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mole percentage of dioleoylglycerol increased the diameter of the micelle 20-25%, as determined by light scattering analysis (R. Wilcox, T. Thuren, and R. Hantgan, unpublished data, 1989). Despite these limitations, this kinetic analysis is extremely valuable in the determination of the substrate specificity of this and potentially other broad specificity (phospho)lipases.

[31] H u m a n P o s t h e p a r i n P l a s m a Lipoprotein Lipase and H e p a t i c T r i g l y c e r i d e Lipase By

RICHARD L . JACKSON a n d LARRY R. M C L E A N

Introduction Lipoprotein lipase (LpL; EC 3.1.1.34) and hepatic triglyceride lipase (H-TGL; triacylglycerol kinase, EC 3.1.1.3) are the major lipolytic enzymes responsible for the metabolism of lipoproteins in the circulation. 1,2 These enzymes catalyze the hydrolysis of the sn-1 ester bond of lipoprotein di- and triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. With lipoprotein substrates, the phospholipase A1 activity is approximately I% of that for triacylglycerols. The major lipoprotein substrates for LpL are chylomicrons and very low-density lipoproteins (VLDL), whereas lipoproteins of intermediate-density (IDL) and highdensity (HDL), particularly the HDL2 subfraction, are the preferred lipoprotein substrates for H-TGL (Fig. 1). These lipolytic enzymes are anchored to the plasma membrane of endothelial cells by electrostatic interactions with heparan sulfate proteoglycans. Intravenous heparin administration releases LpL and H-TGL from these sites, resulting in lipoprotein triglyceride hydrolysis, thus the term postheparin plasma lipolytic activity (PHLA). The major tissue sources of LpL are adipose tissue and muscle; H-TGL is synthesized in the periportal hepatocyte. 3 The primary structures of human LpL and H-TGL have been deduced from the respective cDNAs. 4-7 LpL contains 448 amino acids, whereas 1 R. L. Jackson, "The Enzymes," (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 141. Academic Press, New York, 1983. 2 R. H. Eckel, N. Engl. J. Med. 320, 1060 (1989). 3 A. J. M. Verhoeven and H. Jansen, Biochim. Biophys. Acta 1001, 239 (1989). 4 K. L. Wion, T. G. Kirchgessner, A. J. Lusis, M. C. Schotz, and R. M. Lawn, Science 235, 1638 (1987). 5 G. Stahnke, R. Sprengel, J. Augustin, and H. Will, Differentiation 35, 45 (1987). 6 S. Datta, C. C. Luo, W. H. Li, P. VanTuinen, D. H. Ledbetter, M. A. Brown, S. H. Chen, S. W. Liu, and L. Chan, J. Biol. Chem. 263, 1107 (1988).

METHODS IN ENZYMOLOGY,VOL. 197

Copyright © 1991by Academic Press, Inc. All rights of reproductionin any form reserved.

340

PHOSPHOLIPASEA~ TRIGLYCERIDE-RICH LIPOPROTEIN

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INTERMEDIATE-DENSITY UPOPROTEIN HIGH-DENSITY LIPOPROTEIN

Iflll', llllll ,'" . ~

k"----Apo C-IT

lll/lllll IlllNlllll

MUSCLE -

ADIPOSE TISSUE

HEPATIC TISSUE

FIG. 1. Interaction of LpL and H-TGL with their lipoprotein substrates. The enzymes are shown interacting with heparan sulfate proteoglycans at the endothelial cell surface. Apolipoprotein C-II is shown residing in the monolayer surface of a triglyceride-dch lipoprotein (chylomicron or VLDL) and interacting with a specific domain of LpL. Triacylglycerol substrates reside primarily in the core of the lipoprotein, with a small percentage in the surface monolayer. The enzyme is shown interacting with a triacylglycerol substrate molecule.

