Apolipoprotein C-II deficiency

49

Biochimica et Biophysics Acta, 793 (1984) 49-60

Elsevier BBA 51601

APOLIPOPROTEIN

C-II DEFICIENCY

THE ROLE OF APOLIPOPROTEIN LIPOPRO~INS

C-II IN THE HYDROLYSIS

OF TRIACYLGLYCEROL-RICH

W. HABERBOSCH ‘, A. POLI ‘, G. BAGGIO b, R. FELLIN b, A. GNASSO ’ and J. AUGUSTIN a QKlinisches Heidelberg

Institut

fiir

I (F. R.G.),

Herrinfurktforschung ’ Istituto

di Medicina

Padova, and ’ Istituto di Farmacologia

an der Medizinischen Ctinica,

e Farmacognosia

Gerontologia

Unioersitiitsklinik e Malattie

dell’Universitci,

Heidelberg,

del Ricambio,

Bergheimer

Policlinico,

via A. de1 Sarto, 32, Mikmo

Strasse 58, D - 6900

via Giustiniani

2, I

- 35100

(italy)

(Received April 18th. 1983) (Revised manuscript received December 5th, 1983)

Key words: Lipoprotein turnover; Lipoprotein lipase; Triacylglycerol

lipase; Apolipoprotein

CII;

Enzyme-substrate

interaction

Kinetic studies were performed incubating lipoprotein lipase and hepatic triacylglyceroi lipase from human postheparin plasma with triacylglycerol-rich lipoproteins from two patients with apolipoprotein C-II deficiency. These lipoproteins differed in their lipid and apolipoprotein composition from normal very-low-density lipoproteins and chylomicrons. The addition of isolated apolipoprotein C-II and normal or apolipoprotein C-II-deficient high-density lipoproteins caused an increase of l&,x and a decrease of the K, for lipoprotein Ii~se-India hydrolysis. Hepatic ~acylglycero~ lipase activity was not influenced by the presence of apolipoprotein C-II in the incubation medium, but was inhibited by increasing amounts of high-density lipoproteins. Binding studies were performed in order to analyze the interactions between lipolytic enzymes, apolipoprotein C-II, and triacylglycerol-rich lipoproteins. Apolipoprotein C-II was, as expected, rapidly taken up by apolipoprotein C-II-deficient very-low-density lipoproteins and chylomicrons when they were incubated with normal high-densi~ lipoproteins or with the purified a~ii~p~tein. This uptake was inhibited by the addition of increasing amounts of lipoprotein iipase in conditions in which no lipolysis could occur. Binding of lipoprotein lipase to apolipoprotein C-II-deficient very-low-density lipoproteins or chylomicrons was not affected by the addition of apolipoprotein C-II when an excess of triacylglycerol-rich lipoprotein was present. The stability of lipoprotein lipase was also studied. Apolipoprotein C-II and high-density lipoproteins were unable to prolong the half-life of the enzyme activity, while triacylglycerol-rich particles effectively stabilized li~protein Iipase. We conclude that binding of li~protein lipase to the substrate surface is not affected by apolipoprotein C-II. It is more likely that the peptide catalyzes the conversion of lipoprotein lipase from a less to a more active form.

Introduction Apolipoprotein C-II has been found to activate lipoprotein lipase (EC 3.1.1.3) in vitro [l]. The physiological importance of this activation emerged Abbreviations: HDL, hip-density low-density lipoprotein(s). 0005-2760/84/$03.00

Q 1984

li~proteints);

VLDL, very-

Elsevier Science Publishers B.V.

from the identification of a patient with severe hy~ertriglyceridemia consequent to complete apolipoprotein C-II deficiency in plasma [2-61. The interaction between apolipoprotein C-II and lipoprotein lipase is still poorly understood. A receptor role of apolipoprotein C-II for the enzyme was suggested [7]. The possibility that apolipoprotein C-II might interconvert lipoprotein

50

lipase from a less to a more active form was also taken into account [S-lo]. Studies with synthetic apolipoprotein C-II fragments and apolipoprotein C-II-deficient very-lowdensity lipoproteins or artificial substrates led to the conclusion that residues 55-78 of the peptide sequence are responsible for the activation of lipoprotein lipase [11,12]. Residues below 50 may be involved in binding of apolipoprotein C-II to the lipid phase. The identification of an Italian family with apolipoprotein C-II deficiency allowed us to perform various kinetic studies using very-low-density lipoproteins and chylomicrons from the patients. Lipoprotein lipase and hepatic triacylglycerol lipase from normal human postheparin plasma were used. The aim of the study was to analyze the interrelationships between the enzymes and triacylglycerol-rich lipoproteins from the patients in the presence and in the absence of purified apolipoprotein C-II and high-density lipoproteins. Furthermore, the interactions between lipoprotein lipase and apolipoprotein C-II were studied. Patients The patients were a 35-year-old female and her 37-year-old brother with severe hyperchylomicronemia, who had been previously found to completely lack apolipoprotein C-II in plasma, with lipoprotein lipase and hepatic triacylglycerol lipase activities in the normal range (Table I). Both patients had been on a normal diet for at least 2 months. Blood was taken after a 12-h overnight fast and collected in test tubes with 0.01% EDTA. Plasma was obtained after centrifugation for 30 min at 3000 rpm and 4°C in a Beckman 56 B centrifuge.

0.001% sodium azide (buffer 1) and recentrifuged under identical conditions. The overlay was then taken up in the same buffer, containing in addition 4% defatted albumin, and recentrifuged in order to remove adherent free fatty acids. Finally, the chylomicron suspensions were adjusted to a concentration of 2000 mg% triacylglycerol and stored under nitrogen at 4°C. The isolated chylomicrons were of the same particle size as reported earlier [ 131. Chylomicron-free plasma (10 ml) was overlayered with 3 ml of 0.15 M NaCl in Beckman Quick Seal tubes and centrifuged for 18 h at 50000 rpm in a Beckman 70.1 Ti rotor. Floating very-low-density lipoproteins were removed by slicing, dialyzed extensively against buffer 1, adjusted to a concentration of 2000 mg triacylglycerol and stored at 4°C. The infranatant was adjusted to a density of 1.063 g/ml with solid KBr, and centrifuged for 18 h at 50000 rpm. After removal of low-density lipoproteins the remaining infranatant was adjusted to 1.21 g/ml and centrifuged for 48 h at 65000 rpm. Floating high-density lipoproteins were removed by slicing, dialyzed extensively against buffer 1 and adjusted to a concentration of 10 g/l (total mass). Adjustments of chylomicron, very-low-density lipoprotein and high-density lipoprotein concentrations were performed after ultrafiltration of the lipoprotein solutions by an Amicon pressure cell, equipped with an XM 50 membrane. Chylomicron-containing plasma from healthy young volunteers was obtained with double plasmapheresis 5 h after ingestion of a mixture of 300 g of oil (corn, olive and palm oils, 1 : 1 : 1, v/v). Chylomicron removal and lipoprotein isolation were performed as described above. Isolation of apolipoprotein

