Reduced dimerization of lipoprotein lipase in post-heparin plasma of a patient with hyperchylomicronemia

BB

ELSEVIER

Biochimica et Biophysica Acta 1254 (1995) 30-36

Biochi~ic~a et BiophysicaA~ta

Reduced dimerization of lipoprotein lipase in post-heparin plasma of a patient with hyperchylomicronemia Hiroshi Masuno a,*, Hiroki Nakabayashi b, Junji Kobayashi c, Yasushi Saito Hiromichi Okuda d

c,1,

a Department of Medical Laboratory Technology, Ehime College of Health Science, Takooda, Tobe-cho, lyo-gun, Ehime 791-21, Japan b Medical Research Institute, School of Medicine, Nihon University, ltabashi, Tokyo 173, Japan c The Second Department of Internal Medicine, School of Medicine, Chiba University, lnohana, Chiba 280, Japan d Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-02, Japan

Received 23 May 1994; revised 24 August 1994

Abstract As in post-heparin plasma of control subjects, post-heparin plasma of a patient with hyperchylomicronemia contained lipoprotein lipase (LPL) subunits with M r = 57 000. But although the amount of LPL was the same as in post-heparin plasma of controls, no LPL activity was detectable. Nearly all the LPL in post-heparin plasma of controls bound to heparin-Sepharose and this LPL bound was mainly eluted with 1.5 M NaCI in parallel with the activity. In post-heparin plasma of the patient, 58% of the LPL subunits did not bind to heparin-Sepharose and 23% was eluted with 0.6 M NaCI. Studies by sucrose density gradient centrifugation showed that almost all the LPL in post-heparin plasma of controls was recovered in the peak with a sedimentation coefficient of 6.8 S, corresponding to the position of a dimeric form of LPL, in parallel with the activity; little LPL was recovered in the peak with a sedimentation coefficient of 4.0 S, corresponding to the position of a monomeric form of LPL. In post-heparin plasma of the patient, 35% of the LPL subunits was recovered in fractions with larger sedimentation coefficients at the bottom of the centrifuge tube, indicating the presence of an aggregated form(s) of LPL; the amount of the monomeric form of LPL was increased, while that of the dimeric form was decreased. Thus, defect of LPL activity in post-heparin plasma of the patient with hyperchylomicronemia could result from reduced dimerization of LPL subunits. Keywords: Lipoprotein lipase; Aggregation; Monomer; Dimer; Hyperchylomicronemia

1. Introduction Lipoprotein lipase (LPL) is synthesized by parenchymal cells of extrahepatic tissues and transported to the luminal surfaces of their capillaries, where it hydrolyzes triacylglycerol in chylomicrons and very-low-density lipoproteins to free fatty acid and monoacylglycerol. Defect or dysfunction of L P L on the luminal surface of capillaries causes severe hypertriglyceridemia. The active form of L P L is a noncovalently bound homo-dimer with high affinity for heparin [1-5]. Dissociation of the dimeric form of L P L into the monomeric form results in loss of activity and low affinity for heparin [5].

The active, dimeric form of LPL is eluted from heparinSepharose with a high salt concentration, usually > 1.0 M NaCI, whereas the inactive, monomeric form is eluted with a lower salt concentration, usually 0.6 M NaC1 [5-7]. Ikeda et al. [8], however, reported that the active LPL in human post-heparin plasma is monomeric. This paper reports that post-heparin plasma of a patient with hyperchylomicronemia contained the inactive, aggregated form of LPL. Dysfunction of this patient's LPL could result from reduced dimerization of LPL subunits.

2. Materials and methods 2.1. Materials

* Corresponding author. Fax: +81 0899 58 2177. I Present address: Department of Laboratory Medicine, School of Medicine, Yamagata University, 2-2-2 Iida-Nishi, Yamagata 990-23, Japan. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0005-2760(94)00162-6

Tri[9,10(n)-3H]oleoylglycerol and 125I-protein A were obtained from Amersham. Aldolase ( M r = 160000) and

