Activation of the Phospholipase A1 Activity of Lipoprotein Lipase by Apoprotein C-II J. STOCKS and D.J. GALTON, Lipid Research Laboratory, St. Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, England ABSTRACT
The effect of apo very low density lipoprotein (apo VLDL) and apoprotein C-II on the phospholipase A 1 activity associated with lipoprotein lipase (E.C.220.127.116.11) was studied using purified bovine milk lipoprotein lipase. The enzyme degraded 14C phosphatidylcholine (PC) to 14C 2-acyl lysophosphatidylcholine at a rate of 0.28 • 0.01 nmol/min/ml and triolein at a rate of 20.3 • 0.4 nmol/min/ml in mixed emulsions of PC and triolein. The phospholipase activity and triacylglycerol lipase activity were both increased by the addition of apo VLDL and apoprotein C-II. After maximal activation, the rate of PC degradation was 1.19 • 0.02 nmol/min/ml and triolein degradation 64.4 • 0,4 nmol/ min/ml. Activation of phospholipase A 1 activity and triacylglycerol lipase activity occurred in parallel.
The main function of lipoprotein lipase (E.C.18.104.22.168) is the catabolism and clearance from the circulation of chylomicrons and very low density lipoproteins by hydrolysis of their triacylglycerols (1). Formation of triacylglycerol-depleted remnant particles in vivo appears to be associated with a loss of phospholipid (principally phosphatidylcholine) (2) and much of that lost is hydrolyzed to lysophosphatidylcholine. In addition, hydrolysis of phospholipid occurs when triglyceride-rich lipoproteins are incubated with post-heparin plasma (3,4). This degradation of phospholipids may result from the action of tipoprotein lipase, since it has been demonstrated that lipoprotein lipase has associated phospholipase A 1 activity. Thus, purified bovine milk lipoprotein lipase has been shown to degrade phosphatidylcholine (PC) of both chylomicrons (5) and very low density lipoprotein (VLDL) (6), and this action may account for the removal of surface phospholipid during chylomicron and VLDL catabolism. The phospholipase A 1 activity of lipoprotein lipase may derive from the broad substrate specifity of the enzyme since it can hydrolyze the 1 or 3 acyl ester bonds of partial glycerides and soluble esters (7,8) in addition to triglycerides. A characteristic feature of the enzyme is that it requires a cofactor, apoprotein C-II, for maximal activity toward triglycerides containing long chain fatty acids. A number of investigations have shown that, whatever the source of the enzyme, there is a several-fold increase in activity toward triglycerides in the presence of apoprotein C-II (9-14). Activation is not observed when partial glycerides and
soluble esters are substrates (15). Apoprotein C-II is a constituent of triglyceride-rich lipoproteins and high density lipoprotein (16). Since PC may be a natural substrate for the enzyme (it is the main phospholipid component of the triglyceride-rich lipoproteins), it is important to establish whether activation by apoprotein C-II also is required for maximal hydrolysis of this phospholipid. We have shown phospholipase A 1 activity associated with lipoprotein lipase of rat adipose tissue is activated by serum and VLDL (17). In addition, Groot et al. (18) have shown C-II activation of phosphatidylethanolamine (PE) degradation by rat heart lipoprotein lipase, but reported the enzyme had little activity toward PC. In order to assess whether PC hydrolysis by lipoprotein lipase is activated by apoprotein C-II, we have determined the effect of human apoprotein C-II on PC hydrolysis by highly purified bovine milk lipoprotein lipase. MATERIALS
Glycerol trioleate, fatty-acid-free bovine serum albumin, silicic acid, Co-Enzyme A, (CoA) adenosine 5-triphosphate and 1,acyl-2lyso-phosphatidylcholine (ex egg yolk) were obtained from Sigma, London; 2,5-diphenyloxazole (PPO) and 1,4 [di-2-(5diphenyloxazole)] benzene (POPOP) from Koch Light Ltd.; silica gel impregnated fiber glass chromatography sheets from Gelman Hawksley; [1-14C]linoleic acid (sp act 60 m C i / m m o l ) a n d glycerol tri-[I-laC]oleate (sp act 55 mCi/ mmol) from the Radiochemical Center, Amersham; heparin from Evans Medical Ltd., Liverpool; Sepharose 4B and Sephadex G 100 from Pharmacia, London; and DEAE-cellulose from Whatman Biochemicals. All other chemicals were obtained from BDH Ltd.
