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intervals. The enzyme reactions are terminated in the aliquots by the addition of 3.25 ml of methanol/chloroform/heptane (100:88 : 70, v/v) and 1.0 ml of 0.14 M potassium borate buffer, pH 10.5. Released fatty acids are determined by liquid scintillation counting of the upper phase. Initial rates are determined for 10-15% hydrolysis in both assays.
[32] P h o s p h o l i p a s e A c t i v i t y of M i l k L i p o p r o t e i n L i p a s e B y G U N I L L A BENGTSSON-OLIVECRONA a n d THOMAS OLIVECRONA
Introduction Lipoprotein lipase (LpL; EC 3.1.1.34) is the enzyme which hydrolyzes triglycerides carried in chylomicrons and very low-density lipoproteins (VLDL).I'2 The released fatty acids are taken up by nearby cells for use in metabolic reactions or are recirculated in blood as albumin-bound free fatty acids. This reaction occurs at the endothelial surfaces of blood vessels, where the enzyme is bound by heparan sulfate proteoglycans. 3 The enzyme can be released from these sites by heparin, which has a higher affinity for the lipase than heparan sulfate h a s . 3 The structure of milk LpL is known from cloning of its cDNA 4 and from direct protein sequencing) The enzyme is structurally related to hepatic lipase and to pancreatic lipase. 6'7 An important corollary is that the three enzymes most likely have similar active sites. LpL has a broad substrate specificity: It has been shown to hydrolyze tri-, di-, and monoglycerides, phospholipids, and a variety of model subi A. S. Garfinkel and M. C. Schotz, in "Plasma Lipoproteins" (A. M. Gotto, ed.), p. 335. Elsevier, Amsterdam, 1987. 2 T. Olivecrona and G. Bengtsson-Olivecrona, in "Lipoprotein Lipase" (J. Borensztajn, ed.), p. 15. Evener, Chicago, 1987. 3 T. Olivecrona and G. Bengtsson-Olivecrona, in "Heparin" (D. Lane and U. Lindahl, eds.), p. 335. Edward Arnold, London, 1989. 4 M. Senda, K. Oka, W. V. Brown, P. K. Qasba, and Y. Furuichi, Proc. Natl. Acad. Sci. U.S.A. 84, 4369 (1987). 5 C.-Y. Yang, Z.-W. Gu, H.-X. Yang, M. F. Rohde, A. M. Gotto, Jr., and H. J. Pownall, J. Biol. Chem. 264, 16822 (1989). 6 S. Datta, C.-C. Lou, W.-H. Li, P. VanTuinen, D. H. Ledbetter, M. A. Brown, S.-H. Chen, S.-W. Liu, and L. Chart, J. Biol. Chem. 263, 1107 (1988). 7 F. S. Mickel, F. Weidenbach, B. Swarovsky, K. S. LaForge, and G. A. Scheele, J. Biol. Chem. 294, 12895 (1989). 8 T. Olivecrona and G. Bengtsson, in "Lipases" (B. Borgstr6m and H. L. Brockman, eds.), p. 206. Elsevier, Amsterdam, 1984.
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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strates (e.g., p-nitrophenyl esters). In the action on glycerides it shows specificity for the sn-l,3 ester bonds, with a preference for the sn-1 ester b o n d . 9 It does not show any marked preference for different fatty acids, except that ester bonds involving long polyunsaturated fatty acids (e.g., arachidonic acid and eicosapentaenoic acids) appear to be cleaved more slowly than ester bonds involving saturated or monounsaturated C16 or C18 fatty acids.l° This is true both for tri- and diglycerides and for phospholipids. Ester bonds involving phytanic acid are not cleaved, probably becatise the 3-methyl group causes steric hindrance.H The action of LpL is stimulated by apolipoprotein C-II (apoC-II). This activation appears to occur through binding of the apolipoprotein to the enzyme, which changes its conformation and/or its orientation at the lipid-water interface. Readers are referred to recent reviews for a discussion of the mechanisms and the structural requirements of this activation,2,8,12, ~3
Another characteristic property of LpL is that it is inhibited by fatty acids) 4,15 It has been suggested that this is an important mechanism for feedback control of the enzyme by its main product. The major molecular mechanism is that the enzyme binds to fatty acid aggregates, TM which results in sequestration of the enzyme away from substrate triglycerides. The inhibition is not overcome by activator protein but requires that the fatty acids be removed. In vivo the fatty acids are taken up by the underlying tissues and consumed in metabolic reactions. Albumin has a higher affinity for fatty acids than the lipase has, and it can be used to drive the reaction in oitro. Scow and Egelrud 16 were the first to show that LpL hydrolyzes not only triglycerides but also phospholipids in lipoproteins (rat chylomicrons). They demonstrated that the action on phospholipids shows the same positional specificity as that on triglycerides, namely, the enzyme cleaves only the primary ester bond. Later studies have shown that when large triglyceride-rich lipoproteins are degraded by LpL, phospholipid and triglyceride hydrolysis initially balance each other) 7 As the particle 9 N. Morley and A. Kuksis, J. Biol. Chem. 247, 6389 (1972). t0 B. Ekstr6m,/~ Nilsson, and B./~kesson, Eur. J. Clin. Invest. 19, 259 (1989). 11 S. Laurell, Biochim. Biophys. Acta 152, 80 (1968). 12 R. L. Jackson, in " T h e Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 14, p. 141. Academic Press, New York, 1983. 13 L. R. McLean, R. A. Demel, L. Socorro, M. Shinomiya, and R. L. Jackson, this series, Vol. 129, p. 738. 14 G. Bengtsson and T. Olivecrona, Eur. J. Biochem. 106, 557 (1980). 15 I. Posner and J. DeSanctis, Biochemistry 26, 3711 (1987). 16 R. O. Scow and T. Egelrud, Biochim. Biophys. Acta 431, 538 (1976). 17 S. Eisenberg and T. Olivecrona, J. Lipid Res. 20, 614 (1979).
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shrinks, phospholipid hydrolysis can, however, no longer keep pace with triglyceride depletion, and excess surface is generated which is shed from the particle and ultimately forms high-density lipoproteins (HDL). One factor contributing to this sequence of events is that in chylomicrons, as secreted from the intestine, 10-20% of the phospholipid is phosphatidylethanolamine. 18LpL hydrolyzes phosphatidylethanolamine more rapidly than phosphatidylcholine. LpL acts mainly on chylomicrons and VLDL 12,39and shows low activity against phospholipids in HDL.39 This is a major difference from hepatic lipase, which appears to prefer HDL as a substrate over chylomicrons or VLDL.32 Hepatic lipase and pancreatic lipase also have phospholipase activity. No rigorous comparison of the three enzymes has been made, but available evidence indicates that, for the same triglyceride activity, hepatic lipase has the highest phospholipase activity, and pancreatic lipase has the lowest. The enzymes also catalyze acyl transfer, 2° for example, Phospholipid + monoglyceride--~ lysophospholipid + diglyceride
Detailed studies of the phospholipase activity of LpL have been carded out by Stocks and Galton 23 and by Jackson and collaborators as part of their work on the mechanism by which apolipoprotein C-II stimulates LpL. These studies have been described in detail in an earlier volume in this series 33and are not reviewed here. Our aim is to describe methods to prepare and handle milk LpL and its activator protein, as well as conditions which need to be considered in kinetic studies. We do not discuss different types of substrate presentation (monolayers, mixed emulsions, liposomes, micelles). This is covered by other chapters in this volume. Purification LpL has been purified from postheparin plasma and from animal tissues. A preferred starting material is bovine milk. Milk contains I-2 mg active LpL per liter, and the enzyme can be purified by simple methods in yields approaching 50%. Milk does not contain significant amounts of other lipases or phospholipases. This has made LpL from bovine milk the most used model enzyme in LpL research. 2 In the lactating mammary gland LpL has an important role for uptake of lipids from plasma lipoproIS A. Nilsson, B. Landin, E. Jensen, and B. Akesson, Am. J. Physiol. 252, G817 (1987). 19 S. Eisenberg, D. Schurr, H. Goldman, and T. Olivecrona, Biochim. Biophys. Acta 531, 344 (1978). 20 M. Waite and P. Sisson, J. Biol. Chem. 248, 7985 (1973). ~l j. Stocks and D. Galton, Lipids 15, 186 (1980).