Materials and Methods Isolation of lipoproteins

Lipoproteins were isolated in a Beckman L8-70 preparative ultracentrifuge, at 4°C by sequential ultracentrifugation. Plasma was centrifuged for 40 min at 40000 rpm in a Beckman SW 40 swingingbucket rotor. Floating chylomicrons were carefully aspirated, diluted with a buffer containing 0.01% EDTA and

C-II

Apolipoprotein C-II was isolated from verylow-density lipoproteins of patients with type IV hyperlipoproteinemia by a modification of the method of Herbert et al. [14]. Gel filtration chromatography of delipidated very-low-density lipoprotein-apolipoproteins was carried out on a Sephacryl S-200 column (Pharmacia Fine Chemicals, Uppsala, Sweden), with an equilibration buffer containing 6 M urea and 0.1 M Tris-HCl (pH 8.4). The protein pool with molecular weight between

51

6000 and 10000 then underwent ion-exchange chromatography on DEAE-cellulose (Pharmacia Fine Chemicals), and was eluted in 6 M urea by a linear NaCl gradient from 0.01 to 0.13 M. Homogeneous apolipoprotein C-II preparations could not be achieved by this procedure, because the peptide is usually contaminated by small amounts (up to 5%) of apolipoprotein C-III. Therefore, apolipoprotein C-II was further purified by preparative isoelectric focusing in 4% Ultrodex (LKB, Bromma, Sweden) gels, prepared in 6 M urea and 1.8% ampholines (Servalyt, Serva, Heidelberg, F.R.G.), pH 4-5. Five mg of apolipoprotein C-II, dissolved in the same buffer, were applied to a gel (2d x 8 X 1 cm) and focused for 18 h at 1000 V, amperage free, on a Desaphor (Desaga, Heidelberg, F.R.G.) focusing system. After identification of the protein bands, apolipoprotein C-II was removed from the gel by stirring it in 6 M urea for 10 h, followed by centrifugation for 20 min at 3000 ‘pm. The supernatant containing apolipoprotein C-II was extensively dialyzed, lyop~lized and stored under nitrogen at - 18°C. Apolipoprotein

analysis

Purity of apolipoprotein C-II was controlled by SDS and urea-polyacrylamid< gel electrophoresis j15,16] and amino acid analysis. Analytical separation of very-low-density lipoprotein and chylomicron apolipoproteins was performed either by electrophoresis on urea gels [15) or by isoelectric focusing on 7% acrylamide ultrathin (0.3 mm) flat gels in 6 M urea and 2% ampholines. Lyophilized lipoprotein fractions were delipidated with 3 x 5 ml ethanol/ether (3 : 1, v/v) and 1 x 5 ml ether at - 18°C. Apolipoproteins were dissolved in a buffer containing 6 M urea and 0.1 M Tris-HCl (pH 8.4). Samples were focused for 3 h at 1000 V, amperage free, and the gels stained in 1% Light Green (Serva, Heidelberg, F.R.G.) for 15 min and destained in 7% acetic acid for 35 min. Quantification of apolipoprotein C-II was performed by densitometric determination of the stained protein bands using a LKB 2202 Ultra Scan Laser Densitometer. Peaks were compared with apolipoprotein C-II standards of known concentration [l7]. Enzyme sources

Postheparin

lipolytic activities were obtained

from normal young blood donors 10 min after intravenous injection of heparin (100 U/kg body weight) by procedures described elsewhere [ 181. Purified hepatic triacylglycerol lipase and lipoprotein lipase released 15 000 and 18 000 pmol free fatty acids/h per mg enzyme proteins from a triolein-gum arabic suspension, respectively. Both preparations could be stored for more than 3 months at -80°C without any loss of activity. Enzyme preparations were free of apolipoprotein C-II as proved by SDS-gel electrophoresis and analytical isoelectric focusing. Without addition of isolated apolipoprotein C-II or plasma to the triolein-gum arabic mixture the isolated lipoprotein lipase was not able to release free fatty acids under assay conditions. Lipoprotein lipase, isolated from a patient with apolipoprotein C-II deficiency, had the same kinetic criteria as lipoprotein lipase from healthy volunteers. Unless otherwise stated, assays were conducted for 30 min at 28°C under optimal conditions for hepatic triacylglycerol lipase and lipoprotein lipase with a triolein-gum arabic substrate [18,19]. Lipoprotein

lipase assay

Each milliliter of the final incubation mixture contained: 0.1 M Tris-HCl buffer (pH 8.2), 0.15 M NaCl, 2.3 pmol triolein (5 mCi of i4C/pmol), 4 mg of gum arabic, 5 mg of albu~n and an excess of apolipoprotein C-II or 100 ~1 of human serum [19]. The radiolabeled triolein was mixed with unlabeled triolein to obtain the specific activity indicated above. After 10 min preincubation at 28°C in a water bath, 10 ~1 of enzyme were added to 200 ~1 of the substrate. The reaction was stopped by vigorously mixing (vortex mixer) 1.6 ml of chloroform/heptane/methanol (75 : 60 : 84) and 0.5 M NaOH with 0.1 ml of the enzyme-substrate mixture 161.After centrifugation of the sample for 20 min, 0.6 ml of the upper phase was transferred with a Micro-medic automatic dispenser into counting vials together with 10 ml of the scintillation solution [20]. Hydrolysis of chyIom~crons and VLDL

In vitro hydrolysis of t~acylglycerol-~ch lipoproteins by hepatic triacylglycerol lipase and lipoprotein lipase was performed under optimal conditions for each enzyme. For hepatic triacylglycerol

