26
Biochimica et Biophysics Acta, 409 (1975) 26-38 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BRA 56677
PROPERTIES OF ROOSTER SERUM HIGH DENSITY LIPOPROTEINS
ARTHUR W. KRUSKI* and ANGELO M. SCANU** Departments of Medicine and Biochemistry, University of Chicago Pritzker School of Medicine and The Franklin McLean Memorial Institute, Chicago, Ill. 60637 (U.S.A.) (Received May 16th, 1975)
Summary
The physical and chemical properties of normolipemic rooster serum high density lipoproteins (HDL) were determined and compared with human HDL. Rooster HDL was found to be composed of essentially one class of particles, as determined by flotational analysis at d 1.21 g/ml in the analytical ultracentrifuge. On a weight-percentage basis, it contained 44, 29, 16, 5 and 6% protein, phospholipid, cholesteryl ester, cholesterol, and triacylglycerol, respectively. This distribution resembled that of human HDL2 more closely than that of HDL3. On the other hand, the physical parameters of rooster HDL resembled those of human HDL3. The sedimentation coefficient, diffusion coefficient, molecular weight, partial specific volume and the anhydrous frictional ratio for rooster HDL were 3.99 S, 4.36 * 10e7 cm* . se*, 1.73 . 105, 0.868 ml/g, and 1.24, respectively. However, the circular dichroic spectra of rooster HDL indicated an (Y-helical content 20% greater than that in human HDL. The lipid composition resembled that of human HDL except for a relatively higher content of stearic acid. The HDL protein gave several bands by polyacrylamide gel electrophoresis. The main component, representing almost 90% of the total protein, had a molecular weight of about 26 000, an amino acid composition and an electrophoretic mobility similar to those of human apolipoprotein A-I. A component of mol. wt 15 000 and a group of fast-migrating peptides, resembling the human apo C peptides, were also found. It is concluded that the structure of rooster HDL, although showing similarities to human HDL, can be distinguished from the latter by some physical parameters, but particularly by its polypeptide distribution.
* Present address: Biomedical Division, Lawrence Livermore Laboratory, University of California, Livermore. Calif. 94550. U.S.A. ** To whom correspondence and reprint requests should be addressed. Abbreviations used are: HDL, high density lipoproteins isolated between d 1.063 and 1.21 g/ml: HDk, HDL isolated between d 1.063 and 1.125 g/ml: HDL3. HDLisolated between d 1.125 and 1.21 g/ml; p, solvent density: d, serum density: “2 partial specific volume.
27
Introduction In previous studies, it was shown that the chicken can be a useful experimental animal for the study of cholesterol metabolism [l--5]. In these studies, the large increase in serum cholesterol levels which accompanied cholesterol feeding could be accounted for mainly by the increased concentrations of the serum very low density lipoproteins. Under these experimental conditions, chicken high density lipoproteins (HDL) exhibited much smaller, though significant changes. An understanding of the mechanism of cholesterol-induced changes in the physical and chemical properties of the serum lipoproteins would require an accurate knowledge of these lipoproteins under normolipemic conditions. In the case of the rooster, such information is not available. This laboratory has long been interested in defining the structure of serum HDL from a physical and chemical standpoint in humans as well as in a number of animal species [6-S] . Such investigations, as well as those of others [g--15] have clearly indicated the value of comparative structural studies for the determination of the properties of HDL. In this study, we have investigated the physical and chemical properties of serum HDL from roosters fed a low cholesterol, low fat diet and compared these values with those obtained in humans. The parameters for human HDL were either determined concurrently with those of the rooster or were taken from data found in the literature. Materials and Methods Animals. Six male, white Leghorn roosters (about 2 kg body weight each) were used. The animals, which were obtained commercially, were kept on Purina Chow and water fed ad libitum during the 6 weeks preceding blood withdrawal. Blood was obtained by cardiac puncture and was allowed to clot at room temperature for several hours to give serum upon centrifugation. The serum was made 0.05% in EDTA (pH 7.0) and was used immediately for lipoprotein separation. Isolation of HDL by ultracentrifugal flotation. Rooster HDL, isolated between d 1.063 and 1.21 g/ml, were obtained by preparative ultracentrifugal methods identical to those used for the separation of human serum lipoproteins [ 161. This density range contained essentially all the serum HDL as determined by dodecyl sulfate polyacrylamide gel electrophoresis, agarose column chromatography and by isopycnic density gradient- and analytical ultracentrifugation. The bright yellow-orange HDLs of each animal, after extensive dialysis against a solution of 0.15 M NaCl, 0.05% EDTA, pH 7.0, were analyzed individually. Delipidation of HDL. Dialyzed rooster HDL was delipidated, and the resulting protein and lipid fractions were prepared by methods identical to those previously described for human HDL [ 171. CsCl isopycnic density gradient ultracentrifuga tion. For determination of the purity and homogeneity, as well as the mean density of the HDL prepared as above, the samples were subjected to a CsCl isopycnic density ultracentrifugal technique that was previously described [ 181. Agurose gel chromatography. The HDL samples (about 4 mg/ml of HDL protein) were applied to 6% beaded Agarose gel (Bio-Gel A-5m, 200-400
28
mesh; Bio-Rad Laboratories, Richmond, as previously described [ 181.
CA) at 4°C on a 1.5 X 75 cm column,
Polyacrylamide gel electrophoresis. Dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide) was performed essentially according to the procedure of Weber and Osborn [19], as previously described [18]. The method of Davis [ZO] , with the incorporation of 8 M urea, was followed for making the 8 M urea, 7.5% polyacrylamide gels. After electrophoresis, the gels were stained in 1% Amido Black in 7.5% aqueous acetic acid. Circular dichroic studies. Circular dichroic experiments were performed as previously described [18] . The samples had a protein concentration of 0.05-0.5 mg/ml. Analytical ultracentrifugation. Analytical ultracentrifugal studies of rooster HDL were conducted in a Beckman Model E Analytical Ultracentrifuge at 20°C using an AN-H rotor. Sedimentation experiments were performed at 52 000 rev./min in a double-sector cell. Diffusion experiments were conducted at 4000 rev./min using a synthetic boundary centerpiece. The sedimentation and diffusion coefficients at infinite dilution, ~02s w and D,, w respectively, were calculated by conventional procedures [21]. Fiotational studies of rooster HDL (d 1.063-1.21 g/ml) and human HDL (d 1.063-1.21 g/ml) were performed at 44 000 rev./min at a solvent density of 1.21 g/ml using a doublesector cell. Sedimentation equilibrium experiments were conducted at 5600 rev./min in a synthetic boundary cell using interference optics. The partial specific volume (u2 ) was obtained from density measurements at 2O.O”C using a Mettler/Anton Paar Precision Density Meter (Graz, Austria) equipped with a Tronac Temperature Control Unit (Salt Lake City, UT). Isolation of apo-HDL by gel filtration. Rooster apo-HDL (30 mg) was fractionated by gel filtration in columns (100 X 5 cm) containing Sephadex G-200 at 8°C under previously described conditions [6] . The protein solvent and the eluting buffer were 0.01 M Tris, pH 8.2, 0.001 M EDTA, 0.02% NaN3 in 8 M urea. Amino acid analysis. Protein samples were hydrolyzed for 24 h in a 6 M HCl solution containing P-mercaptoethanol (1 : 2000 v/v) at llO”C, as previously described [22]. Tryptophan was determined by the method of Liu and Chang [23], except that 3 M p-toluene-sulfonic acid was replaced by 4 M methanesulfonic acid. absorption spectra were reUltraviolet absorption studies. Ultraviolet corded on a Beckman DU spectrophotometer with a Car-y recording attachment. Protein samples were dissolved in 0.01 M NH4 HC03, pH 8.2. The pH was changed from 8.2 to 11.6 by the addition of 0.1 M NaOH. Immunodiffusion. Immunodiffusion studies with rooster HDL and rabbit antisera against human low density lipoproteins, human HDL2 , and apo A-I were performed in 1% agarose gels in 0.05 M Verona1 buffer, pH 8.6, as previously described [ 181. Chemical analyses. Protein concentrations were measured by the method of Lowry et al. 1241. Phospholipids were determined as lipid P X 25 [25]. Individual phospholipids were separated and identified by thin-layer chromatography according to the method of Skipski et al. [26] and computed as lipid P X a constant (16.8 for lysolecithin, 25 for phosphatidylcholine and -ethanol-
29
amine and 22.6 for sphingomyelin). By this method, phosphatidyl serine and -inositol are included in the lecithin fraction, however, two-dimentional, thinlayer chromatography indicated that these two phospholipids comprise less than 5% of the total phospholipids. Cholesterol, cholesteryl esters and triacylglycerols were separated by thinlayer chromatography (developing solvent, 90 : 10 : 1, chloroform/methanol/ glacial acetic acid). Cholesterol and cholesteryl esters were determined by the method of Zak et al. [27] , while triacylglycerols were measured by the procedure of Amenta [28]. The fatty acid composition of each phospholipid class, cholesteryl esters and triacylglycerols was determined as previously described [ 181. Results Behavior of HDL after CsCl density gradient ultracentrifugation and agarose column chromatography After agarose column chromatography, rooster HDL as well as human HDL3 emerged as a single, symmetrical peak (Fig. 1). Neither HDL indicated any evidence of higher or lower molecular weight components. Rooster HDL, however, had a slightly larger elution volume than did human HDL, . A single band was also observed after CsCl isopycnic density gradient ultracentrifugation (Fig. 2). In this system, rooster HDL banded at d 1.12 g/ml, whereas human HDL3 banded at d 1 .13 g/ml. 1100,
- 1 35
-125 0.5.