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teins. 22 It is not clear, however, why there is so much LpL in milk. In earlier literature it was generally assumed that the lipase "leaked" into the milk because there was so much in the mammary gland. More recent studies have shown that LPL is secreted into milk as efficiently as other milk proteins are. 23 Hence, the possibility that the enzyme has a useful function in milk may have to be reconsidered. Some possibilities were discussed in a recent review. 23 Several methods to prepare LpL from milk have been described. 24-28 All are based on chromatography on heparinSepharose, with some different additional steps. We describe here a simple method suitable for preparation of LpL for kinetic studies. Heparin-Agarose. Heparin-agarose is commercially available from Pharmacia LKB (Uppsala, Sweden), or it can also be prepared as described by Iverius. 29 Much material from skim milk adsorbs to the gel. To regenerate 100 ml of gel, it is suspended in 200 ml of 0.2 M Na2CO3 in 6 M urea, with gentle stirring for 1 hr. The slurry is filtered through a glass filter and washed with 200 ml of the same buffer and then with 1 liter of 2 M NaC1 followed by at least 2 liters of water. The gel is stored in water with 0.5% (v/v) butanol and is stable for years. Buffers. In earlier purification procedures 24-28 buffers of pH 7.4-8.5 have been used. We now use a 10 mM Bis-Tris/HCl buffer, pH 6.5 (Serva, Heidelberg, FRG). The lower pH enhances the binding of LpL to heparin) ° Make a stock solution of 20 mM Bis-Tris/HCl and mix this with water or 5 M NaC1 to obtain 10 mM Bis-Tris with the desired salt concentration. Milk. There is considerable variation in the amount of LpL in milk from different cows. 23 It is therefore advantageous to test a group of cows and select a few with high LpL activity. For each preparation we use 20 liters of milk obtained by machine-milking. Collect the milk in two 10-1iter plastic containers and chill in ice-water for 1-2 hr, until the temperature is below 10°. Chilling is important for three reasons: (1) LPL is not stable in milk at 37°, but loses activity with a half-time of about 4 hr23; (2) the physical properties of milk lipid droplets (cream) make them easier to remove by centrifugation of cold milk, because they form a mechanically 22 R. O. Scow and S. S. Chernick, in "Lipoprotein Lipase" (J. Borensztajn, ed.), p. 149. Evener, Chicago, 1987. 23 T. Olivecrona and G. Bengtsson-Olivecrona, in "Food Enzymology" (P. F. Fox, ed.), in press. Elsevier, Amsterdam, 1990. 24 T. Egelrud and T. Olivecrona, J. Biol. Chem. 247, 6212 (1972). P.-H. Iverius and A.-M. Ostlund-Lindqvist, J. Biol. Chem. 251, 7791 (1976). 26 P.-H. Iverius and A.-M. 0stlund-Lindqvist, this series, Vol. 129, p. 691. 27 L. Soccorro, G. C. Green, and R. L. Jackson, Prep. Biochem. IS, 133 (1985). 2~ I. Posner, C. S. Wang, and W. J. MacConathy, Arch. Biochem. Biophys. 226, 306 (1983). 29 P.-H. Iverius, Biochem. J. 124, 677 (1971). 30 G. Bengtsson-Olivecrona and T. Olivecrona, Biochem. J. 226, 409 (1985).
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stable fat cake; and (3) there is proteolytic activity in milk which degrades LpL to fragments. 31'32 Procedure. Centrifuge the chilled milk for 15 min at 3500 g, 4°. A swingout rotor makes the floating fat cake easier to handle. Holes are gently made in the fat cakes with a glass rod, and the skim milk (infranatant) is carefully decanted. The skim milk is filtered through glass wool to remove remaining fragments of the fat cake. The total volume is measured, and an aliquot is stored frozen for assay. Solid NaCl is added to the skim milk, 0.34 mol/liter. When the salt is fully dissolved, heparin-agarose is added (100 ml sedimented gel for 20 liters of milk). The mixture is gently stirred with a motor-operated stirring rod for 2 hr (4°). Avoid magnetic stirring since this may damage the agarose beads. The heparin-agarose is collected on a large glass-filter funnel (Schott, Mainz, FRG, pore size 2) by vacuum filtration (10°). The filter is easily clogged if lipid remains in the skim milk. A sample of the filtered skim milk is stored frozen for assay. The agarose gel (still on the filter funnel) is washed with 2 liters of Bis-Tris with 0.5 M NaC1 and then with 1 liter of Bis-Tris with 0.85 M NaC1 to remove proteins which have lower affinity for heparin than LpL has. The gel is then suspended and poured into a short column (diameter - 8 0 mm). The wash is continued with Bis-Tris with 0.9 M NaC1 until the absorbance at 280 nm of the effluent is below 0.03. This may take 1-2 hr at a flow rate of 2-3 ml/min. The salt concentration of this final wash must be adapted to the heparin-agarose preparation used, to remove the maximal amount of contaminating proteins but little or no LpL. LpL is then eluted with Bis-Tris with 1.5 M NaCl. Fractions of about 10 ml are collected, and peak fractions (from absorbance) are collected and pooled (usually 100-150 ml). They can be frozen and stored without significant loss of activity. An aliquot is stored frozen for assay, while the rest is dialyzed for 1 hr at 4° against 1 liter Bis-Tris with 0.5 M NaCI and then applied on a column of about 15 ml heparin-agarose (diameter 15 mm, flow rate 1 ml/min). The column is washed with Bis-Tris with 0.95 M NaC1 until the absorbance is below 0.02. LpL is then eluted by a gradient of Bis-Tris with 0.95 to 2.0 M NaCI (125 plus 125 ml, flow rate 1 ml/min, fraction volume 5 ml). Fractious from the protein peak are assayed for LpL activity and total protein immediately or after storage. The protein peak is usually asymmetrical. The specific activity is initially low but increases and approaches a constant value of about 600 U/rag (one unit is 1/.~mol fatty acid released per minute at 25° and pH 8.5) in the later fractions. The low specific activity in the leading edge of the 31 G. Bengtsson and T. Olivecrona, Eur. J. Biochem. 113, 547 (1981). 32 L. Socorro and R. Jackson, J. Biol. Chem. 2,60, 6324 (1985).