LIPOPROTEIN LIPASE PLA 1 ACTIVITY METHODS Preparation of 14C Phosphatidylcholine 1 - a c yl-2 - [ 1- 14 C ] lin oleyl-sn-gly cerophosph atidylcholine was prepared by re-acylation of 1-acyl-2-1ysophosphatidylcholine with [ 1-14C] linoleic acid using a rat liver homogenate (19). [1-14C]Linoleic acid (250 #Ci) was mixed with 148 mmol of lysophosphatidylcholine, 1.07 mmol ATP, and 26 mmol of CoA in 25 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) and sonicated for 2 rain. The mixture was added to 25 ml of a 25% (w/v) rat liver homogenate in the same buffer and incubated for one hour at 37 C. After incubation, the homogenate was extracted with 250 ml methanol-chloroform (2: 1, v/v) containing 0.1 g/1 butylated hydroxyanisole and then filtered. The filtrate was mixed with t50 ml of water and 150 ml of chloroform. The chloroform phase was removed and dried under reduced pressure at 40 C. The extract was dissolved in diethyl ether and phospholipids precipitated by the addition of cold acetone. The phospholipids were redissolved in chloroform, flushed with nitrogen and stored at -20 C. The specific activities (sp act) of the preparations varied between 6.7 and 11.5 mCi/mmol. The radiochemical purity was checked by thin layer chromatography (TLC) on Silica Gel G in chloroform-methanol-ammonia (70:30: 4, v/v/v). Between 98-99% of the radioactivity applied was recovered in the zone corresponding to PC. Phosphatidycholine
PC was isolated from rat liver using the same technique as for the isolation of the labeled PC from rat liver homogenates. Lipoprotein Lipase
Lipoprotein lipase was prepared from cow's milk by batch absorption and affinity chromatography on heparin-Sepharose (20). The heparin-Sepharose was prepared by the method of Lindahl et al. (21). The final preparation had a sp act of 28,000 mU mg protein (1 mu = 1 nmol fatty acid/min). It was diluted with 0.5 M NaC1 to 200 mU/mI and then stored at -20 C. V L D L C Apoproteins
The d < 1.006 g/ml fraction was prepared from the serum of patients with type IV and type V hyperlipoproteinemia by ultracentrifugation at 40,000 rpm for 18 hr at 10 C in an MSE 8 x 25 ml angle head rotor (22). The VLDL was delipidated with ethanol-ether (3 : 1, v/v) (23). Tris soluble apoproteins were prepared by suspension of apo V L D L in 0.2
mmol/1 tris-HC1, pH 8.1 (24). Individual C apo proteins were prepared by solubilization of apo VLDL in 0.2 mmol/1 sodium dodecyl sulphate followed by gel filtration on Sephadex G-100. After gel filtration, the C apo proteins were separated by DEAE-cellulose chromatography in 8 mmol/1 urea (24). The purity of the apoproteins was checked by polyacrylamide gel electrophoresis in 8 mol/1 urea (25). All preparations gave a single band. Substrates for Phospholipase A 1 and Lipoprotein Lipase