H-TGL has 477 residues; the proteins are 47% homologous. The enzymes belong to a superfamily of lipases that also includes pancreatic lipase. Putative interfacial lipid-binding sites have been identified in both LpL and H-TGL. The sequence of the enzymes near the active site serine residue is homologous to that proposed for pancreatic lipase. 8 Two major differences in the properties of LpL and H-TGL are as follows: (1) LpL requires a 77 amino acid protein cofactor, apolipoprotein C-II, for maximal activity, whereas H-TGL has no cofactor requirements, and (2) C-II, the enzymatic activity of LpL, is inhibited by high salt, whereas H-TGL is active in 1 M NaC1. The difference in salt sensitivity is the basis for many of the reported assays to measure PHLA. As shown in Fig. I, the lipoprotein substrate consists of a neutral lipid core of triacylglycerols and cholesteryl esters and a surface monolayer of lipids and various apolipoproteins. The major lipid constituents of the lipoprotein 7 G. A. Martin, S. J. Busch, G. D. Meredith, A. D. Cardin, D. T. Blankenship, S. J. T. Mao, A. E. Rechtin, C. W. Woods, M. M. Racke, M. P. Schaefer, M. C. Fitzgerald, D. M. Burke, M. A. Flanagan, and R. L. Jackson, J. Biol. Chem. 263, 10907 (1988). 8 A. Guidoni, F. Benkouka, J. De Caro, and M. Rovery, Biochim. Biophys. Acta 660, 148 (1981).

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monolayer are phosphatidylcholine, sphingomyelin, and unesterified cholesterol. The triacylglycerol substrate molecule is distributed between the surface and core of the lipoprotein particle. The concentration of triacylglycerol at the surface of a triglyceride-rich lipoprotein has been estimated at 2-4 mol %, relative to phospholipid. 9 Because of the uncertainty of the true substrate concentration, it is difficult to determine meaningful values for Km and Vm~ with lipoprotein substrates. The purpose of this chapter is to describe several of the methods used in our laboratory for the isolation and characterization of LpL and H-TGL from human postheparin plasma. In addition, we describe assays that have been used for assessing their enzymatic activities. Isolation of Human Postheparin Plasma Lipases Normal fasting (8-12 hr) subjects are given 100 units heparin per kg, and after 15 min blood is collected by venipuncture. After removing cells by low-speed centrifugation, the following are added (final concentrations) to the plasma: sodium azide (0.01%, v/v), EDTA (0.5 mM), glycerol (10%, v/v), and aprotinin (100 kallikrein inhibitory units/ml). The postheparin plasma is immediately mixed with an equal volume of ice-cold 10 mM potassium phosphate, pH 6.8, containing 0.45 M NaC1 and 20% glycerol. Heparin-Sepharose CL-6B (Pharmacia) is added (1.0 ml packed gel per 10 ml of diluted plasma), and the mixture is incubated at 4° for 2 hr with gentle mixing on a rotating mixer; do not vortex the mixture. For plasma volumes of less than 10 ml, heparin-Sepharose is poured into a column and eluted with a NaCI gradient as described below. For large volumes (>100 ml of post-heparin plasma), the gel can be conveniently collected on a sintered glass funnel and washed as previously describedl°; all eluants are added at 4°. After each addition, the gel is gently stirred and the liquid is removed by vacuum suction, care being taken to not let the gel go dry. The eluants added in order are as follows (volumes relative to gel): 10 volumes 5 mM potassium phosphate, pH 6.8, 0.3 M NaCl, and 1 mM EDTA; 10 volumes 5 mM potassium phosphate, pH 6.8, and 1 mM EDTA; 1 volume 5 mM potassium phosphate, pH 6.8, containing 0.2% Triton N-101 (Sigma, St. Louis, MO); 10 volumes 5 mM potassium phosphate, pH 6.8, and 1 mM EDTA; and l0 volumes of 5 mM potassium phosphate, pH 6.8, 0.3 M NaC1, and l mM EDTA. The advantage of washing the gel with detergent is that lipids are removed, facilitating the chromatography step. 9 K. W. Miller and D. M. Small, J. Biol. Chem. 258, 13772 (1983). 10 R. L. Jackson, E. Ponce, L. R. McLean, and R. A. Demel, Biochemistry 25, 1166 (1986).