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peak is due to inactive forms of LpL and sometimes proteins other than LpL. With some milk samples antithrombin has been a major component preceding LpL. 2 In most samples of bovine milk the content of antithrombin is, however, low. LpL preparations always contain some proteolytic fragments.3m2 They are held together by disulfide bonds, and the cleaved molecules migrate together with intact LpL on heparin chromatography. For kinetic experiments the entire LpL peak can usually be pooled and used. In situations when pure LpL protein is desired, one should only use the later part of the peak and consider additional steps of purification (see below). Select fractions from the specific activity. The absorption coefficient (A 1~) for bovine LpL is 16.8 cm-1.33 The overall recovery of LpL activity is 25-40%. Comments. In the first batch adsorption only 50-75% of the LPL activity binds to heparin-agarose. The incomplete binding is presumably due to presence in milk of substances which compete for binding. If the unbound fraction is adsorbed to a new batch of heparin-agarose a similar fraction of the LpL activity binds. In the second step binding to heparinagarose is essentially complete. The amount of agarose gel should be kept low to reduce nonspecific binding. Variations and additions to this basic procedure have been proposed. Iverius and 0stlund-Lindqvist used selective adsorption, size fractionation, and ammonium sulfate precipitation to further purify material from heparin-agarose.25'26 Kinnunen included a nonionic detergent in the buffer to decrease nonspecific adsorption of protein to the gel. 34Ben-Avram et al. used dextran sulfate-agarose instead of heparin-agarose.35Socorro et al.27 advocate the use of phenylmethylsulfonyl fluoride (PMSF) to impede proteolytic cleavage of LpL during the preparation. Storage. The purified lipase (protein concentration 0.2-0.6 mg/ml) can be stored at - 7 0 ° or in liquid nitrogen for several months with minimal loss of lipase activity. The concentrated solution is also rather stable at 4° but is very unstable at higher temperatures, and it cannot be diluted in buffer without grave risk of loss of activity. It is advisable to include albumin (1-2 mg/ml), glycerol (20%, w/v), and/or detergents [e.g., 0.1 Triton X-100 or a combination of 1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS)]. Extensive dialysis (2-3 days) against 5 mM sodium deoxycholate, 10 mM Tris/HC1, pH 8.5, results in a rather stable solution of the enzyme.36 Addition of 0. I M sodium oleate or SDS to this further 33 T. Olivecrona, G. Bengtsson, and J. C. Osborne, Jr., Eur. J. Biochem. 124, 629 (1982). P. K. J. Kinnunen, Med. Biol. 55, 187 (1977). 35 C. M. Ben-Avram, O. Ben-Zeev, T. D. Lee, K. Haaga, J. E. Shively, J. Goers, M. E. Pedersen, J. R. Reeve, and M. C. Schotz, Proc. Natl. Acad. Sci. U.S.A. 83, 4185 (1986). G. Bengtsson and T. Olivecrona, Biochim. Biophys. Acta 575, 471 (1979).
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improves stability, so that the enzyme becomes stable even at room temperature and at low concentrations (e.g., 0.1/.~g/ml). This dramatic stabilization probably occurs through binding of anionic detergents to the enzyme. 36 The activity recovered when aliquots of LpL solutions are pipetted is often higher when the buffer contains detergents. This is due to better transfer of enzyme protein, as can be demonstrated with ~25I-labeled LpL. With dilute solutions of LpL without detergents a large fraction of the enzyme may stick to the pipette. Assay of Lipase Activity. A number of convenient assays have been described which use fatty acid-labeled triglycerides as substrate. The released fatty acids are extracted and then quantitated by liquid scintillation counting. These assays have been discussed in a recent review by NilssonE h l e 37 and are not described here. They suffer from variation between individual preparations of the substrate emulsion (usually prepared by sonication). To calibrate such assays and to monitor the specific activity of lipase preparations over several years, we use the commercial lipid emulsion Intralipid as substrate. 24 Release of fatty acids is quantitated by titration. Preparation of Apolipoprotein C-II ApoC-II is usually prepared from human plasma. For this, VLDL are isolated by ultracentrifugation, delipidated, and the apolipoproteins separated by chromatography. This has been detailed by Jackson and Holdsworth in a previous volume of this series) s Apolipoproteins C are present on HDL as well as on VLDL. If a lipid emulsion is added to plasma, apolipoproteins C will transfer to the emulsion droplets, which can be recovered by centrifugation in a regular centrifuge. This step increases the yield of apolipoproteins C and obviates extensive ultracentrifugation. We have applied this method successfully to human, bovine, 39and guinea pig plasma, TM both fresh and frozen. Lipid Emulsion. We use Intralipid, which is a commercial emulsion of soybean triglycerides in egg yolk phosphatidylcholine used for parenteral nutrition (AB KABI Nutrition, Stockholm, Sweden). This emulsion is provided in two concentrations, 10 and 20%. Both contain the same amount of phospholipids, 12 g/liter, but differ in the amount of triglycerides, 100 or 200 g/liter. Some of the phospholipids are present as liposome37 p. Nilsson-Ehle, in "Lipoprotein Lipase" (J. Borensztajn, ed.), p. 59. Evener, Chicago, 1987. 3s R. L. Jackson and G. Holdsworth, this series, Vol. 128, p. 288. 39 H. N. Astrup and G. Bengtsson, Comp. Biochem. Physiol. 72B, 487 (1982). 39a y . Andersson, L. Thelander, and G. Bengtsson-Olivecrona, J. Biol. Chem., in press (1991).
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like material. This amounts to about two-thirds of total phospholipids in the 10% preparation but only about one-third in the 20% preparation. Therefore, it is preferable to use the 20% preparation. Presumably, other lipid emulsions for parenteral nutrition can also be used. Other reagents required are Tris-buffered saline (TBS: 0.15 M NaCl, 0.1 M Tris/HCl, pH 7.4), sucrose, and chloroform/methanol (1 : 1 and 2 : 1, v/v, cold). Procedure. To separate the emulsion droplets from the excess phospholipid, sucrose is added to 10% (w/v) to increase the density of the Intralipid. The mixture is layered under 2 volumes of TBS and centrifuged at 4° for 90 min at 13,000 g. If possible, use a swing-out rotor. The fat cake is recovered and resuspended in TBS to the original volume. The washing procedure must be repeated twice to remove all excess phospholipid, but this is not necessary for semiquantitative preparative work. Then 5 volumes of bovine plasma is added per volume Intralipid, and the mixture is incubated in a water bath at 25° with gentle stirring for 30 min. Sucrose is then added to 10% (w/v), and the mixture is layered under 2 volumes of TBS and centrifuged for 90 min at 4° and 13,000 g. The fat cakes are resuspended in TBS and washed twice by centrifugation as above, but the final wash should be with water instead of TBS. The final fat cake should be recovered with as little liquid as possible and put into stainless steel centrifugation cups for delipidation. About 10 volume cold chloroform/ methanol (2: 1, v/v) is added. After stirring to a homogeneous sludge, another 20 volumes of chloroform/methanol (1 : 1, v/v) is added. After thorough stirring the mixture is centrifuged. The supernatant is carefully discarded, and the precipitated proteins are suspended in chloroform/ methanol (2:1 and then l : l , v/v) as described above. After another centrifugation the precipitated proteins are further washed with l0 volumes cold diethyl ether. After centrifugation the ether is carefully discarded and the protein left to dry at room temperature. Typically about 150 mg protein is obtained per liter of plasma. The delipidated proteins are separated by chromatography. This has been described by Jackson and Holdsworth in an earlier volume in this series. 38 We dissolve the proteins in 6 M guanidinium chloride in 10 mM Tris/HCl, pH 8.2, which is added directly to the centrifuge cups. The proteins are first separated by size via gel filtration on Sephacryl S-200 (Pharmacia LKB). This results in three major peaks. The first, excluded peak is mainly apolipoprotein B. The second peak contains a variety of plasma proteins including albumin and apolipoproteins A1 and E. The third peak contains the apolipoproteins C. These are further separated by ion-exchange chromatography on DEAE-cellulose in 5 M urea. The main components are isoforms of apolipoprotein C-III. The apoC-II proteins
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are identified by their ability to activate LpL. For this any of the assay systems with radioactive triglyceride substrate 37can be used. Activator is omitted from the assay mix, and a standard amount of LpL is added. The amount of enzyme should be chosen to give about 1% hydrolysis of the substrate in 30 min. Up to 5 /zl of the column fractions can be added directly to a 200-/zl assay. Larger volumes may result in inhibition of lipase by the guanidinium chloride or urea added.