2 ,E z -1.15
zs x
0.1.
“0 40
“T 50
60 TUBE
70
80
90
100
110
NUMBER (0.97ml/tubel
Fig. 1. Agarose (Bio-Gel A-5x11) gel column chromatography elution profile of kooster serum HDL ) and human serum HDL3 (- - - - - 9, with 8 mg of each HDL. The void volume, VO, was 42 ml and (the total solvent volume. VT, was 115 ml. Fig. 2. CsCl isopycnic den&y gradient ultracentrifugation profiles of rooster serum HDL ( -) human serum HDL3 (- - - - - -). The density gradient (o---.-o) was established after 66 h. at 11°C 38 000 rev./min in a Spinco SW-40 rotor.
and and
30
TABLE
I
PROTEIN
AND
LIPID
COMPOSITION
component
OF
ROOSTER
Rooster**
HDL,
Human*
HUMAN
HDL.2.
AND
HUMAN
HDLs*
* *
HDL HDL2
Protein
43.88
i
41
55
Phospholipids
28.66
f 2.21
29.5
22.5
16.03
? 1.63
16.2
11.7
Cholesteryl
esters
2.43
HDL3
Cholesterol
5.01
* 0.34
5.4
2.9
Triacylglycerols
6.42
f 1.38
4.5
4.1
* Percent .** Mean ***
by
weight
average
Scanu
and
t standard
of duplicate
Kruski
deviation.
measurements
for
5 roosters.
[341.
Chemical composition The protein-lipid distribution of rooster HDL gave a composition intermediate between those of human HDLz and HDL3 (Table I). The phospholipid distribution of rooster HDL was found to have a slightly greater percentage of lysolecithin and phosphatidylethanolamine, but a lower content of sphingomyelin and phosphatidylcholine, than human HDL (Table II). The fatty composition of rooster HDL resembled that of human HDL (d 1.063-1.21 g/ml, Table III), although in each lipid class, there was a greater percentage of stearic acid (18 : 0) than in human HDL.
Spectral studies The circular dichroic spectra of both rooster HDL and apo-HDL exhibit minima at 210 and 222 nm (Fig. 3). The depths of the minima indicate that rooster HDL and apo-HDL had a greater amount of a-helix (almost 30%) than that found in the corresponding human fractions (Table IV). The ultraviolet absorption spectrum of rooster HDL at pH 8.2 was generally similar to that of human HDL3, with an absorption maximum at about 278 nm (Fig. 4). When the pH was increased to 11.6, a slight shift to the red occurred. Rooster apo-HDL and human apo-HDL3 had nearly identical spectra TABLE
II
PERCENT
PHOSPHOLIPID
DISTRIBUTION
Phospholipid
IN
HDL
(d
1.063-1.21
R00sl.er
Human
HDL
HDL
g/ml)
OF
ROOSTER
Human*
AND
* HDL3
HDL2 Lysolecithin Sphingomyelin
3.6
+ 0.8
2.1
+ 0.8
2.0
MAN*
.._ 5.4
8.0
+_ 1.4
11.2
+ 1.0
14.5
9.2
Phosphatidylcholine
76.8
_+ 1.6
79.5
_+ 0.8
73.8
77.1
Phosphatidylethanolamine
11.5
f 1.2
7.1
* 1.0
7.9***
_~__.~_ * Mean each
average
* * Scanu
** *
?
in duplicate,
Includes
and
Kruski
standard whereas
deviation. pooled
The human
phospholipids HDL-PL
were
[341.
phosphatidylinositol
and
polyglycerophosphatides.
__~_ from
5 roosters
determined
6.9*** -
were
in triplicate.
~._.
analyzed
separately,
_~
14
0
3.17
0.47 0.86
30.75 20.40
0.55 0.10
3.78 0.89
0.26 0.30
1.20 0.63 2.13 0.90
1.56 2.42
50.33 59.96
1.89 1.17
3.31 2.75
13.61 18.63
2.59 2.37
36.49 29.27
1.61 1.46
2.78 3.62
2.82 2.54
--
:1
9.45 15.72
0.23
1.90 1.29
16
4.97 3.69
16 : 0
OF ROOSTER
23.56 28.23
0.99
:0
0.57 0.36
15
3.31 2.23
:0
COMPOSITION
0.55 1.39
.12:
ACID
FATTY
:0
17.53 30.20
17.14 29.25
5.82 2.38
18.66 20.66
23.16 42.10
2.10 3.67
0.51 0.20
1.77 1.15
:0
5.13 11.95
18
AND HUMAN
0.20 0.40
11
HDL
:1
11.14 7.95
9.69 12.16
9.37 12.41
16.49 13.49
20.87 26.54
37.29 39.28
18
:2
10.36 8.75
16.71 18.55
1.42 1.91
4.19 1.61
48.78 44.86
20.92 13.79
18
-
:4
20.79 18.53
11.45 14.31
0.97
4.58 7.50
5.64 3.90
2.13 2.28
20
HDL TRIACYLGLYCEROLS,
* Each percentage is the mean obtained from 5 different roosters or from a pooled human HDL sample. * * Ass&went not certain.