52

Iipase 300 ~1 of the lipoprotein substrate contained 0.1 M Tris-HCI (pH 8.8), 0.75 M NaCI, 0.01 M CaCI, and 4% albumin. Lipoprotein lipase assays were conducted with a substrate containing 0.1 M Tris (pH 8.2), 0.15 M NaCl, 0.01 M CaCl, and 2% albumin. Isolated apolipoprotein C-II (1 m&ml in 6 M urea) was added to the substrates, carefully mixed and pr~i~cubated for 10 min. Further preincubation had no influence on the reaction rate. Then 60 ~1 of enzyme solutions, which had been previously adjusted to release 5 (*mol free fatty acids/h per ml enzyme solution from a trioleingum arabic emulsion, were added to these substrates. Final volume in each tube was 360 ~1. Incubations were carried out at 3’7OCfor 40 min. The reaction was stopped by addition of 250 ~1 Dole’s solution. Free fatty acid release was measured by the method of Novak 121f. Lipoprotein lipase and hepatic triacylgtjwerol Eipase binding studies 100 ~1 of normal and apolipoprotein C-II-deficient chylomicrons or ver~~low-density lipoproteins were rapidly mixed at room temperature with different amounts of apolipoprotein C-II, albumin or high-density lipoprotein, final volume 140 ~1. After 15 min, 35 (tl enzyme solution were added to these samples. The incubation mixtures (175 pi) were immediately centrifuged in the Beckman Airfuge for 10 min at 30000 ‘pm for chylomicrons and 900~ rpm for very-low-density lipoproteins. This procedure resulted in complete floating of all lipoproteins as controlled by electrophoresis and triacylglycerol determination. A needle was placed in the bottom of each tube and 110 ~1 of the solution were carefully aspirated, representing the bottom fractions of the centrifuged samples. The remaining liquid phase was mixed with the floating lipoprotein cake (65 ~1). Lipoprotein lipase and hepatic triacylglycerol lipase activities of the top and bottom fractions were determined by enzyme assay. Apolipoprotein C-II binding studies Binding of isolated apolipoprotein C-II on apolipoprotein C-II-deficient triacylglycerol~rich lipoproteins was studied in two different ways. (1) 100 ~1 of normal and apolipoprotein C-IIdeficient chyiomicrons or very-low-density lipo-

proteins were incubated with different amounts of dissolved apolipoprotein C-II (1 mg/mI in 6 M urea) and HDL, respectively. To this mixture albumin, lipoprotein lipase or 6 M urea as a control were added (final volume 175 ~1). Samples were then placed on ice to avoid enzymatic removal of C-peptides from the lipoproteins. Centrifugation and separation of the top and bottom fractions followed as described before in a Beckman airfuge. 20 ~1 of each bottom fraction, representing apolipoprotein C-II not bound to lipoproteins, were added to a gum arabic-triolein substrate, with optimal conditions for lipoprotein lipase. Lipoprotein lipase was then added to the assay under regular assay conditions. Activities were compared with a standard curve obtained from identical lipoprotein assays with increasing amounts of apolipoprotein C-II or high-density lipoproteins present. To be sure that apolipoprotein C-II was not released from very-low-density lipoprotein only due to ultracentrifugation, very-low-density lipoproteins from type IV hyperlipoproteine~c donors underwent an identical procedure. The bottom phase did not activate the standard lipoprotein lipnse assay. (2) 1.5 mg of purified apolipoprotein C-II or 100 mg high-density lipoproteins from healthy volunteers were incubated for 1 h at 37°C in 5 ml of a solution containing apolipoprotein C-fl-deficient very-low-density lipoproteins (1200 mg% triacylglycerol), 10 mmol Tris-HCl, pH 7.3, 0.9 M NaCl, 0.5 mmol EDTA and 4 M urea. The verylow-density lipoproteins were isolated and separated from unassociated apolipoFrot~in C-II or high-density lipoproteins by ultracent~fugation for 18 h at 50000 rpm, at 4’C, in a Beckman L8-70 centrifuge using Quick Seal tubes (16 x 76 mm, Beckman) in the 70.1 Ti rotor, The infranatant fraction with the unbound apolipoprotein C-II or high-density lipoproteins and the supernatant fractions were prepared for analytical isoelectric focusing as described before to quantify bound and unbound apolipoprotein C-II. Reisolation of high-density lipoproteins was performed under standard conditions (48 h, 4”C, 65000 rpm). Lipoprotein &pose stabihty To test the influence of apolipoprotein normal and apolipoprotein C-II-deficient

C-II, chyl-

53

omicrons and high-density lipoproteins on enzyme stability, 100 ~1 lipoprotein lipase were incubated at 37°C for different time intervals in solutions with apolipoprotein C-II (15 pg/ml), chylomicrons (7.5 mg triacylglycerol/ml) or high-density lipoproteins (5 mg/ml), final volume 200 ~1. Before and after incubations lasting 5, IO, 20 and 40 min, aliquots of 20 ~1 of the mixture were added to 200 ~1 of a triolein-gum arabic substrate under conditions optimal for lipoprotein lipase. Enzyme activity was measured as described before.

Total cholesterol, triacylglycerol and phospholipids were measured according to the methods of Roschlau et al. [22], Wahlefeld [23] and Zilversmit and Davis [24], respectively, using commercially available kits (Boehringer Mannheim, Mannheim, F.R.G.). Protein concentrations were determined according to the method of Lowry et al. [25]. Materials

All materials were of the best grade available. Delipidated bovine serum albumin was obtained from Serva (Heidelberg, F.R.G.), [‘4C]triolein from Amersham Buchler (Braunschweig, F.R.G.) and triolein from Fluka (Neu-Ulm, F.R.G.), ampholytes, Coomassie brilliant blue and Light Green from Serva, Heidelberg, F.R.G. Results When purified lipoprotein lipase was incubated with apolipoprotein C-II-deficient chylomicrons and increasing amounts of isolated apolipoprotein C-II, maximal hydrolysis occurred at concentrations of 0.35 yg apolipoprotein C-II/mg triacylglycerol. Maximal velocity (I$,,, ) of hydrolysis with increasing concentrations of apolipoprotein C-II-saturated chylo~crons (Fig. 1) from the patients was 1.92 mmol, 20% lower than with normal chylomicrons, but 2.6-fold higher than with apolipoprotein C-II-deficient chylomicrons. The reactions followed Michaelis - Menten kinetics, and the K, for apolipoprotein C-II-deficient chylo~crons decreased from 1.25 mmol to 0.95 mmol upon addition of 0.35 pg apolipoprotein C-II/mg triacylglycerol. The K, of normal