Conditions for Incubations
Enzyme Stability. Enzyme stability is a major consideration for the design of incubation conditions. If one incubates LpL without heparin or lipid substrate the enzymes rapidly loses its activity, under some conditions, within minutes .2 Binding to emulsion droplets stabilizes the enzyme. This is why it is possible to obtain linear release of fatty acids during assay of LpL activity with triglyceride emulsions. Other substrate systems, for example, liposomes, micelles, soluble substrates, may or may not stabilize the enzyme. Factors that improve the stability are as follows: heparin, presumably by forming enzyme-heparin complexes; apoC-II (or serum as a source of the apolipoprotein), probably by forming enzyme-activator complexes; and other proteins (e.g., albumin), probably by covering nonspecific binding sites on glassware. Heparin. The diluted enzyme can usually be stabilized by heparin at concentrations of 1/zg/ml (corresponding to -0.15 IU/ml and - 0 . 1 / x M for most pharmaceutical preparations). The LpL-heparin complex is more stable and soluble than the enzyme alone 3 and is catalytically active. Therefore, heparin is included in most buffers used for extraction of LpL from tissue sources and for incubation of the enzyme in kinetic studies. There are reports that heparin may change the kinetic parameters for the enzyme under some conditions. 4° pH. The pH dependency observed for LpL activity is often a bellshaped curve with optimum around pH 8.5. The shape of the curve varies considerably, however, with the assay conditions. Closer study has revealed that the active site reaction itself increases with pH at least to pH 10. 41'42 The decrease at higher pH can be explained by decreased binding of the enzyme to the lipid-water interface and/or by decreased stability of the enzyme. 42 Lipase activity is usually measured at alkaline pH because the higher 4o I. Posner and J. Desanctis, Arch. Biochem. Biophys. 253, 475 (1987). 41 D. Rapp and T. Olivecrona, Eur. J. Biochem. 91, 379 (1978). 42 G. Bengtsson and T. Olivecrona, Biochim. Biophys. Acta 712, 196 (1982).
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rate makes the assay more sensitive. It should be pointed out, however, that the enzyme has high activity also at physiological pH (7.4). Other aspects of the reaction may be sensitive to pH, for example, lipid packing, lipid-protein interactions, and the rate at which partial glycerides/lysophospholipids are isomerized. Therefore, pH 7.4 is recommended for studies which explore physiological reactions, for example, lipoprotein interconversion. Temperature. Already in 1968 Greten, Levy, and Fredrickson reported that LpL-catalyzed release of fatty acids was linear at room temperature but not at 370. 43 This was because, under their assay conditions, the enzyme was not stable at 37°. This important observation has been overlooked in many subsequent studies. Unless there is a specific reason to use 37° (e.g., to study lipid transitions), it is recommended that incubations be carried at 25 ° or below. Buffer Composition. LpL is sometimes defined as the salt-sensitive lipase. It is now clear, however, that the enzyme can exert full catalytic activity even in the presence of 1 M NaCI. 2m'42 The effect of salt is not exerted at this level but on the physical state and the stability of the enzyme molecule. For most purposes NaCI concentrations of 0.05-0.15 M result in optimal activity and stability for kinetic studies. Albumin. When triglycerides in emulsion droplets/lipoproteins are hydrolyzed by LpL, hydrophobic core molecules are converted to surfaceactive products which locate at the interface. In the absence of an acceptor, the fatty acids and monoglycerides form extensions of the surface film and separate bilayer structures. 44'45 Sequestration of the lipase in these structures is probably the main reason for the inhibition of lipase actionfl In these systems it is necessary to include albumin to bind the fatty acids. The molar ratio of fatty acids to albumin should not exceed 5 at any time. Most incubation systems for triglyceride hydrolysis contain 30-60 mg/ml albumin. For most purposes it is not necessary to use fatty acid-free albumin preparations. With liposomes and micelles the substrate molecules are surface-located, and hydrolysis does not result in overcrowding of the surface. Albumin is less important here. Its main effect will be to impede transesterification reactions. Furthermore, albumin at concentrations of a few milligrams per milliliter is useful to prevent adsorption of the lipase to glassware. Some commercial albumin preparations contain traces of lipids and 43 H. Greten, R. I. Levy, and D. S. Fredrickson, Biochim. Biophys. Acta 164, 185 (1968). 44 R. O. Scow and J. Blanchette-Mackie, Prog. Lipid Res. 24, 197 (1985). 45 D. P. Cistola, J. A. Hamilton, D. Jackson, and D. M. Small, Biochemistry 27, 1881 (1988).
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apolipoproteins. 19.46This can have profound effects on the kinetics of LpLcatalyzed reactions and on the product profiles. We use bovine albumin, fraction V powder from Sigma Chemical Co (St. Louis, MO). For each new batch we check that the albumin, in high concentrations, does not cause inhibition of the lipase. Activator. With triglyceride emulsions as substrate the activity of LpL is increased severalfold by apoC-II. The concentration of apoC-II needed varies, but 1 /.~g/ml (10 -7 M ) is USUally enough for maximal or nearmaximal activation. The relevant parameter is probably the two-dimensional concentration of apoC-II at the lipid-water interface.47 Hence, more apoC-II is needed with higher concentrations of lipid substrate. ApoC-II tends to aggregate in solution. To prevent this we use 3 M guanidinium chloride in stock solutions (4.4 mg/ml, 0.5 mM) of the apolipoprotein. For assay purposes, whole serum or HDL can be used to provide activator. The effect of C-II varies greatly with the substrate system. With most triglyceride emulsions the enzyme displays activity even without the activator. This basal activity is usually 5-20% but can approach 100% of optimal activity, z8 With phospholipid liposomes the basal activity is often low. This aspect must be checked for each substrate system studied and the optimal amount of activator determined. Other Proteins. It has been reported that the activity of LpL is inhibited by a variety of proteins. The mechanism(s) of the inhibition has not been explored in detail. It is probably nonspecific since inhibition is seen with many different proteins. 2 In no case has a specific protein-protein interaction between the lipase and an inhibitory protein been demonstrated. The common denominator seems to be that the proteins bind to the surface of the substrate droplets. Concluding Remarks LpL should be a useful reagent to prepare 2-acyllysophospholipids. Because of its higher relative activity against phospholipids, hepatic lipase could be even better for this purpose. A drawback is that starting material for purification of this enzyme (usually postheparin plasma) is more difficult to obtain. During its action on chylomicrons and VLDL, LpL hydrolyzes most of the triglycerides and some of the phospholipids. Other phospholipids are transferred to the HDL fraction. The balance between these two 46 R. J. Deckelbaum, T. Olivecrona, and M. Fainaru, J. Lipid Res. 21, 425 (1980). 47 R. L. Jackson, S. Tajima, T. Yamamura, S. Yokoyama, and A. Yamamoto, Biochim. Biophys. Acta 875, 211 (1986).
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pathways may be an important determinant of plasma HDL levels. Little is known, however, of what factors govern the relative activity of LpL on phospholipids and triglycerides in emulsions and in lipoproteins. This is an interesting question for future studies. Acknowledgments Our studieson LpL are supportedby GrantB13X-727fromthe SwedishMedicalResearch Council.