Rooster Cholesteryl esters Human Rooster Lysolecithin Human Rooster Sphingomyelin Human Rooster Phosphatidylcholine Human Rooster Phosphatidylethanokunine Human Rooster
Human
TrlacyIgIyceroIs
PERCENT AVERAGE PHOLIPIDS*
TABLE III
2.14 2.77
2.44 1.63
1.67
(20
: 3j**
CHOLESTERYL
: a**
6.54 3.00
(22
ESTERS
PHOS-
7.49 5.83
9.86 4.13
10.69 0.97
5.67 2.00
8.44 0.71
1.36 1.90
Unidentified
AND
\ 8
ii
:,
0.9
_‘L__l
4
CHICKEN APO HDI and HUMAN APO HDL,
i4
‘$4
~
200
210
220
230
240
250
-
240
Xlnm)
260
280
300
320
X lnm)
Fig. 3. Circular dichroic spectra of: A. rooster HDL; B and C, human HDL3 and rooster ape-HDL (these spectra were identical); D, Human ape A-I: E, human ape A-II. The crossover points of the native HDLs are at about 202 nm; they occur at 200 nm for the apoproteins. Fig. 4. Ultraviolet spectra of rooster HDL and ape-HDL, as well as human HDL3 and ape HDL3. at pH 8.2 and 11.6. Top: rooster apo HDL, pH 8.2 ( -) and 11.6 (- - - - - -); human apo HDL3. PH 8.2 (o-----o) and 11.6 (o M>). Bottom: rooster HDL. pH 8.2 ( -) and 11.6 (------): human HDL3, pH 8.2 (0 -) and 11.6 (o-4). The protein concentration of all samples was 0.5 mg/mI.
TABLE
IV
PHYSICAL Physical
PROPERTIES
OF ROOSTER
constants
HDL AND HUMAN
HDL2 AND HDL3
Rooster* HDL
Human* * HDL2
sio,w (S)
D~o,~ (cm2. Mr. 10’
s-l).
10
-1
p(g/mI) ~2
(ml/g)
f//f0 (anhydrous) (assuming [B] 222 . lO+ [01,,,.104
0.2 g HZO/g of lipoprotein) deg . cm2 . dmol-’ deg.cm2.dmol-’
3.99 4.36 1.73 1.152 0.868 1.24 1.16 -3.50 -3.56
+ + * ? +
0.13 0.30 0.13 0.12 0.009
5.45 3.68 3.65 1.105 0.905 1.10 1.02 -2.60 -2.58
HDL3 4.65 3.93 1.75 1.153 0.867 1.25 1.15 -2.52 -2.50
* Mean average f stand deviation of six determinations, one for each rooster, except for molecular weight were three determinations were used. ** From Scanu et al. [S]. except for f/f0 and circular dichroic data which were taken from ref. 35.
33
at pH 8.2 and a comparatively HDLs at pH 11.6.
larger red shift than that observed
with native
In flotational studies at p 1.21 g/ml, rooster HDL gave a single, nearly symmetrical peak. However, human HDL (d 1.063-1.21 g/ml), studied under the same conditions, gave a pattern consisting of a main pe_ak (HDL, ) and a shoulder representing a less dense, faster-floating component (HDLz ) not observed with rooster HDL. In sedimentation studies at p 1.006 g/ml, rooster HDL exhibited a single symmetrical peak. The sedimentation coefficient, siO ,W; diffusion coefficient, D i 0 ,W; partial specific volume, u2 ; and the frictional ratios (f/fO, calculated from the above parameters, refs 29, 30) were generally similar to those of human HDL3 (Table IV). The molecular weight of rooster HDL, calculated from the values for and u2 was 1.71 * 10 ’ which was nearly identical to the value of &,W G0.W 1.73 * 10’ determined directly from the results of the sedimentation equilibrium experiments.
After electrophoresis in 8 M urea gels, the rooster apo-HDL pattern contained a major band (band 3) and a minor band (band 4), which correspond in migration distance to those of human apo A-I and apo A-II, respectively (Fig. 5). Densitometric scans of these gels indicate that the major (band 3) and the minor (band 4) bands found in rooster apo-HDL represented about 91% and 9% of the total protein in comparison to 86% and 14% for human apo A-I and apo A-II respectively. Additional bands were seen when the amount of rooster apo-HDL loaded on the gels was increased: two bands (bands 1 and 2)
Fig. 5. Polwmylamide gel electrophoresis patterns of rooster apo HDL and human apo HDL3: A and B, 8 M urea: C and D. 0.1% dodecyl sulfate. The polyacrylamide concentration in the urea and dodecyl sulfate gels was 7.5 and 10%. respectively. A and C: rooster ape-HDL; B and D: human apo-WLg.
34
with a slower migration, than bands 3 and 4, and four bands (bands 5-8) which had the fastest migration rates. After electrophoresis in the 0.1% dodecyl sulfate-containing gels, three bands were observed for rooster apo-HDL (Fig. 5). The band with the largest molecular weight (about 70 000, band 1) was generally present in variable amounts, depending on the method of preparation of the apo-HDL. The two other bands present in these gels had molecular weights of about 26 000 (band 2) and 15 000 (band 3) which were unaltered by the presence of the reducing agent, dithiothreitol. A representative gel scan of the bands indicated that about 8, 91 and 1% of the observed protein bands were present as bands 1, 2, and 3 respectively. A gel scan of the human apo-HDL3 pattern indicated that about 81 and 19% of apo A-I and apo A-II respectively were present, in general agreement with the distribution reported in the literature [22] . Gel filtration
studies
Two major peaks (peaks I and II) were obtained after elution of rooster apo-HDL by Sephadex G-200 chromatography in 8 M urea (Fig. 6). Peak I was heterogeneous as assessed by dodecyl sulfate-polyacrylamide gel electrophoresis. Peak II (band 2 in Fig. 5 for the dodecyl sulfate gels) eluted at the same volume as did human apo A-I, indicating a similarity in molecular weights. Peak II consisted essentially of one protein, the major rooster protein in apo-HDL, although a small amount of higher molecular weight components was observed
Fig. 6. Sephadex G-200 chromatography in 8 M urea of rooster ape-HDL. Fractions designated by the bar lines indicate the range used for the dodecyl sulfate-polyacrylamide electrophoresis gel.?.. Migration is from top(cathode) to bottom(anode) with the origin designated as 0.
35 TABLE
V
COMPARISON OF THE AMINO ACID COMPOSITION OF THE FRACTIONS OBTAINED FROM SEPHADEX G-200 CHROMATOGRAPHY IN 8 M UREA -Amino acid
-_--
Rooster
OF ROOSTER APO-HDL WITH HUMAN APO A-I*
Human apo A-I * *
Peak I
Peak II
20 1 17 20 11 12 47 11 6 21 N.D.*** 14 5 6 32 6 5 N.D.***
21 1 18 20 11 10 47 12 3 23 N.D.*** 13 5 6 33 7 5 1
___--
LYS His Arg ASX Thr Ser GIX Pro GIY Ala Half-c ys Val Met Be Leu Tyr Phe Trp Total residues
(234)***
(236)***
21 5 16 21 10 14 47 10 10 19 0 13 3 0 39 7 6 4 245 -
* Number of residues per mol, assuming for the rooster fractions a molecular weight of about 26000. i.e., that obtained for the major ape-HDL polypeptide after dodecyl sulfate-gel electrophoresis. Each analysis was the average of at least two determinations. ** Ref. 31. *** Not determined, therefore total residues are tentative.
after dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid analysis of peak II was very similar, though not identical to that of human apo A-I (Table V). For comparison, the amino acid composition of peak I is also included, assuming peak I is mainly an aggregate of peak II. When this comparison is made, the composition of peak I is almost identical to that of peak II. After elution of peak II from the Sephadex column, a very small amount of protein designated as peak III was obtained. This peak contained band 3 in the dodecyl sulfate gels, as well as peak II and small amounts of larger molecular weight components (Fig. 6). Immunology Rooster HDL did not form precipitin lines with human serum low density lipoproteins, HDL2 or apo A-I.