rnnwl FM/h

10 0

12

3‘

Fig. 1. Hydrolysis of apohpoprotein C-II-deficient and normal chylomicrons by lipoprotein lipase. (a) (0) Increasing amounts of apolipoprotein C-II-deficient chylomicrons without apolipoprotein C-II addition; (b) (e) increasing amounts of apolipoprotein C-II deficient chylomicrons after addition of 0.35 J.QI apolipoprotein C-II/mg chylomicron triacylglycerol; (c) (0) increasing amounts of normal chylomicrons. Incubation media contained 0.1 M Tris-HCI (pH 8.2), 0.15 M NaCI, 0.01 M CaCI,, 2% albumin, O-9.5 mmol chylomicron triacylglycerol and 300 ng of lipoprotein hpase (releasing 18000 pmol free fatty acids/h per mg enzyme protein from a triolein-gum arabic suspension). Final volume of the incubation mixture was 360 pl. Enzyme activity was determined from the rate of free fatty acid release at 3’7Y. Points represent mean values of six determinations. The Lineweaver-Burk-plots (inset) show the effect of isolated apolipoprotein C-II on K, and I’#,,,. Values for V,, (mmol free fatty acids/h) and K, (mmol) for normal chylomicrons and lipoprotein lipase were 2.38 f 0.40 and 0.55 + 0.16; for apolipoprotein-C-II deficient chylomicrons without a~lipoprotein C-II, 0.74f0.21 and 1.25 F 0.24; and for apolipoprotein C-II-deficient chylomicrons after addition of isolated apohpoprotein C-II, 1.92 f0.33 and 0.95 & 0.29, respectively. (Values are the mean It SE. of six experiments.)

chylomicrons was 0.55 mmol (Fig. 1). The apolipoprotein C-II/triacylglycerol ratio in normal chylomicrons was 2.02 hg/mg. Incubation of the patients’ very-low-density lipoproteins with lipoprotein lipase followed Michaelis-Menten kinetics only in the range 0.25-12 mmol triacylglycerol in the incubation mixture (Fig. 2). The presence of endogenous lipoprotein lipase inhibitors associated with apolipoprotein C-II-deficient VLDL could be ruled out by addition of patients’ VLDL to an incubation mixture of lipoprotein lipase and the triolein-gum arabic substrate. Results were compared to these obtained with normal VLDL. Maximal free fatty acid release occurred with 2.0 pg apolipoprotein C-II/mg triacylglycerol present. The I%,, of the hydrolysis with apolipoprotein C-II-deficient very-low-density lipoprotein concentrations up to 12.0 mmol was 1.72 mmol in the

54

presence of the optimal amount of apolipoprotein C-II. K, decreased from 7.5 mmol in the absence of apolipoprotein C-II to 5,5 mmol after addition of apolipoprotein C-II. Lineweaver-Burk plots are given in Fig, 2. Normal very-low-density lipoproteins contained 24.6 pg apolipoprotein C-II/mg very-low-density lipoprotein triacylglycerol. There was no significant difference in I$,,, for normal and apolipoprotein C-II-deficient chylomicrons hydrolyzed by hepatic triacylglycerol lipase. K, was 1.8 mmol for normal chylomicrons and 2.4 mmol for apolipoprotein C-II-deficient particles. The addition of apolipoprotein C-II to the latter had no influence on the reaction rate (Fig. 3). For vet-low-density lipoproteins V,, with this enzyme was only 0.72 mmol for normal and 0.65 mmol for the patients’ very-low-density lipoproteins with or without addition of apo-

2

1

6

8

10

12

1L

16 18 2Ommol VLDL- trlocylglycrroi

Fig. 2. Hydrolysis of apo~poprotein C-II-deficient and normal VLDL by lipoprotein lipase. (a) (0) Increasing amounts of apolipoprotein C-II-deficient very-low-density lipoproteins without addition of isolated apolipoprotein C-II; (b) (0) increasing amounts of apolipoprotein C-II-deficient very-lowdensity lipoproteins after addition of 2 cg isolated apolipoprotein C-II/mg very-low-density lipoprotein triacylglycerol; (c) (0) increasing amounts of normal very-lowdensity lipoproteins. Incubation media contained 0.1 M TrisHCl (pH 8.2), 0.15 M NaCl, 0.01 M CaCl,, 2% albumin, O-22 mmol very-low-density lipoprotein triacylglycerol and 300 ng of lipoprotein lipase. Final volume of the incubation mixture was 360 ~1. Mean values and standard deviations were calculated from six different determinations. The Lineweaver-Burk plots (inset) show the effect of isolated apolipoprotein C-II on K, and V,,,. Values of Vmax (mm01 free fatty acids/h} and K, (mmol) of normal VLDL and lipoprotein lipase were 2.95 f 0.44 and 2.77 f 0.36; for apolipoprotein C-II-deficient VLDL without addition of apolipoprotein C-II, 0.66 f 0.19 and 7.5 * 0.47; and for apolipoprotein C-II-deficient VLDL after addition of apohpoprotein C-II, 1.72 & 0.30 and 5.5 rt 0.49, respectively. Values are the mean & S.E. of six experiments.

lipoprotein C-II. High-density lipoproteins from normals and from patients with apolipoprotein C-II deficiency also led to an activation of lipoprotein lipase (Fig. 4). With normal high-density lipoproteins, the hydrolysis rate was nearly 75% of that obtained after apolipoprotein C-II addition, whereas high-density lipoproteins from the patients activated lipoprotein lipase 50% less than isolated apolipoprotein C-II. Increasing amounts of normal and apolipoprotein C-II-deficient high-density lipoproteins inhibited hepatic triacylglycerol lipase activity. With 112 mg% (total mass) high-density lipoproteins in the incubation mixture, which is comparable to low physiological hip-density lipoproteins plasma concentrations, lipolytic activity of hepatic triacylglycerol lipase was completely inhibited (Fig. 5). Short incubation of lipoprotein lipase with apolipoprotein C-II-deficient chylomicrons or very-low-density lipoproteins immediately followed by centrifugation in the airfuge resulted in 95% binding of the activity to the lipoproteins (Fig. 6). This was identical to values found with normal triacylglycerol-rich lipoproteins. The addition of isolated apohpoprotein C-II or albumin to the incubation mixture did not affect lipoprotein lipase binding. With low substrate concentrations (< 5 mmol triacylglycerol) lipoprotein lipase binding was limited by excessive apolipoprotein C-II addition (more than 10 pg apolipoprotein C-II/mg triacylglycerol). In vitro incubations of apolipoprotein C-II-deficient very-low-density lipoproteins with isolated apolipoprotein C-II or normal high-density lipoproteins resulted in the uptake of apolipoprotein C-II by these lipoproteins (Fig. 7). Binding was approx. 10 lug apolipoprotein C-II/mg very-lowdensity lipoprotein triacylglycerol. Excess apolipoprotein C-II was found in the infranatant after centrifugation in the Beckman L8 centrifuge. Normal very-low-density lipoproteins contained 24.6 pg apohpoprotein C-II/mg triacylglycerol. Upon incubation with normal ~~-density lipoproteins 7.5 pg apolipoprotein C-II/mg very-low-density lipoprotein triacylglycerol were transferred after 1 h when the initial high-density lipoprotein apolipoprotein C-II concentration was 2 mg/lOO mg high-density lipoprotein. 1.48 mg apolipoprotein C-11/100 mg high-density lipoproteins remained