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intervals. The enzyme reactions are terminated in the aliquots by the addition of 3.25 ml of methanol/chloroform/heptane (100:88 : 70, v/v) and 1.0 ml of 0.14 M potassium borate buffer, pH 10.5. Released fatty acids are determined by liquid scintillation counting of the upper phase. Initial rates are determined for 10-15% hydrolysis in both assays.
[32] P h o s p h o l i p a s e A c t i v i t y of M i l k L i p o p r o t e i n L i p a s e B y G U N I L L A BENGTSSON-OLIVECRONA a n d THOMAS OLIVECRONA
Introduction Lipoprotein lipase (LpL; EC 3.1.1.34) is the enzyme which hydrolyzes triglycerides carried in chylomicrons and very low-density lipoproteins (VLDL).I'2 The released fatty acids are taken up by nearby cells for use in metabolic reactions or are recirculated in blood as albumin-bound free fatty acids. This reaction occurs at the endothelial surfaces of blood vessels, where the enzyme is bound by heparan sulfate proteoglycans. 3 The enzyme can be released from these sites by heparin, which has a higher affinity for the lipase than heparan sulfate h a s . 3 The structure of milk LpL is known from cloning of its cDNA 4 and from direct protein sequencing) The enzyme is structurally related to hepatic lipase and to pancreatic lipase. 6'7 An important corollary is that the three enzymes most likely have similar active sites. LpL has a broad substrate specificity: It has been shown to hydrolyze tri-, di-, and monoglycerides, phospholipids, and a variety of model subi A. S. Garfinkel and M. C. Schotz, in "Plasma Lipoproteins" (A. M. Gotto, ed.), p. 335. Elsevier, Amsterdam, 1987. 2 T. Olivecrona and G. Bengtsson-Olivecrona, in "Lipoprotein Lipase" (J. Borensztajn, ed.), p. 15. Evener, Chicago, 1987. 3 T. Olivecrona and G. Bengtsson-Olivecrona, in "Heparin" (D. Lane and U. Lindahl, eds.), p. 335. Edward Arnold, London, 1989. 4 M. Senda, K. Oka, W. V. Brown, P. K. Qasba, and Y. Furuichi, Proc. Natl. Acad. Sci. U.S.A. 84, 4369 (1987). 5 C.-Y. Yang, Z.-W. Gu, H.-X. Yang, M. F. Rohde, A. M. Gotto, Jr., and H. J. Pownall, J. Biol. Chem. 264, 16822 (1989). 6 S. Datta, C.-C. Lou, W.-H. Li, P. VanTuinen, D. H. Ledbetter, M. A. Brown, S.-H. Chen, S.-W. Liu, and L. Chart, J. Biol. Chem. 263, 1107 (1988). 7 F. S. Mickel, F. Weidenbach, B. Swarovsky, K. S. LaForge, and G. A. Scheele, J. Biol. Chem. 294, 12895 (1989). 8 T. Olivecrona and G. Bengtsson, in "Lipases" (B. Borgstr6m and H. L. Brockman, eds.), p. 206. Elsevier, Amsterdam, 1984.
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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strates (e.g., p-nitrophenyl esters). In the action on glycerides it shows specificity for the sn-l,3 ester bonds, with a preference for the sn-1 ester b o n d . 9 It does not show any marked preference for different fatty acids, except that ester bonds involving long polyunsaturated fatty acids (e.g., arachidonic acid and eicosapentaenoic acids) appear to be cleaved more slowly than ester bonds involving saturated or monounsaturated C16 or C18 fatty acids.l° This is true both for tri- and diglycerides and for phospholipids. Ester bonds involving phytanic acid are not cleaved, probably becatise the 3-methyl group causes steric hindrance.H The action of LpL is stimulated by apolipoprotein C-II (apoC-II). This activation appears to occur through binding of the apolipoprotein to the enzyme, which changes its conformation and/or its orientation at the lipid-water interface. Readers are referred to recent reviews for a discussion of the mechanisms and the structural requirements of this activation,2,8,12, ~3
Another characteristic property of LpL is that it is inhibited by fatty acids) 4,15 It has been suggested that this is an important mechanism for feedback control of the enzyme by its main product. The major molecular mechanism is that the enzyme binds to fatty acid aggregates, TM which results in sequestration of the enzyme away from substrate triglycerides. The inhibition is not overcome by activator protein but requires that the fatty acids be removed. In vivo the fatty acids are taken up by the underlying tissues and consumed in metabolic reactions. Albumin has a higher affinity for fatty acids than the lipase has, and it can be used to drive the reaction in oitro. Scow and Egelrud 16 were the first to show that LpL hydrolyzes not only triglycerides but also phospholipids in lipoproteins (rat chylomicrons). They demonstrated that the action on phospholipids shows the same positional specificity as that on triglycerides, namely, the enzyme cleaves only the primary ester bond. Later studies have shown that when large triglyceride-rich lipoproteins are degraded by LpL, phospholipid and triglyceride hydrolysis initially balance each other) 7 As the particle 9 N. Morley and A. Kuksis, J. Biol. Chem. 247, 6389 (1972). t0 B. Ekstr6m,/~ Nilsson, and B./~kesson, Eur. J. Clin. Invest. 19, 259 (1989). 11 S. Laurell, Biochim. Biophys. Acta 152, 80 (1968). 12 R. L. Jackson, in " T h e Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 14, p. 141. Academic Press, New York, 1983. 13 L. R. McLean, R. A. Demel, L. Socorro, M. Shinomiya, and R. L. Jackson, this series, Vol. 129, p. 738. 14 G. Bengtsson and T. Olivecrona, Eur. J. Biochem. 106, 557 (1980). 15 I. Posner and J. DeSanctis, Biochemistry 26, 3711 (1987). 16 R. O. Scow and T. Egelrud, Biochim. Biophys. Acta 431, 538 (1976). 17 S. Eisenberg and T. Olivecrona, J. Lipid Res. 20, 614 (1979).
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shrinks, phospholipid hydrolysis can, however, no longer keep pace with triglyceride depletion, and excess surface is generated which is shed from the particle and ultimately forms high-density lipoproteins (HDL). One factor contributing to this sequence of events is that in chylomicrons, as secreted from the intestine, 10-20% of the phospholipid is phosphatidylethanolamine. 18LpL hydrolyzes phosphatidylethanolamine more rapidly than phosphatidylcholine. LpL acts mainly on chylomicrons and VLDL 12,39and shows low activity against phospholipids in HDL.39 This is a major difference from hepatic lipase, which appears to prefer HDL as a substrate over chylomicrons or VLDL.32 Hepatic lipase and pancreatic lipase also have phospholipase activity. No rigorous comparison of the three enzymes has been made, but available evidence indicates that, for the same triglyceride activity, hepatic lipase has the highest phospholipase activity, and pancreatic lipase has the lowest. The enzymes also catalyze acyl transfer, 2° for example, Phospholipid + monoglyceride--~ lysophospholipid + diglyceride
Detailed studies of the phospholipase activity of LpL have been carded out by Stocks and Galton 23 and by Jackson and collaborators as part of their work on the mechanism by which apolipoprotein C-II stimulates LpL. These studies have been described in detail in an earlier volume in this series 33and are not reviewed here. Our aim is to describe methods to prepare and handle milk LpL and its activator protein, as well as conditions which need to be considered in kinetic studies. We do not discuss different types of substrate presentation (monolayers, mixed emulsions, liposomes, micelles). This is covered by other chapters in this volume. Purification LpL has been purified from postheparin plasma and from animal tissues. A preferred starting material is bovine milk. Milk contains I-2 mg active LpL per liter, and the enzyme can be purified by simple methods in yields approaching 50%. Milk does not contain significant amounts of other lipases or phospholipases. This has made LpL from bovine milk the most used model enzyme in LpL research. 2 In the lactating mammary gland LpL has an important role for uptake of lipids from plasma lipoproIS A. Nilsson, B. Landin, E. Jensen, and B. Akesson, Am. J. Physiol. 252, G817 (1987). 19 S. Eisenberg, D. Schurr, H. Goldman, and T. Olivecrona, Biochim. Biophys. Acta 531, 344 (1978). 20 M. Waite and P. Sisson, J. Biol. Chem. 248, 7985 (1973). ~l j. Stocks and D. Galton, Lipids 15, 186 (1980).