rabbit
antiserum
to
Discussion The results of the present study indicate that many chemical parameter8 of rooster HDL are similar to those of in keeping with data in the literature on a number of 8,11,1 3-151. Thus, a typical serum HDL particle from
of the physical and human HDL. This is animal species [6either human or the
36
animal species studied has the following general features: (a) it is composed of almost equal amounts of protein and lipid; (b) about half of the lipid is phospholipid, with most of the remaining lipid present as cholesteryl ester; (c) at least 50% of the fatty acids of the lipids are unsaturated; (d) the particle weight is between 1.7 * lo5 and 3.6 . lo5 ; (e) the major HDL apoprotein has a molecular weight of about 27 000; (f) the apo-HDL has a greater than 50% a-helix conformation; and (g) the major apo-HDL contains no half-cystine, whereas about 20% of the amino acid residues consist of glutamine or glutamic acid. Evidently, these many structural similarities suggest one or more common functions for the serum HDL of different species. What these functions are is not clear at the present time. Rooster serum HDL has a protein-lipid composition which is very similar to that of human HDL2 (Table I); yet the physical parameters more closely resemble those of human HDL3 (Table IV). Of the physical parameters of rooster HDL, the molecular weight, partial specific volume, and the unhydrated or hydrated frictional ratios are practically identical with those of human HDL3, whereas the sedimentation and the diffusion coefficients are consistently different. The lack of reactivity of rooster HDL to rabbit anti-human HDLz or apo A-I, as well as the slight differences noted in the respective ultraviolet absorption spectra, suggest differences in the conformation and/or the amino acid composition (Table V) of the protein moiety of these lipoproteins. Further evidence indicating structural differences between rooster and human HDL comes from circular dichroic studies which reveal that rooster HDL has an almost 30% greater amount of a-helix estimated by the molar ellipticity at 222 nm (ref. 32; Table IV). Also as expected, the content of a-helix in rooster apo-HDL was greater by about 20% than either human apo A-I or apo A-II (Fig, 3). The a-helix content of rooster HDL appears to be the largest yet reported, whether it is compared to man or to the other animal species studied, including the monkey [ 81, rat [7] , pig [ 131, and the pink salmon [ 151. The larger helical content of rooster HDL suggests a structural difference from that of human HDL which may account for the different sedimentation and diffusion coefficients found for these HDLs. The phospholipid class distribution of rooster HDL is slightly different than that of human which may account for additional structural dissimilarities between these lipoproteins (Table II). The major protein found in rooster HDL, comprising about 90% of the total protein as determined by polyacrylamide gel electrophoresis, had a molecular weight of about 26 000 compared to 28 331 [31] for the sequenced major human apo-HDL, apo A-I (Figs 5 and 6). A protein of lower molecular weight (about 15 000 representing band 3 in dodecyl sulfate gels and band 4 in the urea gels, Fig. 5, and peak III, Fig. 6) was also present; but, unlike human apo A-II, it was unaffected by reducing agents. This protein, the relationship of which to human apo A-II remains unknown, comprised about 1 and 9% of the total protein of rooster HDL in dodecyl sulfate or urea gels, respectively. The reason for this consistent difference between the two gel systems may be due to the limitations inherent in the quantitation of stained gels [ 331. Small amounts of polypeptides having migration rates similar to those of the human
37
apo C polypeptides were also present in rooster apo-HDL, as determined by 8 M urea gel electrophoresis (bands 5-8, Fig. 5). Higher molecular weight species were consistently found in all preparations of rooster apo-HDL, as indicated by both dodecyl sulfate (band 1) and urea (bands 1,2) gel electrophoresis (Fig. 5), as well as by gel filtration in 8 M urea (peak I, Fig. 6). Hillyard et al. [12] experienced similar difficulties with aggregates of chicken apo-HDL in several different detergent and denaturing solvents. These authors found a major component of chicken apo-HDL having a molecular weight of 30 100 in a phenol/urea/acetic acid/polyacrylamide gel system which resembles the major component of rooster apo-HDL of molecular weight 26 000 that we find in 0.1% dodecyl sulfate-polyacrylamide gels. Undoubtedly, more detailed studies of rooster apo-HDL will be needed for further characterization of the component polypeptides. A&no wledgments The authors are grateful to Dr Lidia Vitello for valuable advice in the preparation of this manuscript and assistance during the analytical ultracentrifugal studies, and to Mr Jim Foreman for help in performing the amino acid analyses. They are grateful to Mr Randolph Hughes and Mr Lance Lusk for assistance with the animals. A.M. Scanu is a recipient of U.S. Public Health Service Career Development Award No. HL 24867. A.W. Kruski is a recipient of U.S. Public Health Service Postdoctoral Fellowship No. HL 52970. This research was supported by Grants No. HL 08727 and HL 06481 from the U.S. Public Health Service, and by the Illinois and Chicago Heart Association (A 72-6). The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration. References 1 Jones, R.J. and Dobrilovic. L. (1969) Proc. Sot. Exp. Biol. 130. 163-167 2 Kruski. A.W. and Narayan. K.A. (1972) Lipids 7.742-749 3 Kruski. A.W., Kummerow. F.A. and Narayan. K.A. (1972) Protides Biol. Fluids Proc. Colloq. 19, 121-125 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Reveille, G.A.. Tillotson. J.A. and Sauberlich. H.E. (1963) J. Nutr. 81, 357-362 Leveille, G.A. and Sauberlich, H.E. (1963) Proc. Sot. Exp. Biol. 112. 300-303 Edelstein, C.. Lim. C.T. and Scanu. A.M. (1973) J. Biol. Chem. 248,.7653-7660 Koga, S.. Horwtz, D. and Scam& A.M. (1969). J. Lipid Res. 10. 577-588 Swum, A.M., Edelstein, C.. Vitello, L.. Jones, R. and Wissler. R. (1973) J. Biol. Chem. 248, 76487452 Blaton, V.. Vercaemst. R.. Vandecasteele, N.. Caster, H. and Peetea, H. (1974) Biochemistry 13, 1127-1135 Cox. A.C. and Tanford, C. (1968) J. Biol. Chem. 243, 3083--3087 Fidge, N. (1973) Biochim. Biophys. Acta 295. 258-273 HiByard. L.A.. White, H.M. and Pangburn. S.A. (1972) Biochemistry 11, 511-518 Jackson, R.L.. Baker. H.N., Taunton, O.D., Smith, L.C., Garner, C.W. and Gotto, A.M. (1973) J. Biol. Chem. 248, 2639-2644 Jonas, A. (1972) J. Biol. Chem. 247.7767-7772 Nelson, G.J. and’shorn. V. (1974) J. Biol. Chem. 249, 536-542 Scanu, A.M. and Ritter. M.C. (1973) Adv. Cfin. Chem. 16.111-151 Scanu. A. (1965) J. Lipid Res. 7, 295-306 Kruski. A.W. and Scanu, A.M. (1974) Chem. Phys. Lipids 13, 2748