The substrate for both phospholipase A 1 and lipoprotein lipase was an emulsion of glycerol trioleate and rat liver PC that was prepared by dilution of a clear mixture in glycerol (26). For the phospholipase A 1 assays, 5 #Ci of [ 1-14C] PC was mixed with PC to give a total of 6.3 /~mol and then dried under nitrogen at room temperature. Glycerol trioleate (133 #mol) and 2 ml of glycerol were added and the mixture was sonicated on ice for 1 min. The substrate for triglyceride lipase was prepared in the same way except that it contained 10 #Ci of glycerol tri-[ 1-14C] oleate and 6.3 /amol of PC. Before use, the stock solutions were diluted with 4 parts (v/v) of 0.2 mol/1 tris-HC1, pH 8.1, containing 30 g/1 fatty acid free bovine serum albumin. The final concentrations of reagents in the medium were: PC, 0.52 /.tmol/ml; triolein, 11.1 #mol/ml; albumin, 20 mg/ml; glycerol, 4.5 mol/1; and tris-HC1, 0.133 mol/1. Assay of Phospholipase A 1
Phospholipase A 1 activity was determined by the formation of 2-[1-14C]linoleyl lysophosphatidylcholine. Fifty or 100/~1 of enzyme was added to 200/~1 of substrate and incubated at 37 C for 30 min. Then 6 ml of chloroformmethanol (2:1, v/v) was added followed by 2 ml of water. The mixture was vortexed, then centrifuged. The upper phase was removed, transferred to a glass tube and dried under nitrogen at 40 C. The residue was dissolved in 100 /J1 of chloroform containing 0.1 mg/ml lysophosphatidylcholine and then 50 pl was applied to a 20 x 20 cm sheet of glass fiberSilica Gel G . The sheet was developed for 15 cm in chloroform-methanol-ammonia (80:30:2, v/v/v). The lysophosphatidylcholine zone was located with iodine vapor, cut out and transferred to a scintillation vial for counting. Results are means -+ SEM (n = 6). Assay of Lipoprotein Lipase
Lipoprotein lipase was determined by the LIPIDS, VOL. lS, NO. 3
J. S T O C K S A N D D.J. G A L T O N
f o r m a t i o n of [ 1-14C] oleate using assay conditions similar to those described by Nilsson-Ehle and Schotz (26). Fifty or 100/~1 of e n z y m e was added to 200 /sl of substrate and incubated at 37 C for 30 min. then 3.25 ml of c h l o r o f o r m m e t h a n d - h e p t a n e (1: 1.25: 1, v/v/v) was added followed by 1.05 ml of 0.1 mol/1 potassium carbonate. The mixture was v o r t e x e d and centrifuged for 10 min at 1000 g and 1 ml of the u p p e r phase was r e m o v e d for counting.
Tr iolein FFA/~moles/ml
14 C Radioactivity was c o u n t e d in a Packard Tri Carb scintillation s p e c t r o m e t e r using 10 ml toluene-triton X 100, (2: 1, v/v), containing 3.3 g/1 PPO and 0.33 g/1 POPOP as scintillators. RESULTS