H. Masuno et al. /Biochimica et Biophysica Acta 1254 (1995) 30-36

I

2

3

4

5

LPL (57 k) ~

Fig. 1. Western blot of LPL in pre- and post-heparin plasma. Proteins in pre- and post-heparin plasma were separated by SDS-PAGE. After transfer to a nitrocellulose membrane, the separated proteins were blotted with chicken antiserum to bovine LPL. All blots were then exposed to rabbit antichicken IgG and 125I-protein A as described under Section 2. An acetone/ether powder of human adipose tissue was prepared using a 25% homogenate of the tissue as descibed previously [7]. Lane 1, LPL in an extract of the acetone/ether powder of human adipose tissue; lane 2, LPL in pre-heparin plasma of the patient; lane 3, LPL in post-heparin plasma of the patient; lane 4, LPL in pre-heparin plasma of the control; lane 5, LPL in post-heparin plasma of the control. Arrow, location of LPL; arrowhead, location of degraded LPL.

bovine serum albumin (M r = 67000) were obtained from Serva Feinbiochemica, Germany. Rabbit antichicken IgG was from Pel-Freez. Heparin-Sepharose CL-6B was from

0.3 M N a C I

0.6 M NaCI

31

Pharmacia, Sweden. Chicken antiserum to bovine LPL and chicken anti-LPL IgG were gifts from Drs. Thomas Olivecrona and Gunilla Bengtsson-Olivecrona, Department of Physiological Chemistry, University of Ume~, Sweden. All other chemicals were of the highest quality commercially available. Solution A, used for elution of LPL from a heparinSepharose column, consisted of 20 mM Tris, 20% glycerol, and 0.1% Triton X-100, pH 7.4. Solution B, used for separating the monomeric and dimeric forms of LPL by sucrose density gradient centrifugation, consisted of 20 mM Tris, 0.1 M NaC1, and 10% glycerol, pH 7.4. Solution C, used for incubation of nitrocellulose membranes with antibodies, consisted of 20 mM Tris and 137 mM NaC1, pH 7.6. Solution D, used for washing nitrocellulose membranes, consisted of 0.1% Tween-20 in solution C.

2.2. Subjects The patient studied was a 16-year-old girl (height, 153.7 cm; body weight, 52.2 kg). Throughout childhood she had suffered from eruptive xanthomas. Her fasting plasma lipid and lipoprotein values were as follows: triacylglycerol, 2499 mg/dl; total cholesterol, 295 mg/dl; chylomicrons, 2097 mg/dl; high-density lipoprotein-cholesterol, 17.0 mg/dl. Her fasting plasma apolipoprotein values were as follows: A-I, 106 mg/dl; A-II, 24 mg/dl; B, 96 mg/dl; C-II, 7.8 mg/dl; C-Ill, 33.3 mg/dl; E, 23.1 mg/dl. Her renal and liver function tests were normal. She did not

1,5 M NaCI

6

250

0.3 M N a C l

0.6 M NaCI

1 .5 M N a C I

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2

O~

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150

X .1 1

100 .,4

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20

30

40

50

60

70

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F P a c t ion N u m b e r

Fig. 2. Chromatography on heparin-Sepharose of lipolytic activity in post-heparin plasma. A mixture of 0.8 ml of post-heparin plasma and 1.6 ml of 0.3 M NaCI in solution A was applied to a heparin-Sepharose column equilibrated with the same buffer. The column was washed with the same buffer, and then developed first with 0.6 M NaC1 in solution A and then with 1.5 M NaC1 in solution A. The lipolytic activities in the fractions eluted with 0.3 M and 1.5 M NaCI were assayed in the presence of heat-inactivated serum from starved rats and that in the fractions eluted with 0.6 M NaCI was assayed in the absence of the serum. (O), Lipolytic activity in post-heparin plasma of the control; ( 0 ) , lipolytic activity in post-heparin plasma of the patient.

10

20

30

Fraction

40

50

60

70

Number

Fig. 3. Chromatography on heparin-Sepharose of LPL in post-heparin plasma. Proteins in post-heparin plasma were eluted from a heparin-Sepharose column as described in Fig. 2. The LPL mass of each fraction was measured as described previously [9]. (©), LPL mass in post-heparin plasma of the control; ( 0 ) , LPL mass in post-heparin plasma of the patient.