19 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 44064412 20 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404427 21 Chervenka, C.H. (1970) A Manual of Methods for the Analytical Ultracentrifuge, Beckman Instruments, Inc., Palo Alto, CA. 22 Scanu, A., Toth, J., Edelstein, C., Koga, S. and Stiller, E. (1969) Biochemistry 8. 3309-3316 23 Liu, T.-Y. and Chang. Y.H. (1971) J. Biol. Chem. 246, 2842-2848 24 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-2’7t 25 Bartlett, G.F. (1959) J. Biol. Chem. 234, 466-468 26 Skipski, V.P.. Peterson, RF. and Barclay, M. (1962) J. Lipid Res. 3, 467-470 27 Zak, B., Dickenman, R.C., White. E.G., Burnett, H. and Cberney, P.J. (1954) Am. J. Clin. Patbol. 24, 1307-1315 28 Amen&, J.S. (1964) J. Lipid Res. 5, 270-272 29 Hazelwood. R.N. (1957) J. Am. Chem. Sot. 80, 2152-2156 30 Tanford. C. (1967) Physical Chemistry of Macromolecules John Wiley and Sons, New York 31 Baker, N.H.. Delahunty, T., Gotto, A.M. and Jackson, R.L. (1974) Proc. Nat]. Acad. Sci. U.S. 71, 3631-3634 32 Scanu, A.M. (1969) Biochim. Biophys. Acta 181, 268-274 33 Kruski, A.W. and Narayan, K.A. (1974) Anal. Biochem. 60.431-440 34 Scanu, A.M. and Kruski, A.W. (1975) International Encyclopedia &arm. Therapeutics, Chap. 2, Sect. 24, Pergamon Press, London 35 Scanu, A.M., ViteRo, L. and DeGanello, S. (1974) CRC Cnt. Rev. Biochem. March, 175-196
Biochimica et Biophysics Acta, 409 (1975) 26-38 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BRA 56677
PROPERTIES OF ROOSTER SERUM HIGH DENSITY LIPOPROTEINS
ARTHUR W. KRUSKI* and ANGELO M. SCANU** Departments of Medicine and Biochemistry, University of Chicago Pritzker School of Medicine and The Franklin McLean Memorial Institute, Chicago, Ill. 60637 (U.S.A.) (Received May 16th, 1975)
Summary
The physical and chemical properties of normolipemic rooster serum high density lipoproteins (HDL) were determined and compared with human HDL. Rooster HDL was found to be composed of essentially one class of particles, as determined by flotational analysis at d 1.21 g/ml in the analytical ultracentrifuge. On a weight-percentage basis, it contained 44, 29, 16, 5 and 6% protein, phospholipid, cholesteryl ester, cholesterol, and triacylglycerol, respectively. This distribution resembled that of human HDL2 more closely than that of HDL3. On the other hand, the physical parameters of rooster HDL resembled those of human HDL3. The sedimentation coefficient, diffusion coefficient, molecular weight, partial specific volume and the anhydrous frictional ratio for rooster HDL were 3.99 S, 4.36 * 10e7 cm* . se*, 1.73 . 105, 0.868 ml/g, and 1.24, respectively. However, the circular dichroic spectra of rooster HDL indicated an (Y-helical content 20% greater than that in human HDL. The lipid composition resembled that of human HDL except for a relatively higher content of stearic acid. The HDL protein gave several bands by polyacrylamide gel electrophoresis. The main component, representing almost 90% of the total protein, had a molecular weight of about 26 000, an amino acid composition and an electrophoretic mobility similar to those of human apolipoprotein A-I. A component of mol. wt 15 000 and a group of fast-migrating peptides, resembling the human apo C peptides, were also found. It is concluded that the structure of rooster HDL, although showing similarities to human HDL, can be distinguished from the latter by some physical parameters, but particularly by its polypeptide distribution.
* Present address: Biomedical Division, Lawrence Livermore Laboratory, University of California, Livermore. Calif. 94550. U.S.A. ** To whom correspondence and reprint requests should be addressed. Abbreviations used are: HDL, high density lipoproteins isolated between d 1.063 and 1.21 g/ml: HDk, HDL isolated between d 1.063 and 1.125 g/ml: HDL3. HDLisolated between d 1.125 and 1.21 g/ml; p, solvent density: d, serum density: “2 partial specific volume.
27
Introduction In previous studies, it was shown that the chicken can be a useful experimental animal for the study of cholesterol metabolism [l--5]. In these studies, the large increase in serum cholesterol levels which accompanied cholesterol feeding could be accounted for mainly by the increased concentrations of the serum very low density lipoproteins. Under these experimental conditions, chicken high density lipoproteins (HDL) exhibited much smaller, though significant changes. An understanding of the mechanism of cholesterol-induced changes in the physical and chemical properties of the serum lipoproteins would require an accurate knowledge of these lipoproteins under normolipemic conditions. In the case of the rooster, such information is not available. This laboratory has long been interested in defining the structure of serum HDL from a physical and chemical standpoint in humans as well as in a number of animal species [6-S] . Such investigations, as well as those of others [g--15] have clearly indicated the value of comparative structural studies for the determination of the properties of HDL. In this study, we have investigated the physical and chemical properties of serum HDL from roosters fed a low cholesterol, low fat diet and compared these values with those obtained in humans. The parameters for human HDL were either determined concurrently with those of the rooster or were taken from data found in the literature. Materials and Methods Animals. Six male, white Leghorn roosters (about 2 kg body weight each) were used. The animals, which were obtained commercially, were kept on Purina Chow and water fed ad libitum during the 6 weeks preceding blood withdrawal. Blood was obtained by cardiac puncture and was allowed to clot at room temperature for several hours to give serum upon centrifugation. The serum was made 0.05% in EDTA (pH 7.0) and was used immediately for lipoprotein separation. Isolation of HDL by ultracentrifugal flotation. Rooster HDL, isolated between d 1.063 and 1.21 g/ml, were obtained by preparative ultracentrifugal methods identical to those used for the separation of human serum lipoproteins [ 161. This density range contained essentially all the serum HDL as determined by dodecyl sulfate polyacrylamide gel electrophoresis, agarose column chromatography and by isopycnic density gradient- and analytical ultracentrifugation. The bright yellow-orange HDLs of each animal, after extensive dialysis against a solution of 0.15 M NaCl, 0.05% EDTA, pH 7.0, were analyzed individually. Delipidation of HDL. Dialyzed rooster HDL was delipidated, and the resulting protein and lipid fractions were prepared by methods identical to those previously described for human HDL [ 171. CsCl isopycnic density gradient ultracentrifuga tion. For determination of the purity and homogeneity, as well as the mean density of the HDL prepared as above, the samples were subjected to a CsCl isopycnic density ultracentrifugal technique that was previously described [ 181. Agurose gel chromatography. The HDL samples (about 4 mg/ml of HDL protein) were applied to 6% beaded Agarose gel (Bio-Gel A-5m, 200-400
28
mesh; Bio-Rad Laboratories, Richmond, as previously described [ 181.