In m o s t experiments, a m i x e d emulsion of PC and triolein was used that was stable for several hours at 37 C. A d d i t i o n of lipoprotein lipase (final c o n c e n t r a t i o n 66 m u / m l ) to the emulsion resulted in the degradation of b o t h PC and triolein. Over 30 min incubation, the rate of l y s o p h o s p h a t i d y l c h o l i n e f o r m a t i o n was 0.28 -+ 0.01 n m o l / m i n / m l (mean +-- SEM, n = 6). When serum was added to the substrate to a c o n c e n t r a t i o n of 1.5% v/v, conditions k n o w n to result in the activation of triolein hydrolysis by lipoprotein lipase (26), there was an increase in the rate of hydrolysis of b o t h PC and triolein. F o r a 30 min incubation, the rate of lysophosphatidylcholine f o r m a t i o n rose to 1.19 -+ 0.02 n m o l / m i n / m l and oleate f o r m a t i o n rose to 64.4 +- 0.4 n m o l / m i n / m l . The degradation of b o t h PC and triolein in the m i x e d emulsion f o l l o w e d
I n c u b a t i o n t i m e rains
FIG. 1. Hydrolysis of PC- and triolein by lipoprotein lipase. Aliquots of enzyme (100 ~1) were incubated at 37 C with 200/sl of a mixed emulsion of PC (0.52 mmol/1) and triolein (11.1 mol/1)containing e i t h e r 14C PC o r 14C triolein and added serum (1.5% v/v). Aliquots were removed at intervals for lysolecithin formation (o) or oleate formation (o). a similar time course (Fig. 1). Soluble apoproteins prepared by suspension of apo V L D L in 0.2 M tris-HC1 buffer (pH 8.2) stimulated b o t h the phospholipase A 1 and the triglyceride lipase activity when added to the substrate at concentrations up to 12.5 g g / m l (Table I). The activation of triolein degradation p r o d u c e d by the addition of increasing quantities of apo V L D L to the substrate paralleled the activation of PC degradation. Individual soluble apoproteins prepared from V L D L - a p o C-I, arginine-rich, apo C-II and apo C-III-1 - w e r e e x a m i n e d for activation of PC hydrolysis by lipoprotein lipase. A p o p r o t e i n C-II was the only apoprotein that p r o d u c e d a significant stimulation of PC degradation (Table
TABLE I E f f e c t o f A p o p r o t e i n s o f H u m a n V L D L on P h o s p h a t i d y l c h o l i n e (PC) and Triolein H y d r o l y s i s by L i p o p r o t e i n Lipase a
Control Apo-VLDL A p o C-I A p o C-II A p o C-Ill Arg. rich
Triacylglycerol hydrolysis (nmol/min/ml)
0.32 1.40 0.35 1.84 0.34 0.41
§ 0.02 +_ 0.02 +- 0.01 -+ 0.01 +- 0.01 + 0.02
100 437 109 575 106 128
13.0 65.1 11.9 62.4 14.6 14.1
-+ 0.5 +- 0.7 + 0.6 +- 0.7 +- 0.5 -+ 0.1
100 501 99 480 112 108
a A p o V L D L at a final c o n c e n t r a t i o n o f 12.5 ~ g ] m l a n d individual a p o p r o t e i n s isolated f r o m a p o V L D L at a final c o n c e n t r a t i o n o f 5 ~ug/ml w e r e a d d e d to s u b s t r a t e c o n t a i n i n g 14C PC or 14C triolein. E n z y m e ( 1 0 0 ktl) w a s i n c u b a t e d w i t h 200 /al o f s u b s t r a t e f o r 30 rain at 37 C a n d l y s o l e c i t h i n or oleate f o r m a t i o n w a s d e t e r m i n e d (see M e t h o d s ) . Results are m e a n + S.E.M. o f six assays.