H. Masuno et al. / Biochimica et Biophysica Acta 1254 (1995) 30-36

32

I 7

Aldolase

BSA

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(4.3 S)

0

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Fig. 4. Sucrose density gradient sedimentation of lipolytic activity and LPL protein in post-heparin plasma of the control. Proteins in post-heparin plasma of the control were separated in a 5-15% sucrose gradient, and recovered in 44 fractions. The lipolytic activity of each fraction was assayed in the presence of heat-inactivated serum from starved rats. The bands corresponding to LPL in Fig. 6 were cut out and their radioactivities were measured. (O), Lipolytic activity; (O), radioactivity in the band corresponding to LPL. Arrows: positions of the sedimentation markers, aldolase (s = 7.5 S) and bovine serum albumin (BSA) (s = 4.3 S), run in parallel.

show any evidence of diabetes mellitus, and her plasma hormone levels were normal. The plasma triacylglycerol and cholesterol levels of controls (age, 30 _+ 10 years) were < 150 m g / d l and < 220 m g / d l , respectively. Figs. 2 - 4 show typical chromatograms of LPL in post-heparin plasma of a 21-year-old man.

2.3. Quantitation of LPL activity and mass in post-heparin plasma Heparin (60 units/kg body weight) was injected intravenously into controls and the patient with hyperchylomicronemia, and venous blood was obtained before and 15 min after heparin injection. Selective measurement of LPL activity in post-heparin plasma was carried out as follows:

20

30

40

Controls Patient

50

F r a c t i o n Number

Fig. 5. Sucrose density gradient sedimentation of lipolytic activity and

LPL protein in post-heparin plasma of a patient with hyperchylomicronemia. Proteins in post-heparin plasma of the patient were separated in a 5-15% sucrose gradient, and recovered in 44 fractions. The lipolytic activity of each fraction was assayed in the presence of heat-inactivated serum from starved rats. The bands corresponding to LPL in Fig. 6 were cut out and their radioactivities were measured. (O), Lipolytic activity; (O), radioactivity in the band corresponding to LPL. Arrows: positions of the sedimentation markers, aldolase ( s = 7.5 S) and bovine serum albumin BSA ( s = 4.3 S), run in parallel.

An aliquot (5 /zl) of post-heparin plasma with or without 15 /xg of chicken anti-LPL IgG in a final volume of 100 /xl, adjusted with 50 mM N H a / N H 4 C 1 buffer, pH 8.2, containing 20 / x g / m l heparin was stood for 1 h at 0°C. Then the lipase activity was measured with phosphatidylcholine-stabilized tri[9,10(n)-3H]oleoylglycerol as substrate in the presence of heat-inactivated (56°C, 10 min) serum from starved rats [7]. The mixture was incubated for 15 min at 37°C, and LPL activity was calculated by subtracting the lipase activity in the presence of antibody from that in its absence. The lipase activity eluted with 0.6 M NaC1 from a heparin-Sepharose column (Fig. 2) was measured in the absence of heat-inactivated rat serum. One milliunit of lipolytic activity was defined as that releasing 1 nmol of fatty acid per min at 37°C. LPL mass was determined by a sandwich enzyme-linked immunosorbent assay as described previously [9].

Table 1 LPL activity and mass in pre- and post-heparin plasma of control subjects and patients with hyperchylomicronemia Subject

''~

LPL in pre-heparin plasma

LPL in post-heparin plasma

Activity

Mass

Activity

Mass

/ t m o l / h per ml 0 (3) a 0 (1)

ng/ml 133 + 47 (14) 219 (1)

/ z m o l / h per ml 5.5 + 0.3 (3) 0 (1)

ng/ml 444 + 67 (15) 392 (1)

LPL activity and mass were assayed in duplicate. Values are given as means + S.D. aNumbers in parentheses show numbers of samples.

H. Masuno et aL / Biochimica et Biophysica Acta 1254 (1995) 30-36

2.4. Chromatography of post-heparin plasma lipolytic activity on a heparin-Sepharose column

(200 000 c p m / m l ) , washed with solution D, and exposed to Kodak X-Omat film. The band corresponding to LPL was cut out and its radioactivity was determined in an Aloka ARC-600 y-counter.

A mixture of 0.8 ml of post-heparin plasma and 1.6 ml of 0.3 M NaCI in solution A was applied to a heparin-Sepharose column (column size, 1 ml) equilibrated with 0.3 M NaCI in solution A. The column was washed with 0.3 M NaC1 in solution A and then developed first with 0.6 M NaC1 in solution A and then with 1.5 M NaC1 in solution A. Fractions of 0.52 ml were collected.