CA) at 4°C on a 1.5 X 75 cm column,
Polyacrylamide gel electrophoresis. Dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide) was performed essentially according to the procedure of Weber and Osborn [19], as previously described [18]. The method of Davis [ZO] , with the incorporation of 8 M urea, was followed for making the 8 M urea, 7.5% polyacrylamide gels. After electrophoresis, the gels were stained in 1% Amido Black in 7.5% aqueous acetic acid. Circular dichroic studies. Circular dichroic experiments were performed as previously described [18] . The samples had a protein concentration of 0.05-0.5 mg/ml. Analytical ultracentrifugation. Analytical ultracentrifugal studies of rooster HDL were conducted in a Beckman Model E Analytical Ultracentrifuge at 20°C using an AN-H rotor. Sedimentation experiments were performed at 52 000 rev./min in a double-sector cell. Diffusion experiments were conducted at 4000 rev./min using a synthetic boundary centerpiece. The sedimentation and diffusion coefficients at infinite dilution, ~02s w and D,, w respectively, were calculated by conventional procedures [21]. Fiotational studies of rooster HDL (d 1.063-1.21 g/ml) and human HDL (d 1.063-1.21 g/ml) were performed at 44 000 rev./min at a solvent density of 1.21 g/ml using a doublesector cell. Sedimentation equilibrium experiments were conducted at 5600 rev./min in a synthetic boundary cell using interference optics. The partial specific volume (u2 ) was obtained from density measurements at 2O.O”C using a Mettler/Anton Paar Precision Density Meter (Graz, Austria) equipped with a Tronac Temperature Control Unit (Salt Lake City, UT). Isolation of apo-HDL by gel filtration. Rooster apo-HDL (30 mg) was fractionated by gel filtration in columns (100 X 5 cm) containing Sephadex G-200 at 8°C under previously described conditions [6] . The protein solvent and the eluting buffer were 0.01 M Tris, pH 8.2, 0.001 M EDTA, 0.02% NaN3 in 8 M urea. Amino acid analysis. Protein samples were hydrolyzed for 24 h in a 6 M HCl solution containing P-mercaptoethanol (1 : 2000 v/v) at llO”C, as previously described [22]. Tryptophan was determined by the method of Liu and Chang [23], except that 3 M p-toluene-sulfonic acid was replaced by 4 M methanesulfonic acid. absorption spectra were reUltraviolet absorption studies. Ultraviolet corded on a Beckman DU spectrophotometer with a Car-y recording attachment. Protein samples were dissolved in 0.01 M NH4 HC03, pH 8.2. The pH was changed from 8.2 to 11.6 by the addition of 0.1 M NaOH. Immunodiffusion. Immunodiffusion studies with rooster HDL and rabbit antisera against human low density lipoproteins, human HDL2 , and apo A-I were performed in 1% agarose gels in 0.05 M Verona1 buffer, pH 8.6, as previously described [ 181. Chemical analyses. Protein concentrations were measured by the method of Lowry et al. 1241. Phospholipids were determined as lipid P X 25 [25]. Individual phospholipids were separated and identified by thin-layer chromatography according to the method of Skipski et al. [26] and computed as lipid P X a constant (16.8 for lysolecithin, 25 for phosphatidylcholine and -ethanol-
29
amine and 22.6 for sphingomyelin). By this method, phosphatidyl serine and -inositol are included in the lecithin fraction, however, two-dimentional, thinlayer chromatography indicated that these two phospholipids comprise less than 5% of the total phospholipids. Cholesterol, cholesteryl esters and triacylglycerols were separated by thinlayer chromatography (developing solvent, 90 : 10 : 1, chloroform/methanol/ glacial acetic acid). Cholesterol and cholesteryl esters were determined by the method of Zak et al. [27] , while triacylglycerols were measured by the procedure of Amenta [28]. The fatty acid composition of each phospholipid class, cholesteryl esters and triacylglycerols was determined as previously described [ 181. Results Behavior of HDL after CsCl density gradient ultracentrifugation and agarose column chromatography After agarose column chromatography, rooster HDL as well as human HDL3 emerged as a single, symmetrical peak (Fig. 1). Neither HDL indicated any evidence of higher or lower molecular weight components. Rooster HDL, however, had a slightly larger elution volume than did human HDL, . A single band was also observed after CsCl isopycnic density gradient ultracentrifugation (Fig. 2). In this system, rooster HDL banded at d 1.12 g/ml, whereas human HDL3 banded at d 1 .13 g/ml. 1100,
- 1 35
-125 0.5.
2 ,E z -1.15
zs x
0.1.
“0 40
“T 50
60 TUBE
70
80
90
100
110
NUMBER (0.97ml/tubel
Fig. 1. Agarose (Bio-Gel A-5x11) gel column chromatography elution profile of kooster serum HDL ) and human serum HDL3 (- - - - - 9, with 8 mg of each HDL. The void volume, VO, was 42 ml and (the total solvent volume. VT, was 115 ml. Fig. 2. CsCl isopycnic den&y gradient ultracentrifugation profiles of rooster serum HDL ( -) human serum HDL3 (- - - - - -). The density gradient (o---.-o) was established after 66 h. at 11°C 38 000 rev./min in a Spinco SW-40 rotor.
and and
30
TABLE
I
PROTEIN
AND
LIPID
COMPOSITION
component
OF
ROOSTER
Rooster**
HDL,
Human*
HUMAN
HDL.2.
AND
HUMAN
HDLs*
* *
HDL HDL2
Protein
43.88
i
41
55
Phospholipids
28.66
f 2.21
29.5
22.5
16.03
? 1.63
16.2
11.7
Cholesteryl
esters
2.43
HDL3
Cholesterol
5.01
* 0.34
5.4
2.9
Triacylglycerols
6.42
f 1.38
4.5
4.1
* Percent .** Mean ***
by
weight
average
Scanu
and
t standard
of duplicate
Kruski
deviation.
measurements
for
5 roosters.
[341.
Chemical composition The protein-lipid distribution of rooster HDL gave a composition intermediate between those of human HDLz and HDL3 (Table I). The phospholipid distribution of rooster HDL was found to have a slightly greater percentage of lysolecithin and phosphatidylethanolamine, but a lower content of sphingomyelin and phosphatidylcholine, than human HDL (Table II). The fatty composition of rooster HDL resembled that of human HDL (d 1.063-1.21 g/ml, Table III), although in each lipid class, there was a greater percentage of stearic acid (18 : 0) than in human HDL.
Spectral studies The circular dichroic spectra of both rooster HDL and apo-HDL exhibit minima at 210 and 222 nm (Fig. 3). The depths of the minima indicate that rooster HDL and apo-HDL had a greater amount of a-helix (almost 30%) than that found in the corresponding human fractions (Table IV). The ultraviolet absorption spectrum of rooster HDL at pH 8.2 was generally similar to that of human HDL3, with an absorption maximum at about 278 nm (Fig. 4). When the pH was increased to 11.6, a slight shift to the red occurred. Rooster apo-HDL and human apo-HDL3 had nearly identical spectra TABLE
II
PERCENT
PHOSPHOLIPID
DISTRIBUTION
Phospholipid
IN
HDL
(d
1.063-1.21
R00sl.er
Human
HDL
HDL
g/ml)
OF
ROOSTER
Human*
AND
* HDL3
HDL2 Lysolecithin Sphingomyelin
3.6
+ 0.8
2.1
+ 0.8
2.0
MAN*
.._ 5.4
8.0
+_ 1.4
11.2
+ 1.0
14.5
9.2
Phosphatidylcholine
76.8
_+ 1.6
79.5
_+ 0.8
73.8
77.1
Phosphatidylethanolamine
11.5
f 1.2
7.1
* 1.0
7.9***
_~__.~_ * Mean each
average
* * Scanu
** *
?
in duplicate,
Includes
and
Kruski
standard whereas
deviation. pooled
The human
phospholipids HDL-PL
were
[341.
phosphatidylinositol
and
polyglycerophosphatides.
__~_ from
5 roosters
determined
6.9*** -
were
in triplicate.