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The heparin-Sepharose is then poured into a column, and the protein is eluted with a NaCI gradient (0.3-2.5 M); the buffer is 10 mM Tris-HC1, pH 7.4, containing 1 mM EDTA and 10% glycerol. The volume of the gradient should not exceed 2 gel volumes. With these conditions, H-TGL elutes between 0.6 and 0.8 M NaC1 and LpL between 1.2 and 1.6 M. As an alternative to elution with a salt gradient, the enzymes can be eluted batchwise, first with 0.8 M NaCI (H-TGL) and then with 2.5 M NaC1 (LpL). Fractions should be assayed for lipolytic activity immediately and the appropriate fractions pooled; if no further purification is required, the enzymes should be stored in 50% glycerol at - 20°. With these conditions of storage, LpL and H-TGL are stable for several months. Typically, this simple one-step procedure results in purifications exceeding 10,000-fold. If purer preparations are required, H-TGL is further purified by chromatography on Phenyl-Sepharose. The heparin-Sepharose fractions described above are diluted with 5 volumes of 50 mM TrisHCI, pH 8.6, 0.4 M NaCI, 1 mM EDTA and applied to a column of Phenyl-Sepharose CL-4B (Sigma). The volume of Phenyl-Sepharose is 1 ml per 100 ml of starting postheparin plasma. The column is then washed with 10 volumes of 50 mM Tris-HC1, pH 8.6, 0.4 M NaC1, and H-TGL is eluted with 25 mM sodium deoxycholate in 5 mM potassium phosphate, pH 6.8. To remove the detergent, fractions containing H-TGL activity are diluted with 10 volumes of 5 mM potassium phosphate, pH 6.8, and applied immediately to a column (same volume as the Phenyl-Sepharose) of heparin-Sepharose. After the sample enters the resin, the column is washed with 10 volumes of 5 mM potassium phosphate, pH 6.8, and then 10 volumes of 5 mM potassium phosphate, pH 6.8, containing 0.4 M NaC1. H-TGL is eluted with 50 mM Tris-HC1, pH 7.4, containing 1.0 M NaC1, 1 mM EDTA. The enzyme is stored in 50% (v/v) glycerol at - 2 0 °. The lipolytic activities of postheparin plasma and purified LpL and H-TGL are routinely determined with a detergent-solubilized trioleoylglycerol emulsion. The substrate is prepared as follows: 200 mg of unlabeled trioleoylglycerol in chloroform and 200/~Ci of tri[1-14C]oleoylglycerol (~50 Ci/mol) are mixed and then evaporated to dryness in a 50-ml conical glass tube. Then 15 ml of 0.2% Triton N-101 (Sigma), 15 ml of 1 M Tris-HC1, pH 8.0, and 15 ml of 20% fatty acid-free bovine serum albumin (Sigma) are added. The mixture is emulsified by sonication (Branson 350 with microtip) for 4 min at 4°. The substrate can be aliquoted into 5-ml portions and stored at - 20°. Once thawed the substrate should be vortexed and used immediately; it should not be refrozen. For PHLA measurements, each assay is performed in triplicate tubes, namely, no-plasma blank, plus plasma, and plus plasma with 1 M NaC1.

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All tubes (on ice) contain 75/zl substrate and 50 ~1 normal human plasma (as a source of apoC-II). For the plus-plasma tube (total PHLA), 10 to 50 tzl postheparin plasma or the appropriate amount of purified enzyme is added. For the tubes with salt (H-TGL activity), 50/zl of 5 M NaCI is added. The final volume for all tubes is 250/zl. The tubes are then incubated at 37°. Typically, the amount of enzyme chosen for assay should give less than 10% hydrolysis in 10-30 min. The reaction is stopped by adding 3 ml of methanol/chloroform/heptane (140:125 : 100, v/v) and 1 ml of 140 mM potassium carbonate, 140 mM boric acid, pH 10.6. After vortexing (30 sec), the samples are centrifuged at 3000 rpm for 20 min (-2000 g) at room temperature; 1 ml of the top fraction (2.2 ml) is removed and radioactivity is determined. Enzyme activity is expressed as micromoles oleic acid released per hour per milliliter plasma or, for purified enzyme, per milligram protein. LpL activity is determined by subtracting H-TGL activity (plus 1 M NaC1) from total PHLA. The specific activity of pure LpL and H-TGL is approximately 30 mmol oleic acid released/hr/mg protein. Synthetic Phospholipid Substrates The primary advantage of synthetic phospholipid substrates in examining the phospholipase activity of LpL and H-TGL is the ease of manipulation of the physical characteristics of the substrates, which are generally well-defined. Two types of substrates are discussed: (1) phosphatidylcholines (PC) with relatively high critical micellar concentrations (CMC), which allow comparison of activities toward monomeric and micellar substrates, 1~ and (2) detergent-solubilized PC in which the physical form of the substrate may be varied from bilayer structures to micelles by increasing the detergent to PC ratio. 12-~4 Short-Chain Phosphatidylcholines. Dihexanoyl-PC (di-C6PC) and diheptanoyl-PC (di-C7PC) have sufficiently high CMC values to allow comparison of the rates of phospholipase-catalyzed hydrolysis above and below the CMC of the substrate. The CMC is 9.3 mM for di-C6PC and 1.0 mM for di-C7PC. The reaction mixture contains 0.1 M Tris-HCl, pH 8.0, heparin (0.5 /zg), and 3.5-60 mM PC in a total volume of 0.1 ml; the reaction is initiated by addition of enzyme. Heparin is added to stabilize the enzyme. Rates of enzyme catalysis are determined at 30° for 15-60 11 M. 12 M. 13 M. i4 M.