L I P I D S , V O L . 15, NO. 3
Phosphatidyl choline -LPC nmoles/ml
LIPOPROTEIN LIPASE PLA1 ACTIVITY I). As expected, apo C-II also produced a marked activation of triolein degradation. Addition of apoprotein C-II to the substrate at a concentration of 5 /lg/ml produced a four-fold increase in PC hydrolysis that was similar to the degree of activation achieved with apo VLDL. The activation of phospholipase A 1 activity paralleled the activation of triglyceride lipase activity (Fig. 2). PC degradation by lipoprotein lipase and the activation of phospholipase A1 activity were not restricted to PC-coated triolein particles. When micellar dispersions of 0.52 mmol/1 PC were used as substrhte, the rate of degradation of PC (0.37 + 0.12 nmol/min/ml) was similar to that in mixed emulsions of 0.52 mmol/1 PC and 11.1 tool/1 triolein (0.43 -+ '0.17 nmol/min/ml). Similarly, the activation of PC degradation by apo C-II occurred to the same degree in either PC dispersions, where it increased to 1.63 -+ 0.19 nmol/min/ml after the addition of C-II to 5 /lg/ml, or in mixed emulsions, where it increased to 1.71 + 0.36 nmol/min/ml. DISCUSSION
The activation of lipoprotein lipase by apoprotein C-II is essential for maximal hydrolysis of triacylglycerols containing long chain fatty .acids (9-14). This peptide is a constituent of the natural substrates of lipoprotein lipase, i.e., chylomicrons and VLDL, and suggests activation of lipoprotein lipase by apoprotein C-II is of physiological importance in the catabolism of these triacylglycerol-rich lipoproteins. This suggestion is supported by the demonstrated absence of C-II in one individual, resulting in a marked hypertriglyceridemia caused by defective clearance of triacylglycerol-rich lipoproteins from the circulation (27). Previous studies have shown lipoprotein lipase has associated phospholipase A 1 activity degrading PC in lipoproteins (5,6), and this is confirmed by our observation that in emulsions of PC- and triolein-containing serum activator both components are degraded in parallel during incubation with a purified preparation of the enzyme. This contrasts with previous reports that lipoprotein lipase has little or no activity toward PC in synthetic substrates (18,28). The differences may result from the use of egg lecithin, which has a markedly different fatty acid composition to PC in plasma lipoproteins, or alternatively, to the lack of an activator of lipoprotein lipase in the substrate. The rate of triolein hydrolysis was greater than PC hydrolysis (3). However, the relative
Triolein F FA nmoles/ min/ml
Phosphatidyl choline LPC n m o l e s / min/ml
apo Cll /zg/ml
FIG. 2. Effect of VLDL apo C-II on triolein and PC hydrolysis. Apoprotein C-II was isolated from apo VLDL by gel filtration and DEAE-cellulose chromatography (see Methods). The apoprotein was added to the substrate and activation of phospholipase A t activity (o) and triacylglycerol lipase activity (o) was determined by lysolecithin or oleate formation after 30 min at 37 C. rates of hydrolysis must be interpreted by observing that they may depend on relative substrate concentrations and by considering the chain length and degree of unsaturation of the PC fatty acids, as well as the physical state of the emulsion. In this study, the 1-acyl fatty acid in the labeled PC was primarily stearic acid, which is a major fatty acid component of VLDL PC (29), and the relative proportion of PC to triolein in the substrate was similar to the proportions in chylomicra (26). Removal of PC during VLDL and chylomicron metabolism may be necessary for the maintenance of the appropriate ratio of polar surface components to apolar core components during degradation of core triacylglycerol by lipoprotein lipase (30). Although loss of phospholipid from these particles can occur by transfer to HDL (31,32), the observation that lipoprotein lipase can degrade PC at greatly increased rates in the presence of C-II activator supports the view that hydrolysis of PC is an important factor in the catabolism of triglyceride-rich lipoproteins. While C-II activation of lipoprotein lipase may be a requirement for PC hydrolysis, other factors may limit phospholipid degradation by the enzyme since PC of HDL is not degraded by lipoprotein lipase, even though the lipoprotein contains apo C-II. Although the activating property of apo C-II has been localized to specific regions of its primary structure (33), the precise mechanism of activation is unknown. It may act by increasing enzyme substrate affinity (34). Since in mixed emulsions of PC and triolein the activation of triacylglycerol lipase and phosphoLIPIDS, VOL. 15, NO. 3
J. STOCKS AND D.J. GALTON
lipase A 1 activity by C-II occurs in parallel, it is likely that a similar mechanism of activation is involved.
17. 18. 19.