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teins. 22 It is not clear, however, why there is so much LpL in milk. In earlier literature it was generally assumed that the lipase "leaked" into the milk because there was so much in the mammary gland. More recent studies have shown that LPL is secreted into milk as efficiently as other milk proteins are. 23 Hence, the possibility that the enzyme has a useful function in milk may have to be reconsidered. Some possibilities were discussed in a recent review. 23 Several methods to prepare LpL from milk have been described. 24-28 All are based on chromatography on heparinSepharose, with some different additional steps. We describe here a simple method suitable for preparation of LpL for kinetic studies. Heparin-Agarose. Heparin-agarose is commercially available from Pharmacia LKB (Uppsala, Sweden), or it can also be prepared as described by Iverius. 29 Much material from skim milk adsorbs to the gel. To regenerate 100 ml of gel, it is suspended in 200 ml of 0.2 M Na2CO3 in 6 M urea, with gentle stirring for 1 hr. The slurry is filtered through a glass filter and washed with 200 ml of the same buffer and then with 1 liter of 2 M NaC1 followed by at least 2 liters of water. The gel is stored in water with 0.5% (v/v) butanol and is stable for years. Buffers. In earlier purification procedures 24-28 buffers of pH 7.4-8.5 have been used. We now use a 10 mM Bis-Tris/HCl buffer, pH 6.5 (Serva, Heidelberg, FRG). The lower pH enhances the binding of LpL to heparin) ° Make a stock solution of 20 mM Bis-Tris/HCl and mix this with water or 5 M NaC1 to obtain 10 mM Bis-Tris with the desired salt concentration. Milk. There is considerable variation in the amount of LpL in milk from different cows. 23 It is therefore advantageous to test a group of cows and select a few with high LpL activity. For each preparation we use 20 liters of milk obtained by machine-milking. Collect the milk in two 10-1iter plastic containers and chill in ice-water for 1-2 hr, until the temperature is below 10°. Chilling is important for three reasons: (1) LPL is not stable in milk at 37°, but loses activity with a half-time of about 4 hr23; (2) the physical properties of milk lipid droplets (cream) make them easier to remove by centrifugation of cold milk, because they form a mechanically 22 R. O. Scow and S. S. Chernick, in "Lipoprotein Lipase" (J. Borensztajn, ed.), p. 149. Evener, Chicago, 1987. 23 T. Olivecrona and G. Bengtsson-Olivecrona, in "Food Enzymology" (P. F. Fox, ed.), in press. Elsevier, Amsterdam, 1990. 24 T. Egelrud and T. Olivecrona, J. Biol. Chem. 247, 6212 (1972). P.-H. Iverius and A.-M. Ostlund-Lindqvist, J. Biol. Chem. 251, 7791 (1976). 26 P.-H. Iverius and A.-M. 0stlund-Lindqvist, this series, Vol. 129, p. 691. 27 L. Soccorro, G. C. Green, and R. L. Jackson, Prep. Biochem. IS, 133 (1985). 2~ I. Posner, C. S. Wang, and W. J. MacConathy, Arch. Biochem. Biophys. 226, 306 (1983). 29 P.-H. Iverius, Biochem. J. 124, 677 (1971). 30 G. Bengtsson-Olivecrona and T. Olivecrona, Biochem. J. 226, 409 (1985).
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stable fat cake; and (3) there is proteolytic activity in milk which degrades LpL to fragments. 31'32 Procedure. Centrifuge the chilled milk for 15 min at 3500 g, 4°. A swingout rotor makes the floating fat cake easier to handle. Holes are gently made in the fat cakes with a glass rod, and the skim milk (infranatant) is carefully decanted. The skim milk is filtered through glass wool to remove remaining fragments of the fat cake. The total volume is measured, and an aliquot is stored frozen for assay. Solid NaCl is added to the skim milk, 0.34 mol/liter. When the salt is fully dissolved, heparin-agarose is added (100 ml sedimented gel for 20 liters of milk). The mixture is gently stirred with a motor-operated stirring rod for 2 hr (4°). Avoid magnetic stirring since this may damage the agarose beads. The heparin-agarose is collected on a large glass-filter funnel (Schott, Mainz, FRG, pore size 2) by vacuum filtration (10°). The filter is easily clogged if lipid remains in the skim milk. A sample of the filtered skim milk is stored frozen for assay. The agarose gel (still on the filter funnel) is washed with 2 liters of Bis-Tris with 0.5 M NaC1 and then with 1 liter of Bis-Tris with 0.85 M NaC1 to remove proteins which have lower affinity for heparin than LpL has. The gel is then suspended and poured into a short column (diameter - 8 0 mm). The wash is continued with Bis-Tris with 0.9 M NaC1 until the absorbance at 280 nm of the effluent is below 0.03. This may take 1-2 hr at a flow rate of 2-3 ml/min. The salt concentration of this final wash must be adapted to the heparin-agarose preparation used, to remove the maximal amount of contaminating proteins but little or no LpL. LpL is then eluted with Bis-Tris with 1.5 M NaCl. Fractions of about 10 ml are collected, and peak fractions (from absorbance) are collected and pooled (usually 100-150 ml). They can be frozen and stored without significant loss of activity. An aliquot is stored frozen for assay, while the rest is dialyzed for 1 hr at 4° against 1 liter Bis-Tris with 0.5 M NaCI and then applied on a column of about 15 ml heparin-agarose (diameter 15 mm, flow rate 1 ml/min). The column is washed with Bis-Tris with 0.95 M NaC1 until the absorbance is below 0.02. LpL is then eluted by a gradient of Bis-Tris with 0.95 to 2.0 M NaCI (125 plus 125 ml, flow rate 1 ml/min, fraction volume 5 ml). Fractious from the protein peak are assayed for LpL activity and total protein immediately or after storage. The protein peak is usually asymmetrical. The specific activity is initially low but increases and approaches a constant value of about 600 U/rag (one unit is 1/.~mol fatty acid released per minute at 25° and pH 8.5) in the later fractions. The low specific activity in the leading edge of the 31 G. Bengtsson and T. Olivecrona, Eur. J. Biochem. 113, 547 (1981). 32 L. Socorro and R. Jackson, J. Biol. Chem. 2,60, 6324 (1985).