i

i

i

Fig. 3. (left-hand figure) Lineweaver-Burk plots for the hydrolysis of apolipoprotein C-II-deficient and normal chylomicrons by hepatic triacylglycerol lipase. Incubation mixture contaized: 0.1 M Tris-HCI (pH 8.8), 0.75 M NaCI, 0.01 M CaCl,, 4% albumin, O-9.5 mmol chylomicron triacylglycerol and 350 ng hepatic triacylglycerol lipase (releasing 15000 pmol free fatty acids/h per mg enzyme protein from a triolein-gum arabic suspension). Final volume was 360 ~1. (a) (0) Values of six different determinations with apolipoprotein C-II-deficient chylomicrons and hepatic triacylglycerol lipase. Addition of isolated apolipoprotein C-II to the incubation mixture had no effect on the reaction rate. (b) (0) Values of six different determinations with normal chylomicrons and hepatic triacylglycerol lipase. The Lineweaver-Burk plot represents the kinetic parameters of the reaction. V,,, (mmol free fatty acids/h) and K, (mmol) for normal chylomicrons and hepatic triacylglycerol lipase were 2.83kO.36 and 1.8+0.24; for apolipoprotein C-II-deficient chylomicrons with and without addition of isolated apolipoprotein, C-II 2.27 +0.41 and 2.4kO.31, respectively. Values are the mean f SE. of six experiments. Fig. 4. Hydrolysis of apolipoprotein C-II-deficient chylomicrons by lipoprotein lipase. Effect of normal and apolipoprotein C-II-deficient high-density lipoproteins on enzyme activity. The incubation mixtures contained: 0.1 M Tris-HCl (pH 8.2), 0.15 M NaCl, 0.01 M CaCl,, 2% albumin, 9 mmol of chylomicron triacylglycerol, O-l.5 mg high-density lipoproteins and 300 ng lipoprotein lipase. (a) (0) Free fatty acid release after addition of increasing amounts of apolipoprotein C-II-deficient high-density lipoproteins; (b) (0) hydrolysis rates after addition of increasing amounts of normal high-density lipoproteins. Values are given as mean f SD. from six different determinations. Fig. 5. The inhibitory effect of normal and apohpoprotein C-II-deficient high-density lipoproteins on hepatic triacylglycerol lipase activity. Each sample contained: 0.1 M Ttis-HCI (pH 8.8), 0.75 M NaCl, 0.01 M CaCI,, 4% albumin, 9 mmol chylomicron triacylglycerol, 350 ng hepatic triacylglycerol lipase and different amounts of normal (0) or apolipoprotein C-II-deficient (0) high-density lipoproteins (O-O.55 mg). Values are given as mean f SD. from six different determinations.

with the reisolated high-density lipoproteins. The apolipoprotein C-II binding studies with the airfuge yielded the following results. After addition of 20 pg apolipoprotein C-II to the incubation mixture, which contained apolipoprotein C-II-deficient chylomicrons, 10 pg were found in

% of total activity

a.

b.

C

Fig. 6. Binding of lipoprotein hpase on apolipoprotein C-II-deficient chylomicrons. Effect of isolated apolipoprotein C-II. Each tube contained: 20 mmol chylomicron triacylglycerol(lO0 pl) or, as a control, 100 pl destilled water (panel C), 20 pl of 6 M urea and the following amounts of isolated apohpoprotein C-II, and lipoprotein Iipase. (a) No apolipoprotein C-II, 200 ng lipoprotein lipase protein; (b) 20 pg isolated apolipoprotein C-II and 200 ng lipoprotein lipase-protein; (c) 20 pg isolated apolipoprotein C-II and destilled water instead of chylomicrons and 200 ng lipoprotein lipase Final volume of each sample was 170 pl. The enzyme was added after 10 min, preincubation of the apolipoprotein C-II-chylomicron mixtures and the samples were immediately centrifuged for 10 min at 30000 ‘pm in the airfuge. Incubation temperature was 4’C. After centrifugation 20 pl of each bottom and top fraction were added to a gum arabic-triolein mixture in order to measure the enzyme activity under assay conditions. The bars represent the percentage of total enzyme activity which was recovered in the top fractions (open bars) and bottom fractions (dotted bars). Values are given as meanf SD from six different determinations. Total activity in panel C is far below lOO%, because the stabilizing effect of apolipoprotein C-II-deficient chylomicrons on lipsprotein Iipase activity was not present.

56

A

!