Shinomiya and R. L. Jackson, Biochem. Biophys. Res. Shinomiya, L. R. McLean, and R. L. Jackson, J. Biol. Shinomiya, R. L. Jackson, and L. R. McLean, J. Biol. Shinomiya and R. L. Jackson, Biochim. Biophys. Acta

Commun. 113, 811 (1983). Chem. 258, 14178 (1983). Chem. 259, 8724 (1984). 794, 177 (1984).

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min at a concentration of enzyme which results in less than 20% hydrolysis. At the appropriate time points, the reaction mixture is applied directly to silica gel G thin-layer chromatography plates. The plates are developed in chloroform/methanol/water (65 : 35 : 5, v/v). The lipids are visualized with iodine vapor, the spots corresponding to lyso-PC and PC are scraped from the plate, and the lipid content is determined by the method of Bartlett 15 for phospholipid phosphorus. F l u o r e s c e n t P h o s p h a t i d y l c h o l i n e . 16 LPL and H-TGL catalyze the hydrolysis of C6-NBD-PC {1-acyl-2-[6-(7-nitro-2,1,3-benzoxadiazol-4yl)amino]caproylphosphatidylcholine} in the sn-1 position, yielding the fluorescent product lyso-NBD-PC. Associated with catalysis of the C6NBD-PC is a 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm. The increase in fluorescence intensity allows continuous monitoring of enzyme catalysis. The rate of catalysis may be measured above and below the CMC (0.2/zM) of the lipid. The reaction mixture contains 10 mM Tris-HCl, pH 7.4, 0.I M KCI, and 5 × 10 -8 to 10 -6 M C6-NBD-PC (Avanti Biochemical Corp., Birmingham, AL) with or without apoC-II (2/xg) in a total volume of 1.0 ml. The reaction is initiated by the addition of enzyme and is monitored continuously with excitation at 470 nm and emission at 540 nm. P h o s p h a t i d y l c h o l i n e - D e t e r g e n t M i x t u r e s . Two methods are used to follow the LpL-catalyzed hydrolysis of PC in Triton: the pH-stat assay 17'18 and release of radioactive fatty acid products. 12-14Substrates are prepared as follows: the phosphatidylcholine of interest (1.6/.~mol) is mixed with PC radiolabeled in the fatty acyl chain in chloroform and dried first under N2 and then in a lyophilizer. To the dry lipid is added 1.0 ml of 0.5 mM Bicine, pH 8.0, 0.15 M NaCI, 1 m m CaCI2 (to bind released fatty acids) containing various amounts (up to 8/zmol) of Triton X-100. After vortexing, the lipid mixtures are incubated at 37° for 30 min. The assay mixtures are diluted to 0.16 mM PC. ApoC-II (0-200 /xg/ml) is added for LpL assays. The substrate is split into two parts of 5 ml each. One part is used for measurement of hydrolysis by pH stat on a Radiometer automatic titrator. The temperature is maintained constant with a jacketed reaction vial and a recirculating water bath; the pH is maintained at 8.0 with 10 mM NaOH. The other part is incubated and 0.25-ml aliquots are taken at 15G. R. Bartlett,J. Biol. Chem. 234, 466 (1959). t6 L. A. Wittenauer, K. Shirai, R. L. Jackson, and J. D. Johnson,Biochem. Biophys. Res. Commun. 118, 894 (1984). 17L. R. McLean,S. Best, A. Balasubramaniam,and R. L. Jackson,Biochim. Biophys. Acta 878, 446 (1986). 18A. Balasubramaniam,A. Rechtin, L. R. McLean,and R. L. Jackson,Biochem. Biophys. Res. Commun. 137, 1041 (1986).

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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.

Human postheparin plasma lipoprotein lipase and hepatic triglyceride lipase.

[31] POSTHEPARIN PLASMA LIPASES 339 mole percentage of dioleoylglycerol increased the diameter of the micelle 20-25%, as determined by light scatte...
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