~._.
analyzed
separately,
_~
14
0
3.17
0.47 0.86
30.75 20.40
0.55 0.10
3.78 0.89
0.26 0.30
1.20 0.63 2.13 0.90
1.56 2.42
50.33 59.96
1.89 1.17
3.31 2.75
13.61 18.63
2.59 2.37
36.49 29.27
1.61 1.46
2.78 3.62
2.82 2.54
--
:1
9.45 15.72
0.23
1.90 1.29
16
4.97 3.69
16 : 0
OF ROOSTER
23.56 28.23
0.99
:0
0.57 0.36
15
3.31 2.23
:0
COMPOSITION
0.55 1.39
.12:
ACID
FATTY
:0
17.53 30.20
17.14 29.25
5.82 2.38
18.66 20.66
23.16 42.10
2.10 3.67
0.51 0.20
1.77 1.15
:0
5.13 11.95
18
AND HUMAN
0.20 0.40
11
HDL
:1
11.14 7.95
9.69 12.16
9.37 12.41
16.49 13.49
20.87 26.54
37.29 39.28
18
:2
10.36 8.75
16.71 18.55
1.42 1.91
4.19 1.61
48.78 44.86
20.92 13.79
18
-
:4
20.79 18.53
11.45 14.31
0.97
4.58 7.50
5.64 3.90
2.13 2.28
20
HDL TRIACYLGLYCEROLS,
* Each percentage is the mean obtained from 5 different roosters or from a pooled human HDL sample. * * Ass&went not certain.
Rooster Cholesteryl esters Human Rooster Lysolecithin Human Rooster Sphingomyelin Human Rooster Phosphatidylcholine Human Rooster Phosphatidylethanokunine Human Rooster
Human
TrlacyIgIyceroIs
PERCENT AVERAGE PHOLIPIDS*
TABLE III
2.14 2.77
2.44 1.63
1.67
(20
: 3j**
CHOLESTERYL
: a**
6.54 3.00
(22
ESTERS
PHOS-
7.49 5.83
9.86 4.13
10.69 0.97
5.67 2.00
8.44 0.71
1.36 1.90
Unidentified
AND
\ 8
ii
:,
0.9
_‘L__l
4
CHICKEN APO HDI and HUMAN APO HDL,
i4
‘$4
~
200
210
220
230
240
250
-
240
Xlnm)
260
280
300
320
X lnm)
Fig. 3. Circular dichroic spectra of: A. rooster HDL; B and C, human HDL3 and rooster ape-HDL (these spectra were identical); D, Human ape A-I: E, human ape A-II. The crossover points of the native HDLs are at about 202 nm; they occur at 200 nm for the apoproteins. Fig. 4. Ultraviolet spectra of rooster HDL and ape-HDL, as well as human HDL3 and ape HDL3. at pH 8.2 and 11.6. Top: rooster apo HDL, pH 8.2 ( -) and 11.6 (- - - - - -); human apo HDL3. PH 8.2 (o-----o) and 11.6 (o M>). Bottom: rooster HDL. pH 8.2 ( -) and 11.6 (------): human HDL3, pH 8.2 (0 -) and 11.6 (o-4). The protein concentration of all samples was 0.5 mg/mI.
TABLE
IV
PHYSICAL Physical
PROPERTIES
OF ROOSTER
constants
HDL AND HUMAN
HDL2 AND HDL3
Rooster* HDL
Human* * HDL2
sio,w (S)
D~o,~ (cm2. Mr. 10’
s-l).
10
-1
p(g/mI) ~2
(ml/g)
f//f0 (anhydrous) (assuming [B] 222 . lO+ [01,,,.104
0.2 g HZO/g of lipoprotein) deg . cm2 . dmol-’ deg.cm2.dmol-’
3.99 4.36 1.73 1.152 0.868 1.24 1.16 -3.50 -3.56
+ + * ? +
0.13 0.30 0.13 0.12 0.009
5.45 3.68 3.65 1.105 0.905 1.10 1.02 -2.60 -2.58
HDL3 4.65 3.93 1.75 1.153 0.867 1.25 1.15 -2.52 -2.50
* Mean average f stand deviation of six determinations, one for each rooster, except for molecular weight were three determinations were used. ** From Scanu et al. [S]. except for f/f0 and circular dichroic data which were taken from ref. 35.
33
at pH 8.2 and a comparatively HDLs at pH 11.6.
larger red shift than that observed
with native
In flotational studies at p 1.21 g/ml, rooster HDL gave a single, nearly symmetrical peak. However, human HDL (d 1.063-1.21 g/ml), studied under the same conditions, gave a pattern consisting of a main pe_ak (HDL, ) and a shoulder representing a less dense, faster-floating component (HDLz ) not observed with rooster HDL. In sedimentation studies at p 1.006 g/ml, rooster HDL exhibited a single symmetrical peak. The sedimentation coefficient, siO ,W; diffusion coefficient, D i 0 ,W; partial specific volume, u2 ; and the frictional ratios (f/fO, calculated from the above parameters, refs 29, 30) were generally similar to those of human HDL3 (Table IV). The molecular weight of rooster HDL, calculated from the values for and u2 was 1.71 * 10 ’ which was nearly identical to the value of &,W G0.W 1.73 * 10’ determined directly from the results of the sedimentation equilibrium experiments.
After electrophoresis in 8 M urea gels, the rooster apo-HDL pattern contained a major band (band 3) and a minor band (band 4), which correspond in migration distance to those of human apo A-I and apo A-II, respectively (Fig. 5). Densitometric scans of these gels indicate that the major (band 3) and the minor (band 4) bands found in rooster apo-HDL represented about 91% and 9% of the total protein in comparison to 86% and 14% for human apo A-I and apo A-II respectively. Additional bands were seen when the amount of rooster apo-HDL loaded on the gels was increased: two bands (bands 1 and 2)
Fig. 5. Polwmylamide gel electrophoresis patterns of rooster apo HDL and human apo HDL3: A and B, 8 M urea: C and D. 0.1% dodecyl sulfate. The polyacrylamide concentration in the urea and dodecyl sulfate gels was 7.5 and 10%. respectively. A and C: rooster ape-HDL; B and D: human apo-WLg.
34
with a slower migration, than bands 3 and 4, and four bands (bands 5-8) which had the fastest migration rates. After electrophoresis in the 0.1% dodecyl sulfate-containing gels, three bands were observed for rooster apo-HDL (Fig. 5). The band with the largest molecular weight (about 70 000, band 1) was generally present in variable amounts, depending on the method of preparation of the apo-HDL. The two other bands present in these gels had molecular weights of about 26 000 (band 2) and 15 000 (band 3) which were unaltered by the presence of the reducing agent, dithiothreitol. A representative gel scan of the bands indicated that about 8, 91 and 1% of the observed protein bands were present as bands 1, 2, and 3 respectively. A gel scan of the human apo-HDL3 pattern indicated that about 81 and 19% of apo A-I and apo A-II respectively were present, in general agreement with the distribution reported in the literature [22] . Gel filtration
studies
Two major peaks (peaks I and II) were obtained after elution of rooster apo-HDL by Sephadex G-200 chromatography in 8 M urea (Fig. 6). Peak I was heterogeneous as assessed by dodecyl sulfate-polyacrylamide gel electrophoresis. Peak II (band 2 in Fig. 5 for the dodecyl sulfate gels) eluted at the same volume as did human apo A-I, indicating a similarity in molecular weights. Peak II consisted essentially of one protein, the major rooster protein in apo-HDL, although a small amount of higher molecular weight components was observed
Fig. 6. Sephadex G-200 chromatography in 8 M urea of rooster ape-HDL. Fractions designated by the bar lines indicate the range used for the dodecyl sulfate-polyacrylamide electrophoresis gel.?.. Migration is from top(cathode) to bottom(anode) with the origin designated as 0.