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peak is due to inactive forms of LpL and sometimes proteins other than LpL. With some milk samples antithrombin has been a major component preceding LpL. 2 In most samples of bovine milk the content of antithrombin is, however, low. LpL preparations always contain some proteolytic fragments.3m2 They are held together by disulfide bonds, and the cleaved molecules migrate together with intact LpL on heparin chromatography. For kinetic experiments the entire LpL peak can usually be pooled and used. In situations when pure LpL protein is desired, one should only use the later part of the peak and consider additional steps of purification (see below). Select fractions from the specific activity. The absorption coefficient (A 1~) for bovine LpL is 16.8 cm-1.33 The overall recovery of LpL activity is 25-40%. Comments. In the first batch adsorption only 50-75% of the LPL activity binds to heparin-agarose. The incomplete binding is presumably due to presence in milk of substances which compete for binding. If the unbound fraction is adsorbed to a new batch of heparin-agarose a similar fraction of the LpL activity binds. In the second step binding to heparinagarose is essentially complete. The amount of agarose gel should be kept low to reduce nonspecific binding. Variations and additions to this basic procedure have been proposed. Iverius and 0stlund-Lindqvist used selective adsorption, size fractionation, and ammonium sulfate precipitation to further purify material from heparin-agarose.25'26 Kinnunen included a nonionic detergent in the buffer to decrease nonspecific adsorption of protein to the gel. 34Ben-Avram et al. used dextran sulfate-agarose instead of heparin-agarose.35Socorro et al.27 advocate the use of phenylmethylsulfonyl fluoride (PMSF) to impede proteolytic cleavage of LpL during the preparation. Storage. The purified lipase (protein concentration 0.2-0.6 mg/ml) can be stored at - 7 0 ° or in liquid nitrogen for several months with minimal loss of lipase activity. The concentrated solution is also rather stable at 4° but is very unstable at higher temperatures, and it cannot be diluted in buffer without grave risk of loss of activity. It is advisable to include albumin (1-2 mg/ml), glycerol (20%, w/v), and/or detergents [e.g., 0.1 Triton X-100 or a combination of 1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS)]. Extensive dialysis (2-3 days) against 5 mM sodium deoxycholate, 10 mM Tris/HC1, pH 8.5, results in a rather stable solution of the enzyme.36 Addition of 0. I M sodium oleate or SDS to this further 33 T. Olivecrona, G. Bengtsson, and J. C. Osborne, Jr., Eur. J. Biochem. 124, 629 (1982). P. K. J. Kinnunen, Med. Biol. 55, 187 (1977). 35 C. M. Ben-Avram, O. Ben-Zeev, T. D. Lee, K. Haaga, J. E. Shively, J. Goers, M. E. Pedersen, J. R. Reeve, and M. C. Schotz, Proc. Natl. Acad. Sci. U.S.A. 83, 4185 (1986). G. Bengtsson and T. Olivecrona, Biochim. Biophys. Acta 575, 471 (1979).
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improves stability, so that the enzyme becomes stable even at room temperature and at low concentrations (e.g., 0.1/.~g/ml). This dramatic stabilization probably occurs through binding of anionic detergents to the enzyme. 36 The activity recovered when aliquots of LpL solutions are pipetted is often higher when the buffer contains detergents. This is due to better transfer of enzyme protein, as can be demonstrated with ~25I-labeled LpL. With dilute solutions of LpL without detergents a large fraction of the enzyme may stick to the pipette. Assay of Lipase Activity. A number of convenient assays have been described which use fatty acid-labeled triglycerides as substrate. The released fatty acids are extracted and then quantitated by liquid scintillation counting. These assays have been discussed in a recent review by NilssonE h l e 37 and are not described here. They suffer from variation between individual preparations of the substrate emulsion (usually prepared by sonication). To calibrate such assays and to monitor the specific activity of lipase preparations over several years, we use the commercial lipid emulsion Intralipid as substrate. 24 Release of fatty acids is quantitated by titration. Preparation of Apolipoprotein C-II ApoC-II is usually prepared from human plasma. For this, VLDL are isolated by ultracentrifugation, delipidated, and the apolipoproteins separated by chromatography. This has been detailed by Jackson and Holdsworth in a previous volume of this series) s Apolipoproteins C are present on HDL as well as on VLDL. If a lipid emulsion is added to plasma, apolipoproteins C will transfer to the emulsion droplets, which can be recovered by centrifugation in a regular centrifuge. This step increases the yield of apolipoproteins C and obviates extensive ultracentrifugation. We have applied this method successfully to human, bovine, 39and guinea pig plasma, TM both fresh and frozen. Lipid Emulsion. We use Intralipid, which is a commercial emulsion of soybean triglycerides in egg yolk phosphatidylcholine used for parenteral nutrition (AB KABI Nutrition, Stockholm, Sweden). This emulsion is provided in two concentrations, 10 and 20%. Both contain the same amount of phospholipids, 12 g/liter, but differ in the amount of triglycerides, 100 or 200 g/liter. Some of the phospholipids are present as liposome37 p. Nilsson-Ehle, in "Lipoprotein Lipase" (J. Borensztajn, ed.), p. 59. Evener, Chicago, 1987. 3s R. L. Jackson and G. Holdsworth, this series, Vol. 128, p. 288. 39 H. N. Astrup and G. Bengtsson, Comp. Biochem. Physiol. 72B, 487 (1982). 39a y . Andersson, L. Thelander, and G. Bengtsson-Olivecrona, J. Biol. Chem., in press (1991).
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like material. This amounts to about two-thirds of total phospholipids in the 10% preparation but only about one-third in the 20% preparation. Therefore, it is preferable to use the 20% preparation. Presumably, other lipid emulsions for parenteral nutrition can also be used. Other reagents required are Tris-buffered saline (TBS: 0.15 M NaCl, 0.1 M Tris/HCl, pH 7.4), sucrose, and chloroform/methanol (1 : 1 and 2 : 1, v/v, cold). Procedure. To separate the emulsion droplets from the excess phospholipid, sucrose is added to 10% (w/v) to increase the density of the Intralipid. The mixture is layered under 2 volumes of TBS and centrifuged at 4° for 90 min at 13,000 g. If possible, use a swing-out rotor. The fat cake is recovered and resuspended in TBS to the original volume. The washing procedure must be repeated twice to remove all excess phospholipid, but this is not necessary for semiquantitative preparative work. Then 5 volumes of bovine plasma is added per volume Intralipid, and the mixture is incubated in a water bath at 25° with gentle stirring for 30 min. Sucrose is then added to 10% (w/v), and the mixture is layered under 2 volumes of TBS and centrifuged for 90 min at 4° and 13,000 g. The fat cakes are resuspended in TBS and washed twice by centrifugation as above, but the final wash should be with water instead of TBS. The final fat cake should be recovered with as little liquid as possible and put into stainless steel centrifugation cups for delipidation. About 10 volume cold chloroform/ methanol (2: 1, v/v) is added. After stirring to a homogeneous sludge, another 20 volumes of chloroform/methanol (1 : 1, v/v) is added. After thorough stirring the mixture is centrifuged. The supernatant is carefully discarded, and the precipitated proteins are suspended in chloroform/ methanol (2:1 and then l : l , v/v) as described above. After another centrifugation the precipitated proteins are further washed with l0 volumes cold diethyl ether. After centrifugation the ether is carefully discarded and the protein left to dry at room temperature. Typically about 150 mg protein is obtained per liter of plasma. The delipidated proteins are separated by chromatography. This has been described by Jackson and Holdsworth in an earlier volume in this series. 38 We dissolve the proteins in 6 M guanidinium chloride in 10 mM Tris/HCl, pH 8.2, which is added directly to the centrifuge cups. The proteins are first separated by size via gel filtration on Sephacryl S-200 (Pharmacia LKB). This results in three major peaks. The first, excluded peak is mainly apolipoprotein B. The second peak contains a variety of plasma proteins including albumin and apolipoproteins A1 and E. The third peak contains the apolipoproteins C. These are further separated by ion-exchange chromatography on DEAE-cellulose in 5 M urea. The main components are isoforms of apolipoprotein C-III. The apoC-II proteins
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are identified by their ability to activate LpL. For this any of the assay systems with radioactive triglyceride substrate 37can be used. Activator is omitted from the assay mix, and a standard amount of LpL is added. The amount of enzyme should be chosen to give about 1% hydrolysis of the substrate in 30 min. Up to 5 /zl of the column fractions can be added directly to a 200-/zl assay. Larger volumes may result in inhibition of lipase by the guanidinium chloride or urea added.