3. Results

3.1. LPL in pre- and post-heparin plasma Heparin (60 units/kg body weight) was injected intravenously into controls and a patient with hyperchylomicronemia, and the lipolytic activity in plasma obtained 15 min after heparin injection was measured. The lipolytic activity in post-heparin plasma of controls increased linearly with the volumes of plasma up to 5 /xl, whereas that in post-heparin plasma of the patient was not detected even when 50 /xl of plasma was added to the assay mixture (data not shown). Based on these findings, we used 5/xl of plasma to measure the lipolytic activity. Table 1 shows the activity and mass of LPL in pre- and post-heparin plasma of controls and the patient. Heparin injection into controls caused 2.3-fold increase in LPL mass and release of active LPL. The amount of LPL in plasma of the patient increased 0.8-fold to 392 n g / m l after heparin injection. In spite of the same LPL mass as that in post-heparin plasma of controls, no activity was detectable in post-heparin plasma of the patient. The proteins in pre- and post-heparin plasma were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with antiserum to bovine LPL. Antiserum identified bands, which co-migrated with human adipose tissue LPL with M r = 57 000 [11], in pre- and post-heparin plasma samples of the patient as in those of controls (Fig. 1). Thus the LPL subunits of the patient were normal in size ( M r = 57000)

2.5. Sucrose density gradient centrifugation A mixture of 0.2 ml of post-heparin plasma and 0.4 ml of solution B was layered on a linear 5 - 1 5 % sucrose gradient (11.2 ml) in solution B overlaid on 1.0 ml of a cushion of 40% sucrose in solution B. The gradient was centrifuged at 40000 rpm for 24 h at 4°C in a Hitachi RPS-40T rotor, and 0.28-ml fractions were then collected by aspiration from the bottom of the tube. Aldolase (s = 7.5 S) and bovine serum albumin (s = 4.3 S) were run in parallel as markers.

2.6. Western blotting An aliquot of each fraction from the gradient was mixed with an equal volume of 0.125 M Tris buffer (pH 6.8) containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.002% bromophenol blue and heated for 5 min at 95°C. Proteins in the fraction were separated by SDSPAGE in a Laemmli type system [10] with 10% acrylamide resolving gel and 3% acrylamide stacking gel. The proteins separated were then transferred electrophoretically to a nitrocellulose membrane and blotted. Nonspecific binding was blocked by incubating the membranes in solution D containing 5% bovine serum albumin for 1 h. The blots were washed with solution D and then incubated for 1 h with chicken antiserum to bovine LPL at 1:6000 dilution in solution C. The blots were washed with solution D and exposed for 1 h to rabbit antichicken IgG at 1:20000 dilution in solution C. They were then washed with solution D, incubated for 1 h with 125I-protein A

Fraction

33

3.2. Chromatography of LPL in post-heparin plasma on heparin-Sepharose Heparin-Sepharose has been used to separate LPL from hepatic triacylglycerol lipase (HTGL) in post-heparin

NO.

Control

Bottom

Fraction

No.

I

Top

5

10

15

20

25

30

35

40

Patient

tB o t t o m

1

Top

Fig. 6. Western blots of LPL in fractions from a sucrose density gradient. Proteins in each fraction separated in Figs. 4 and 5 were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted as described in Section 2.

34

H. Masuno et al. / Biochimica et Biophysica Acta 1254 (1995) 30-36

plasma [12]. Both lipases bind to heparin and can be eluted with NaCI solution. HTGL is eluted with a low salt concentration, usually 0.6 M, whereas LPL is eluted with a higher concentration, usually > 1.0 M. Fig. 2 shows the chromatograms of the lipolytic activities in post-heparin plasma on heparin-Sepharose. In the patient, the activities eluted with 0.6 M and 1.5 M NaC1 were less than 10 and 15%, respectively, of those of controls. Heparin-Sepharose is also used to separate the activedimeric form of LPL from the inactive-monomeric form [5-7]. The inactive-monomeric form of LPL is eluted with a low salt concentration, usually 0.6 M NaC1, whereas the active-dimeric LPL is eluted with a higher salt concentration, usually > 1.0 M NaC1. Fig. 3 shows the chromatograms of the LPL mass in post-heparin plasma on heparin-Sepharose. Almost all the LPL in post-heparin plasma of controls bound to the column, and 74% of the bound LPL was eluted with 1.5 M NaC1 in parallel with the activity, 26% being eluted with 0.6 M NaC1. These results suggest that most of LPL subunits in post-heparin plasma of controls was in the dimeric form. In contrast, in post-heparin plasma of the patient 58% of LPL did not bind to heparin-Sepharose, and 23% and 19% were eluted with 0.6 M and 1.5 M NaC1, respectively (Fig. 3). These results suggest that 81% of LPL subunits in post-heparin plasma of the patient was not in the dimeric form. 3.3. Oligomeric structure and activity o f LPL in postheparin plasma