Robinson, D.S., and D.R. Wing, in "Plasma Lipoproteins," Edited by R.M.S. Smellie, Academic Press, London, 1971, pp. 123-135. 2. Mj~s, O., O. Faergeman, R.L. Hamilton, and R.J. Havel, J. Clin. Invest. 56:603 (1975). 3. Eisenberg, S., and D. Rachmilewtz, J. Lipid Res. 16:341 (1975). 4. Vogel, W.C., and E.L. Bierman, Proc. Soc. Exp. Biol. Med., 127:77 (1968). 5. Scow, R.O., and T. Egelrud, Biochim. Biophys. Acta 431:538 (1976). 6. Eisenberg, S., Biochim. Biophys. Acta 489:337 (1977). 7. Nilsson-Ehle, P., P. Belfrage, and B. Bergstrom, Biochim. Biophys. Acta 248:114 (1971). 8. Nilsson-Ehle, P., T. Egelrud, P. Belfrage, T. Olivecrona, and B. Bergstrom, J. Biol. Chem. 248:6734 (1973). 9. Havel, R.J., C.J. Fielding, T. Olivecrona, V.G. Shore, P.E. Fielding, and J.C. Egelrud, Biochemistry 12:1828 (1973). 10. Brown, W.V., and M . L . Baginsky, Biochem. Biophys. Res. Commun. 46:375 (1972). 11. La Rosa, J.C., R.I. Levy, S.E. Herbert, and D.S. Fredrickson, Biochem. Biophys. Res. Commun. 41:57 (1970). 12. Havel, R.J., V.G. Shore, B. Shore, and D.M. Bier, Circ. Res. 27:595 (1970). 13. Ekman, R., and P. Nilsson-Ehle, Clin. Chem. Acta 63:29 (1973). 14. Kraus, R.M., P.N. Herbert, R.J. Levy, and D.S. Fredrickson, Circ. Res. 33:403 (1973). 15. Egelrud, T., and T. Olivecrona, Biochim. Biophys. Acta 306:115 (1973). 16. Jackson, R.L., J.D. Morrisett, and A.M. Gotto, Physiol. Rev. 56:259 (1976).
LIPIDS, VOL. 15, NO. 3
23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
Stocks, J., and D.J. Galton, Biochem. Soc. Trans. 6:103 (1978). Groot, P.H.E., M.C. Oerlemans, and L.M. Scheck, Biochim. Biophys. Acta 530:99 (1978). Waite, M., and L.L.M. van Deenen, Biochim. Biophys. Acta 137:498 (1967). Bengtsson, G., and T. Olivecrona, Biochem. J. 167:109 (1977). Lindahl, U., J.A. Cifonelli, B. Lindahl, and L. Roden, J. Biol. Chem. 240:2817 (1965). Lindgren, F.T., L.C. Jensen, and F.T. Hatch, in "Blood Lipids and Lipoproteins," Edited by G.J. Nelson, J. Wiley, New York, 1972, pp. 181-274. Scanu, A.M., and C. Edelstein, Anal. Biochem. 44:576 (1971). Brown, V.G., R.I. Levy, and D.S. Fredrickson, J. Biol. Chem. 245:6588 (1970). Kane, J.P., Anal. Biochem. 53:350 (1973). Nilsson-Ehle, P., and M.C. Schotz, J. Lipid Res. 17:536 (1976). Breckenridge, W.C., J.A. Little, G. Steiner, A. Chow, and M. Poapst, New Eng. J. Med. 298:1265 (1978). Fielding, C.J., and P.E. Fielding, J. Lipid Res. 17:248 (1976). Nelson, G.J., and N.K. Freeman, J. Biol. Chem. 235:578 (1960). Eisenberg, S., and T. Olivecrona, J. Lipid Res. 20:614 (1979). Eisenberg, S., and D. Schurr, J. Lipid Res. 17:578 (1976). Eisenberg, S., D. Schurr, H. Goldman, and T. Olivecrona, Biochim. Biophys. Acta 531:344 (1978). Pownall, H.J., J.P. Morrisett, J.T. Sparrow, L.C. Smith, J. Shepherd, R.L. Jackson, and A.M. Gotto, Lipids 14:428 (1979). Schrecker, O., and H. Greten, Biochim. Biophys. Acta 572:244 (1979).