The purified enzyme is notoriously labile and must be handled with care. This may, in large part, be due to a strong tendency to aggregate. Certain maneuvers with the native enzyme, such as concentration by ultrafiltration, electrophoresis under nondenaturing conditions, and sedimentation equilibrium ultracentrifugation, are therefore precluded. How-

23 P.-H. Iverius, U. Lindahl, T. Egelrud, and T. Olivecrona, J. Biol. Chem. 247, 6610 (1972). z4 E. D. K o r n , J. Biol. Chem. 215, 1 0955). 25 T. Olivecrona and G. B e n g t s s o n , Biochim. Biophys. Acta 752, 38 (1983). 26 j. p. Segrest and R. L. Jackson, this series, Vol. 28B, p. 54.

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METABOLISM OF PLASMA LIPOPROTEINS

[42]

ever, aggregation and inactivation can be at least partially prevented in the presence of high concentrations of glycerol,6,27 glycine,6 and protein.27 Furthermore, the enzyme is also stabilized by heparin 23 and detergents such as Triton X - 1 0 0 , 7 Nonidet P - 4 0 , 6 and sodium deoxycholate. Acknowledgments This work was supported by grants from the American Diabetes Association, the National Institutes of Health (AM 02456), and the Swedish Medical Research Council (19X 07193). 27 J.-S. Twu, A. S. Garfinkel, and M. C. Schotz, Atherosclerosis 24, 119 (1976).

[42] A s s a y s of t h e in Vitro M e t a b o l i s m of V e r y - L o w - D e n s i t y L i p o p r o t e i n s in W h o l e P l a s m a b y P u r i f i e d Lipoprotein Lipase By BYUNG HONG CHUNG and JERE P. SEGREST Introduction Very-low-density lipoproteins (VLDL) are the major transport vesicles of triglycerides of hepatic origin, endogenous lipid. VLDL are the spherical particles consisting of triglyceride (60-70% of total mass) and small amounts of cholesteryl ester in the core, and phospholipid, free cholesterol, and apolipoproteins on the surface monolayer film. The metabolism of VLDL, in vivo, occurs on the capillary wall through the activity of lipoprotein lipase. 2 Lipoprotein lipase (triacylglycerol lipase, E.C. 3.1.1.3) is located on the endothelial surface of several extrahepatic tissues 3,4 and is released into the blood stream by heparin. 5 Lipoprotein lipase is also present in high concentration in the bovine raw milk of many mammals. 6 I L. E. Smith, H. J. Pownall, and A. M. Gotto, Annu. Rev. Biochem. 47, 751 (1978). 2 D. W. Bilheimer, S. Eisenberg, and R. I. Levy, Biochim. Biophys. Acta 260, 212 (1972). 3 C. F. Chung, G. M. Ousta, A. Bensadoun, and R. D. Rosenberg, J. Biol. Chem. 256, 12893 (1981). 4 K. Shimada, P. J. Gill, J. E. Silbert, W. H. J. Douglas, and B. L. Fanburg, J. Clin. Invest. 68, 995 (1981). 5 E. D. Korn, J. Biol. Chem. 215, I (1955). 6 T. Egelrud and T. Olivercrona, J. Biol. Chem. 247, 6212 (1972).

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