The relationship between the oligomeric structure and activity of LPL in post-heparin plasma was examined by sucrose density gradient centrifugation. After centrifugation, the lipolytic activity in each fraction of the gradient was assayed. The lipolytic activity in post-heparin plasma of controls was detected as a broad peak (Fig. 4), which could be due to both LPL and HTGL activities. But in post-heparin plasma of the patient, no lipolytic activity was detected in any fraction (Fig. 5). Proteins in each fraction were separated by SDS-PAGE, transferred to a nitrocellulose membrane and blotted with antiserum to bovine LPL. The LPL subunits recovered in each fraction of the gradient all migrated as a single band with M r = 57 000 (Fig. 6). Next, the amounts of radioactivity in the bands corresponding to LPL were measured to determine the sedimentation coefficient of LPL (Figs. 4 and 5). In post-heparin plasma of controls, almost all the radioactivity was recovered in a peak (fraction No. 19-34) with a sedimentation coefficient of 6.8 S (Fig. 4). The M r of LPL in this peak was estimated to be 136 000 from the sedimentation coefficient of 6.8 S by the method of Martin and Ames [13], which corresponds to that of a dimeric form of LPL. Little radioactivity was recovered in the peak (fraction No. 3 6 39) with a sedimentation coefficient of 4.0 S. The M r of LPL was estimated to be 61000 from the sedimentation

coefficient of 4.0 S, which corresponds to that of the monomeric form of LPL. No radioactivity was found at the bottom of the centrifuge tube. In post-heparin plasma of the patient, 35% of radioactivity was recovered in fractions with larger sedimentation coefficients at the bottom of the centrifuge tube (fraction No. 1-10) (Fig. 5), and the radioactive bands in these fractions migrated on the gel with the same mobility as those corresponding to the monomeric form of LPL (Fig. 6). 9% and 40% of radioactivities were recovered in the peaks corresponding to the monomeric form and dimeric form of LPL, respectively.

4. Discussion

Studies by radiation inactivation [1,2] and sedimentation equilibrium analysis [3] of bovine milk LPL, and a study with monoclonal antibody of human LPL [5] showed that active LPL is a dimer. However, in a study by gel filtration [8] active LPL in human post-heparin plasma was found to be a monomer. In the present study, we found by sucrose density gradient centrifugation that over 90% of LPL in post-heparin plasma of controls was recovered in fractions corresponding to the position of the dimeric form in parallel with the activity (Fig. 4). The study with heparinSepharose showed that active LPL had a high affinity for heparin (Fig. 2). These results indicate that active LPL in normal human post-heparin plasma is a dimer. LPL deficiency is a relatively rare autosomal recessive disorder, occurring at a frequency of one in a million. As shown in Table 1, we also found that LPL in post-heparin plasma of a patient with hyperchylomicronemia was inactive. Some LPL subunits of the patient had larger sedimentation coefficients, being recovered at the bottom of the centrifuge tube (Fig. 5). Analysis by SDS-PAGE showed that LPL subunits recovered in the fractions with larger sedimentation coefficients migrated as a single band on the gel with the same mobility as the monomeric form of LPL (Fig. 6). Thus some LPL subunits in post-heparin plasma of the patient were in an aggregated form(s). Results also indicated increase in the monomeric form of LPL and decrease in the dimeric form (Fig. 5). Active LPL is a dimer with a high affinity for heparin, and conversion of the dimeric form to the monomeric form results in loss of activity and low affinity for heparin [1-5]. Fig. 3 showed that about 70% of LPL in post-heparin plasma of controls was active and had a high affinity for heparin, whereas about 80% of that in post-heparin plasma of the patient was inactive and had lower affinity for heparin. These findings corresponds with those in Fig. 5 that the dimeric form of LPL was decreased in post-heparin plasma of the patient. Thus dysfunction of LPL in this patient could result from reduced dimerization of LPL subunits.