Fig. 7. Apohpoprotein C-II binding on apolipoprotein C-II-deficient very-low-density lipoprotein. Analytical isoelectric focusing of the reisolated vex-loo-density lipoprotein apolipoproteins. A, normal very-low-density fipoprotein apohpoproteins; B, very-low-density lipoprotein apolipoproteins of the apohpoprotein C-II-deficient patients; C, isolated apohpoprotein C-II; D, apolipoproteins of reisokted apolipoprotein C-II-deficient very-low-density lipoprotein after 1 h incubation with isolated apolipoprotein C-II. Incubation was carried out in a buffer containing 10 mM Tris-HCl fpH 7.3) 0.9 M N&I, 0.5 mM EDTA and 50 mM urea, 1.5 mg purified apolipoprotein C-II and 12.7 mmol very-low-density Ijpoprotein tri~cylglycerol from the patients. Final volume was 5 ml. Very-tow-density lipoprotein was reisolated under standard conditions (50000 rpm, 18 h, 4°C) and prepared for isoelectric focusing as described in the method section.

the infranatant after centrifugation (Fig. 8). When the same amount of apolipoprotein C-II was added to normal chylo~~ro~s under the same conditions, I8 pg remained in the bottom fractions and densitometric measurements of apolipoprotein C-II after isoelectric focusing of these reisolated chylomicrons showed no increase in their apolipoprotein C-II content, whereas the reisolated ~hylomi&rons of the patients contained between 7 and 9 pg apulipoprotein C-II after the incubation procedure. In the presence of lipoprotein lipase 15 pg apolipoprotein C-II remained in the infranatant and only a minor amount could be found in the reisolated lipoproteins. Apolipoprotein C-II-deficient vex-low-density lipoproteins showed a lower binding capacity for isolated apolipoprotein C-II than chylomicrons. 18-22 pg ~polipoprotein C-II were recovered in the infranatants after addition of 40 pg to the incubation mixture. With lipoprotein lipase present, 28-30 yg a~lipoprotein remained in the

2

3

Fig 8. Binding of apolipoprotein C-II on triacylglycerol-rich lipoproteins with apohpoprotein C-II deficiency: studies with the airfuge. A. Apolipoprotei~ C-II-deficient ~hylo~crons Incubation mixtures contained: 1.20 mmol triacylglycerot, 20 gg isolated apohpoprotein C-II; 2, 20 mmol triacylglycerol, 40 pg isolated apoli~protein C-II; 3, 20 mmol trjacylgiyceroi, 200 ng lipoprotein tipase and 40 gg isolated apofipoprotein C-II. B. ApoIipo~rote~n C-II-deficient very-low-density lipoprotein. 1, 20 mmol triacylglycerol, 20 ng isolated apolipoprotein C-II; 2, 20 mmol triacylglycerol, 40 ~(g isolated apolipoprotein C-II; 3, 20 mmol triacylglycerol, 200 ng lipoprotein lipase and 40 pg isolated apoI~poprot~~ C-II. Volume of each sample was 175 pl. The mixtures were incubated for 20 min at 4°C. After centrifugation in the airfuge, 20 ~1 of each bottom fraction representing apolipoprotein C-II not bound to the lipoproteins, were added to a lipoprotein hpase assay instead of plasma or isolated apolipoprotein C-II, which is normally used to activate lipoprotein lipase. Resulting activities were compared with a standard curve obtained from identical lipoprotein iipase assays with increasing amount of a~li~oprotein C-II present. The bars represent the amount of apohpoprotein C-If which remained in the bottom fractions after centrifugation. Values are given as mean + SD from six different determinations.

bottom fractions and only Y-10 pg remained in the reisolated very-low-density lipoproteins (Fig. 8). The basal values of free fatty acids in the infranatant and those bound to the reisolated Vera-low-density lipoproteins were not increased, showing that under these conditions no hydrolysis had taken place. The reduced apolipoprotein C-II binding consequently may not be attributed to a reduction of very-low-density lipoprotein surface by lipoprotein lipase. Albumin in different concentrations had no inluence on the binding of apolipoprotein G-II to chylomicrons and very-low-density lipoproteins of the patients. Such experiments could not be performed with high-density lipoproteins, because it is not possible to separate high-density lipoproteins from chylomicrons and very-low-density

51

2cm

mu

I 5

IO

M

Jo

. LO min

Fig. 9. Effects of isolated apolipoprotein C-II, chylomicrons and high-density lipoproteins on the stability of lipoprotein lipase. Incubation mixtures contained 0.1 M Tris-HCl (pH 8.2), 0.15 M NaCl, 28 albumin, 350 ng lipoprotein lipase and, in addition, 15 pg apolipoprotein C-II (O), normal chylomicrons (A) or chylomicrons from the patients (A) (10 mmol triacylglycerol), normal (m) or apolipoprotein C-II-deficient (0) high-density lipoproteins (0.6 mg) or only buffer (- -- --). Final volume in each tube was 200 pl. Incubation was carried out at 37°C. At the times indicated 20 pl of each sample were added to 200 ~1 of a labeled triolein medium under optimal conditions for lipoprotein lipase. Remaining enzyme activity was measured as described in the method section. In order to determine lipoprotein lipase activity at zero time the incubation mixture without enzyme was mixed with the assay medium and lipoprotein lipase was then added. Values are given as mean of six different determinations. Standard deviations were in the range of 5-10% of mean values.

lipoproteins during a 10 min centrifugation in the airfuge. The stability of lipoprotein lipase was not affected by addition of isolated apolipoprotein C-II (Fig. 9). The enzyme activity with or without apolipoprotein C-II addition was almost lost after 10 min. With normal and apolipoprotein C-II-deficient high-density lipoproteins the initial activity of lipoprotein lipase increased, but there was no effect on the life-time of this enzyme. Only little activation of lipoprotein lipase was found with chylomicrons from patients and normals, but these suspensions extended the life-time of the enzyme more than 3-fold. After 20 min the activity was still 55% of the initial activity at 37’C in the incubation mixtures. Discussion The present investigations had two aims. 1. To analyze triacylglycerol-rich lipoproteins

from

two patients with apolipoprotein C-II deficiency and to test their substrate characteristics for human lipoprotein lipase and hepatic triacylglycerol lipase. 2. To determine the influence of apolipoprotein C-II and high-density lipoproteins on human lipoprotein lipase and hepatic triacylglycerol lipase. Chylomicrons and very-low-density lipoproteins from the two patients were poorly hydrolyzed by human lipoprotein lipase. Addition of isolated apolipoprotein C-II led to decreases of K, from 1.25 mmol to 0.95 mmol and from 7.5 mmol to 5.5 mmol, respectively, but also to an increase in maximal velocity. The decrease of K, is in agreement with results of other authors [7,10,13], whereas contrary results exist concerning the increase of V&x [7,10,26,27]. Schrecker and Greten [7] inferred from the decrease of K, after addition of apolipoprotein C-II to triolein substrates a receptor function of apolipoprotein C-II for lipoprotein lipase. In most enzymatic reactions in fact K, corresponds to the dissociation constant K, of the enzyme-substrate complex and is a measure of the affinity of the enzyme for the substrate [28]. In the case of a changing V,,,, this conclusion cannot be drawn [28]. Furthermore, in several of these studies artificial substrates were used to investigate the effect of apolipoprotein C-II on lipoprotein lipase from bovine milk. This may explain some of the controversial kinetic results published. Experiments with apolipoprotein-deficient chylomicrons and human lipoprotein lipase have not been reported so far. Optimal hydrolysis of the patients’ chylomicrons and very-low-density lipoproteins with lipoprotein lipase occurred after addition of 0.35 pg apolipoprotein C-II/mg triacylglycerol or 2.0 pg apolipoprotein C-II, respectively. This corresponds to a molar ratio of apolipoprotein C-II to triacylglycerol of 1: 3225 for chylomicrons and 1: 564 for very-low-density lipoproteins. Fitzharris et al. [9] found a ratio of 1 : 1210-3770 for very-lowdensity lipoproteins from guinea pigs, and Lukens and Borenztajn [29] one of 1 : 963 for trypsinyzed chylomicrons. These data are consistent with our results. Higher than physiological very-low-density lipoprotein concentrations in the incubation mixtures inhibited the activity of lipoprotein lipase.