Conditions for Incubations
Enzyme Stability. Enzyme stability is a major consideration for the design of incubation conditions. If one incubates LpL without heparin or lipid substrate the enzymes rapidly loses its activity, under some conditions, within minutes .2 Binding to emulsion droplets stabilizes the enzyme. This is why it is possible to obtain linear release of fatty acids during assay of LpL activity with triglyceride emulsions. Other substrate systems, for example, liposomes, micelles, soluble substrates, may or may not stabilize the enzyme. Factors that improve the stability are as follows: heparin, presumably by forming enzyme-heparin complexes; apoC-II (or serum as a source of the apolipoprotein), probably by forming enzyme-activator complexes; and other proteins (e.g., albumin), probably by covering nonspecific binding sites on glassware. Heparin. The diluted enzyme can usually be stabilized by heparin at concentrations of 1/zg/ml (corresponding to -0.15 IU/ml and - 0 . 1 / x M for most pharmaceutical preparations). The LpL-heparin complex is more stable and soluble than the enzyme alone 3 and is catalytically active. Therefore, heparin is included in most buffers used for extraction of LpL from tissue sources and for incubation of the enzyme in kinetic studies. There are reports that heparin may change the kinetic parameters for the enzyme under some conditions. 4° pH. The pH dependency observed for LpL activity is often a bellshaped curve with optimum around pH 8.5. The shape of the curve varies considerably, however, with the assay conditions. Closer study has revealed that the active site reaction itself increases with pH at least to pH 10. 41'42 The decrease at higher pH can be explained by decreased binding of the enzyme to the lipid-water interface and/or by decreased stability of the enzyme. 42 Lipase activity is usually measured at alkaline pH because the higher 4o I. Posner and J. Desanctis, Arch. Biochem. Biophys. 253, 475 (1987). 41 D. Rapp and T. Olivecrona, Eur. J. Biochem. 91, 379 (1978). 42 G. Bengtsson and T. Olivecrona, Biochim. Biophys. Acta 712, 196 (1982).
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rate makes the assay more sensitive. It should be pointed out, however, that the enzyme has high activity also at physiological pH (7.4). Other aspects of the reaction may be sensitive to pH, for example, lipid packing, lipid-protein interactions, and the rate at which partial glycerides/lysophospholipids are isomerized. Therefore, pH 7.4 is recommended for studies which explore physiological reactions, for example, lipoprotein interconversion. Temperature. Already in 1968 Greten, Levy, and Fredrickson reported that LpL-catalyzed release of fatty acids was linear at room temperature but not at 370. 43 This was because, under their assay conditions, the enzyme was not stable at 37°. This important observation has been overlooked in many subsequent studies. Unless there is a specific reason to use 37° (e.g., to study lipid transitions), it is recommended that incubations be carried at 25 ° or below. Buffer Composition. LpL is sometimes defined as the salt-sensitive lipase. It is now clear, however, that the enzyme can exert full catalytic activity even in the presence of 1 M NaCI. 2m'42 The effect of salt is not exerted at this level but on the physical state and the stability of the enzyme molecule. For most purposes NaCI concentrations of 0.05-0.15 M result in optimal activity and stability for kinetic studies. Albumin. When triglycerides in emulsion droplets/lipoproteins are hydrolyzed by LpL, hydrophobic core molecules are converted to surfaceactive products which locate at the interface. In the absence of an acceptor, the fatty acids and monoglycerides form extensions of the surface film and separate bilayer structures. 44'45 Sequestration of the lipase in these structures is probably the main reason for the inhibition of lipase actionfl In these systems it is necessary to include albumin to bind the fatty acids. The molar ratio of fatty acids to albumin should not exceed 5 at any time. Most incubation systems for triglyceride hydrolysis contain 30-60 mg/ml albumin. For most purposes it is not necessary to use fatty acid-free albumin preparations. With liposomes and micelles the substrate molecules are surface-located, and hydrolysis does not result in overcrowding of the surface. Albumin is less important here. Its main effect will be to impede transesterification reactions. Furthermore, albumin at concentrations of a few milligrams per milliliter is useful to prevent adsorption of the lipase to glassware. Some commercial albumin preparations contain traces of lipids and 43 H. Greten, R. I. Levy, and D. S. Fredrickson, Biochim. Biophys. Acta 164, 185 (1968). 44 R. O. Scow and J. Blanchette-Mackie, Prog. Lipid Res. 24, 197 (1985). 45 D. P. Cistola, J. A. Hamilton, D. Jackson, and D. M. Small, Biochemistry 27, 1881 (1988).
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apolipoproteins. 19.46This can have profound effects on the kinetics of LpLcatalyzed reactions and on the product profiles. We use bovine albumin, fraction V powder from Sigma Chemical Co (St. Louis, MO). For each new batch we check that the albumin, in high concentrations, does not cause inhibition of the lipase. Activator. With triglyceride emulsions as substrate the activity of LpL is increased severalfold by apoC-II. The concentration of apoC-II needed varies, but 1 /.~g/ml (10 -7 M ) is USUally enough for maximal or nearmaximal activation. The relevant parameter is probably the two-dimensional concentration of apoC-II at the lipid-water interface.47 Hence, more apoC-II is needed with higher concentrations of lipid substrate. ApoC-II tends to aggregate in solution. To prevent this we use 3 M guanidinium chloride in stock solutions (4.4 mg/ml, 0.5 mM) of the apolipoprotein. For assay purposes, whole serum or HDL can be used to provide activator. The effect of C-II varies greatly with the substrate system. With most triglyceride emulsions the enzyme displays activity even without the activator. This basal activity is usually 5-20% but can approach 100% of optimal activity, z8 With phospholipid liposomes the basal activity is often low. This aspect must be checked for each substrate system studied and the optimal amount of activator determined. Other Proteins. It has been reported that the activity of LpL is inhibited by a variety of proteins. The mechanism(s) of the inhibition has not been explored in detail. It is probably nonspecific since inhibition is seen with many different proteins. 2 In no case has a specific protein-protein interaction between the lipase and an inhibitory protein been demonstrated. The common denominator seems to be that the proteins bind to the surface of the substrate droplets. Concluding Remarks LpL should be a useful reagent to prepare 2-acyllysophospholipids. Because of its higher relative activity against phospholipids, hepatic lipase could be even better for this purpose. A drawback is that starting material for purification of this enzyme (usually postheparin plasma) is more difficult to obtain. During its action on chylomicrons and VLDL, LpL hydrolyzes most of the triglycerides and some of the phospholipids. Other phospholipids are transferred to the HDL fraction. The balance between these two 46 R. J. Deckelbaum, T. Olivecrona, and M. Fainaru, J. Lipid Res. 21, 425 (1980). 47 R. L. Jackson, S. Tajima, T. Yamamura, S. Yokoyama, and A. Yamamoto, Biochim. Biophys. Acta 875, 211 (1986).
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pathways may be an important determinant of plasma HDL levels. Little is known, however, of what factors govern the relative activity of LpL on phospholipids and triglycerides in emulsions and in lipoproteins. This is an interesting question for future studies. Acknowledgments Our studieson LpL are supportedby GrantB13X-727fromthe SwedishMedicalResearch Council.