H. Masuno et al. / Biochimica et Biophysica Acta 1254 (1995) 30-36

The formation of a dimer is essential for expression of the LPL activity [1-3,5]. However, all the dimeric LPL does not seem to be active. Although over 90% of LPL in post-heparin plasma of controls was recovered in fractions corresponding to the position of the dimeric form of LPL (Fig. 4), the study with heparin-Sepharose showed that about 70% of that of controls was active and dimeric (Figs. 2 and 3). In addition, the patient's LPL recovered in fractions corresponding to the position of a dimeric form of LPL was inactive (Fig. 5). Thus, some of the dimeric LPL may be inactive. Pre-heparin plasma of the patient contained a large amount of LPL, and increment of LPL released into plasma by heparin was small (Table 1). Fig. 3 showed that about 60% of LPL in post-heparin plasma of the patient did not bind to heparin-Sepharose and 53% of the bound LPL was eluted with 0.6 M NaC1. These results indicate that the affinity of the patient's LPL for heparin was very low. There are many reports on mutations in the LPL gene of patients with LPL deficiency. Beg et al. [14] reported that a single amino acid substitution (Ala-176 ~ Thr) in an LPL molecule led to abnormal heparin binding and loss of enzyme activity. Moreover, Emi et al. [15] found that LPL with a missense mutation (Gly-188 ~ Glu) was catalytically inactive and displayed lower affinity for heparin than the normal enzyme. The present patient had only a single base substitution (C ~ A) at nucleotide 1338 in exon 8 of the LPL gene and was heterozygous for this mutation (data not shown). But this mutation had no effect on the primary structure of LPL. Thus dysfunction of the present patient's LPL was not due to the abnormal primary structure of LPL. The present study showed that the patient with lipase deficiency exhibited defects of both HTGL and LPL activities in post-heparin plasma (Fig. 2). This combined lipase deficiency is very rare. Auwerx et al. [16] reported a large family with familial HTGL deficiency showing reduced levels of LPL similar to that in the heterozygous state of LPL deficiency. Analysis of genomic DNA by restrictionenzyme digestion revealed no major abnormalities in the structure of either the HTGL or LPL gene. Kihara et al. [17] reported a patient with marked hyperchylomicronemia and reduced activities of LPL and HTGL. They found antibodies to LPL and HTGL in plasma of the patient and concluded that these autoantibodies were responsible for the hyperchylomicronemia. In the present study, plasma of the patient did not suppress the post-heparin plasma lipolytic activity of controls (data not shown). This result indicates that dysfunction of LPL and HTGL in the patient was not due to the presence of antibodies to LPL and HTGL in plasma. In mice, combined lipase deficiency is a recessive mutation within the T / t complex of chromosome 17, and mice homozygous for this defect develop massive hyperchylomicronemia and die within 3 days if allowed to suckle [18]. The HTGL and LPL activities in their post-

35

heparin plasma [18] and tissues [7,18-20] are both defective. Dysfunction of these enzymes is due to incomplete processing of their oligosaccharide chains in the cells [7,20]. Human LPL has two N-linked oligosaccharide chains [21], a high mannose type and a complex type chain [11]. However, whether defect of LPL activity from postheparin plasma of the present patient resulted in abnormal processing of oligosaccharide chains of an LPL subunit remains to be resolved. LPL activity was not detected in 5 /xl of post-heparin plasma of the patient (Table 1). However, when LPL in a large volume (800 /zl) of post-heparin plasma was absorbed to heparin-Sepharose and eluted with 1.5 M NaC1, a small amount of active LPL with near normal specific activity was detected (Figs. 2 and 3). The reason for this discrepancy is probably the different volumes of postheparin plasma used.

Acknowledgements We thank Drs. Thomas Olivecrona and Gunilla Bengtsson-Olivecrona, University of Ume~, Ume~, Sweden, for gifts of chicken antiserum to bovine LPL and chicken anti-LPL IgG. We also thank Dr. Kohji Shirai, Toho University, Tokyo, for valuable discussion.

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