58 TABLE la CHOLESTEROL, TRIACYLGLYCEROL AND PHOSPHOLIPIDS OF THE TWO PATIENTS AND IN 10 NORMALS

IN PLASMA AND IN THE LIPOPROTEIN

FRACTIONS

Levels in normals were measured 5 h after ingestion of 300 g oil (corn oil/olive oil/palm oil, 1: 1: 1, v/v). All values are expressed as mg%. Chot, cholesterol; TG, triacylglycerol; PL, phospholipids. Patients

S.A. SF. Normals

Plasma

Chylomicrons

VLDL

LDL

HDL

Chol

TG

PL

Chol

TG

PL

Chol

TG

PL

Chol

TG

PL

Chol

TG

PL

235 183 198

2020 1985 309

304 296 241

116 96 16

1113 1175 180

60 86 4

64 40 23

790 764 96

178 173 48

21 10 101

21 28 12

23 12 82

12 9 49

_ _ 10

28 17 101

TABLE lb POSTHEPARIN PATIENTS

LIPGLYTIC ACTIVITIES OF THE TWO

Data for the two patients are the results of three different determinations on three different occasions. Results are expressed as pmol/ml per h. LPL, lipoprotein lipase; HTGL, hepatic triacylglycerol lipase. Patient

LPL

HTGL

S.A. S.F. Normals (n =lO)

6.4 f 3.9 4.5 * 3.2

32.3i2.5 17.4+ 5.2

9.8+4.3

24.0 rt 7.4

The patients’ very-low-density lipoproteins were also poorly hydrolyzed by the enzyme of hepatic origin. Bengtsson and Olivecrona [S] recently reported that very-low-density lipoproteins were better hydrolyzed by lipoprotein lipase after their incubation with phospholipa~ A *, suggesting that the equilibrium between a less active lipoprotein lipase form EX and the maximal active form E may depend on the quality of the interface. It is noteworthy that the very-low-density lipoproteins of the patients with apolipoprotein C-II deficiency are relatively enriched in phospholipids (Table I). Type I hyperlipoproteinemia is characterized by a selective deficiency of lipoprotein lipase with an excess of plasma chylomicrons and usually normal very-low-density lipoprotein levels [30,31]. Although lipoprotein lipase is supposed to be responsible for intravasal degradation of chylomicrons and very-low-density lipoproteins, the undisturbed very-low-density lipoprotein catabolism in these patients is poorly understood. It has

been suggested that hepatic triacylglycerol lipase in this situation may remove very-low-density lipoprotein triacylglycerol. In patients with apolipoprotein C-II deficiency elevated very-lowdensity lipoprotein concentrations indicate a disturbance in the clearance of chylomicrons and very-low-density lipoproteins. These findings are in accordance with those of other authors [2-6,321 and suggest that apolipoprotein C-II not only activates lipoprotein lipase but plays a role in lipoprotein metabolism. This is further supported by the fact that apolipoprotein concentrations in plasma from normals by far exceed leveis neccessary for optimal activation of lipoprotein lipase. High-density lipoproteins of healthy volunteers led to an activation of the hydrolysis of triacylglycerol by lipoprotein lipase. This reaction can be explained by a transfer of apolipoprotein C-II from the high-density lipoprotein particles to the apolipoprotein C-II-deficient lipoproteins. However, an increase in enzyme activity was also observed after addition of high-density lipoproteins from our patients with apolipoprotein C-II deficiency. The nature of this activation remains to be established. On the other hand the maximal hydrolysis rate of apolipoprotein C-II-deficient chylomicrons was higher after the addition of purified apolipoprotein C-II than with normal ~~-density lipoproteins because apolipoprotein C-II is only in part transferred from high-density lipoproteins to triacylglycerol-rich lipoprotein in our experiments. measurements of the stabthty of lipoprotein lipase demonstrated that the initial activity of the enzyme was increased by normal and to the same extent by apolipoprotein C-II-deficient high-den-

59

sity lipoproteins with no effect on the life-time of the enzyme. The latter was markedly increased by normal and apolipoprotein C-II-deficient chylomicrons. Isolated apolipoprotein C-II appeared to have no influence on the stability of lipoprotein lipase as found for this enzyme from bovine milk [S]. Only t~acylglycerol emulsions, such as chylomicrons, were able to enhance efficiently the life-time of lipoprotein lipase. Hepatic triacylglycerol lipase activity on apolipoprotein C-II-deficient triacylglycerol-~ch lipoproteins did not change after the addition of isolated apolipoprotein C-II, but was less than that found with normal lipoproteins. Increasing amounts of high-density lipoproteins inhibited hepatic triacylglycerol lipase progressively. The mechanism of this inhibition is not clear. In earlier studies 1131we showed that the major ~~-density lipoprotein apolipoproteins, AI and AII, decrease hepatic triacylglycerol lipase activity under assay conditions. On the other hand, high-density lipoprotein phospholipid and t~acyl~ycerol hydrolysis by the hepatic enzyme has been demonstrated [33,34]. However, in our experiments no further release of free fatty acids occurred when high-density lipoprotein lipids were present in the assay system. In addition, maximal inhibition of the hydrolysis of chylomicron triacylglycerol by hepatic triacylglycerol lipase was detected at very low high-density lipoprotein concentrations. Therefore a competition of substrates for hepatic triacylglycerol lipase activity under these conditions could be ruled out. In patients with lipoprotein lipase deficiency and hyperchylomicronemia extremely low highdensity lipoprotein concentrations were found 12%6,321. This may underline the importance of hepatic triacylglycerol lipase in the clearance of chylomicrons and very-low-density lipoproteins in such metabolic situations. In patients with apolipoprotein C-II deficiency high-density lipoprotein levels were comparably low, but very-low-density lipoprotein concentrations were above normal. This fits with our in vitro studies demonstrating that the abnormal verylow-density lipoproteins from our apolipoprotein C-II-deficient patients are poorly hydrolyzed by the enzyme of hepatic origin. Therefore the role of hepatic triacylglycerol

lipase for the in vivo degradation of triacylglycerol-rich lipoproteins in these patients needs further investigation. Apolipoprotein C-II binding studies with the airfuge showed that in the presence of lipoprotein lipase the capacity for apolipoprotein C-II binding was markedly reduced. On the other hand, lipoprotein lipase binding to the lipoproteins was inhibited by high apolipoprotein C-II concentrations when substrate was limited. The latter is in agreement with observations made by Shirai et al. [35], who studied lipoprotein lipase binding on artificial phospholipid particles. In our experiments nearly all of the added lipoprotein lipase was bound to the triacylglycerol-rich lipoproteins, even when apolipoprotein C-II was absent. We postulate that apolipoprotein C-II does not favor the adsorption of lipoprotein lipase to the lipoprotein particles. On the contrary, there seems to be a competitive binding mechanism between apolipoprotein C-II and lipoprotein lipase, as found for other apolipoproteins 1351.The limited binding of lipoprotein lipase in the presence of high apolipoprotein C-II concentrations may explain a recently reported case of hypertriglyceridemia caused by excessive amounts of apolipoprotein C-II in the triacylglycerol-rich lipoproteins [36]. In addition, Jackson et al. [37] found that high apolipoprotein C-II concentrations inhibit the activity of lipoprotein lipase. In conclusion, our results demonstrate that apolipoprotein C-II has no influence on the binding of lipoprotein lipase to t~acyl~ycerol-~ch lipoproteins and on the stability of this enzyme. The latter is completely guaranteed by the triacylglycerol phase of the particles. The data even favor the hypothesis that lipoprotein lipase and apolipoprotein C-II to some extent compete for binding sites on the surface of chylomicrons and very-low-density lipoproteins with a specific apolipoprotein C-II/t~acylglycerol ratio for optimal hydrolysis. Interaction between lipoprotein lipase and apolipoprotein C-II therefore is likely to occur directly at the triacylglycerol phase after the enzyme is bound to the particles independent of apolipoprotein C-II. Apolipoprotein C-II might be able to convert the enzyme into a more active form. This process, however, needs to be demonstrated in further studies.

60

Ac~owl~gements

We are indebted to Ingrid Geldmacher, Evelyn Glatting, Doris Schraube and Margret Miltner for their asisstance in these studies. This project was supported by a grant from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 90). References P.N., Lux, S.E. and 1 LaRosa, J.C., Levy, RI., Herbert, Fredrickson, D.S. (1970) Biochem. Biophys. Res. Commun. 42, 57-72 W.C., Little, J. Steiner, G., Chow, A. and 2 Breckenridge, Poapst, M. (1978) N. EngI. J. Med. 298,1265-1273 3 Capurso, A., Pace, L. Bonomo, L. et al (1980) Lancet I, 268 W.C. and Little J.A. (1978) N. 4 Cox, D.W., Breckenridge, Engl. J. Med. 299, 1421-1424 5 Miller, M.E., Rao, S.N., Alaupovic, P., Noble, N. Slack, J. BrunzeII, J.S. and Lewis, B. (1981) Eur. J. Clin. Invest. 11, 69-76 K. et al. (1979) T., Sudo. H,., Ishikawa, 6 Yamamura, Atherosclerosis 34, 53-65 7 Schrecker, 0. and Greten H. (1979) B&him. Biophys. Acta 572, 244256 T. (1980) Eur. J. Biochem. 8 Bengtsson, G. and Olivecrona, 106, 549-555 T.J. Quinn, D.M., Goh, E.H. et al. (1981) J. 9 Fitzharris, Lipid Res. 22, 921-933 N., Shirai, K., Johnson, J.D. et al. (1981) 10 Matsuoka, Metabolism 30, 818-824 A.L., Kinnunen, P.K.J., Breckenridge, W.C. et 11 Catapano, al. (1979) Biocbim. Biophys. Res. Commun. 89, 951-957 Smith, L.C., Voyta, J.C., Catapano, A.L. et al. (1980) N.Y. Acad. Sci. 213-221 Haberbosch, W., Poli, A. and Augustin, J. (1982) Biochim. Biophys. Acta 713, 365-374 Herbert, P.N., Schulman, R.S., Levy, RI. and Fredrickson, D.S. (1973) J. Biol. Chem. 248, 4941-4946 Kane, J.P., Sata, T., Hamilton, R.L. and Havel, R.J. (1975) J. Clin. Invest. 56. 1622-1634

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29 Lukens, T.W. and Borensztajn, J. (1978) B&hem. J. 175, 1143-1146 30 Fredrickson, D.S., Goldstein, J.L. and Brown, MS. (1978) in The Metabolic Basis of Inherited Disease, 4th Edn. (Stanbury, J.B., Wyngaarden J.B. and Fredrickson D.S., eds.), pp. 604-655, McGraw-Hill, New York 31 Greten, H. Levy, R.I. and Fredrickson, D.S. (1969) J. Lipid Res. 10, 326-330 32 Breckenridge, W.C., Alaupovic, P., Cox, P.W. and Little, J.A. (1982) Atherosclerosis 44, 223-235 33 Groot, P.H.E., Jansen, H. and Van Tol, A. (1981) FEBS Lett. 129, 269-272 34 Shirai, K. Barnhart, R.L. and Jackson, R.L. (1981) Biochem. Biophys. Res. Commun, 100, 591-599 35 Shirai, K. Matsuoka, N. and Jackson, R.L. (1981) B&him. Biophys. Acta 665, 504-510 36 Stocks, J. Holdsworth, G., Dodson, P. and Galton, D.J. (1981) Atherosclerosis 38, l-9 37 Jackson, R.L., Pattus, F., and De Hass, G. (1981) Biochemistry 19, 373-378