35 TABLE
V
COMPARISON OF THE AMINO ACID COMPOSITION OF THE FRACTIONS OBTAINED FROM SEPHADEX G-200 CHROMATOGRAPHY IN 8 M UREA -Amino acid
-_--
Rooster
OF ROOSTER APO-HDL WITH HUMAN APO A-I*
Human apo A-I * *
Peak I
Peak II
20 1 17 20 11 12 47 11 6 21 N.D.*** 14 5 6 32 6 5 N.D.***
21 1 18 20 11 10 47 12 3 23 N.D.*** 13 5 6 33 7 5 1
___--
LYS His Arg ASX Thr Ser GIX Pro GIY Ala Half-c ys Val Met Be Leu Tyr Phe Trp Total residues
(234)***
(236)***
21 5 16 21 10 14 47 10 10 19 0 13 3 0 39 7 6 4 245 -
* Number of residues per mol, assuming for the rooster fractions a molecular weight of about 26000. i.e., that obtained for the major ape-HDL polypeptide after dodecyl sulfate-gel electrophoresis. Each analysis was the average of at least two determinations. ** Ref. 31. *** Not determined, therefore total residues are tentative.
after dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid analysis of peak II was very similar, though not identical to that of human apo A-I (Table V). For comparison, the amino acid composition of peak I is also included, assuming peak I is mainly an aggregate of peak II. When this comparison is made, the composition of peak I is almost identical to that of peak II. After elution of peak II from the Sephadex column, a very small amount of protein designated as peak III was obtained. This peak contained band 3 in the dodecyl sulfate gels, as well as peak II and small amounts of larger molecular weight components (Fig. 6). Immunology Rooster HDL did not form precipitin lines with human serum low density lipoproteins, HDL2 or apo A-I.
rabbit
antiserum
to
Discussion The results of the present study indicate that many chemical parameter8 of rooster HDL are similar to those of in keeping with data in the literature on a number of 8,11,1 3-151. Thus, a typical serum HDL particle from
of the physical and human HDL. This is animal species [6either human or the
36
animal species studied has the following general features: (a) it is composed of almost equal amounts of protein and lipid; (b) about half of the lipid is phospholipid, with most of the remaining lipid present as cholesteryl ester; (c) at least 50% of the fatty acids of the lipids are unsaturated; (d) the particle weight is between 1.7 * lo5 and 3.6 . lo5 ; (e) the major HDL apoprotein has a molecular weight of about 27 000; (f) the apo-HDL has a greater than 50% a-helix conformation; and (g) the major apo-HDL contains no half-cystine, whereas about 20% of the amino acid residues consist of glutamine or glutamic acid. Evidently, these many structural similarities suggest one or more common functions for the serum HDL of different species. What these functions are is not clear at the present time. Rooster serum HDL has a protein-lipid composition which is very similar to that of human HDL2 (Table I); yet the physical parameters more closely resemble those of human HDL3 (Table IV). Of the physical parameters of rooster HDL, the molecular weight, partial specific volume, and the unhydrated or hydrated frictional ratios are practically identical with those of human HDL3, whereas the sedimentation and the diffusion coefficients are consistently different. The lack of reactivity of rooster HDL to rabbit anti-human HDLz or apo A-I, as well as the slight differences noted in the respective ultraviolet absorption spectra, suggest differences in the conformation and/or the amino acid composition (Table V) of the protein moiety of these lipoproteins. Further evidence indicating structural differences between rooster and human HDL comes from circular dichroic studies which reveal that rooster HDL has an almost 30% greater amount of a-helix estimated by the molar ellipticity at 222 nm (ref. 32; Table IV). Also as expected, the content of a-helix in rooster apo-HDL was greater by about 20% than either human apo A-I or apo A-II (Fig, 3). The a-helix content of rooster HDL appears to be the largest yet reported, whether it is compared to man or to the other animal species studied, including the monkey [ 81, rat [7] , pig [ 131, and the pink salmon [ 151. The larger helical content of rooster HDL suggests a structural difference from that of human HDL which may account for the different sedimentation and diffusion coefficients found for these HDLs. The phospholipid class distribution of rooster HDL is slightly different than that of human which may account for additional structural dissimilarities between these lipoproteins (Table II). The major protein found in rooster HDL, comprising about 90% of the total protein as determined by polyacrylamide gel electrophoresis, had a molecular weight of about 26 000 compared to 28 331 [31] for the sequenced major human apo-HDL, apo A-I (Figs 5 and 6). A protein of lower molecular weight (about 15 000 representing band 3 in dodecyl sulfate gels and band 4 in the urea gels, Fig. 5, and peak III, Fig. 6) was also present; but, unlike human apo A-II, it was unaffected by reducing agents. This protein, the relationship of which to human apo A-II remains unknown, comprised about 1 and 9% of the total protein of rooster HDL in dodecyl sulfate or urea gels, respectively. The reason for this consistent difference between the two gel systems may be due to the limitations inherent in the quantitation of stained gels [ 331. Small amounts of polypeptides having migration rates similar to those of the human
37
apo C polypeptides were also present in rooster apo-HDL, as determined by 8 M urea gel electrophoresis (bands 5-8, Fig. 5). Higher molecular weight species were consistently found in all preparations of rooster apo-HDL, as indicated by both dodecyl sulfate (band 1) and urea (bands 1,2) gel electrophoresis (Fig. 5), as well as by gel filtration in 8 M urea (peak I, Fig. 6). Hillyard et al. [12] experienced similar difficulties with aggregates of chicken apo-HDL in several different detergent and denaturing solvents. These authors found a major component of chicken apo-HDL having a molecular weight of 30 100 in a phenol/urea/acetic acid/polyacrylamide gel system which resembles the major component of rooster apo-HDL of molecular weight 26 000 that we find in 0.1% dodecyl sulfate-polyacrylamide gels. Undoubtedly, more detailed studies of rooster apo-HDL will be needed for further characterization of the component polypeptides. A&no wledgments The authors are grateful to Dr Lidia Vitello for valuable advice in the preparation of this manuscript and assistance during the analytical ultracentrifugal studies, and to Mr Jim Foreman for help in performing the amino acid analyses. They are grateful to Mr Randolph Hughes and Mr Lance Lusk for assistance with the animals. A.M. Scanu is a recipient of U.S. Public Health Service Career Development Award No. HL 24867. A.W. Kruski is a recipient of U.S. Public Health Service Postdoctoral Fellowship No. HL 52970. This research was supported by Grants No. HL 08727 and HL 06481 from the U.S. Public Health Service, and by the Illinois and Chicago Heart Association (A 72-6). The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration. References 1 Jones, R.J. and Dobrilovic. L. (1969) Proc. Sot. Exp. Biol. 130. 163-167 2 Kruski. A.W. and Narayan. K.A. (1972) Lipids 7.742-749 3 Kruski. A.W., Kummerow. F.A. and Narayan. K.A. (1972) Protides Biol. Fluids Proc. Colloq. 19, 121-125 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Reveille, G.A.. Tillotson. J.A. and Sauberlich. H.E. (1963) J. Nutr. 81, 357-362 Leveille, G.A. and Sauberlich, H.E. (1963) Proc. Sot. Exp. Biol. 112. 300-303 Edelstein, C.. Lim. C.T. and Scanu. A.M. (1973) J. Biol. Chem. 248,.7653-7660 Koga, S.. Horwtz, D. and Scam& A.M. (1969). J. Lipid Res. 10. 577-588 Swum, A.M., Edelstein, C.. Vitello, L.. Jones, R. and Wissler. R. (1973) J. Biol. Chem. 248, 76487452 Blaton, V.. Vercaemst. R.. Vandecasteele, N.. Caster, H. and Peetea, H. (1974) Biochemistry 13, 1127-1135 Cox. A.C. and Tanford, C. (1968) J. Biol. Chem. 243, 3083--3087 Fidge, N. (1973) Biochim. Biophys. Acta 295. 258-273 HiByard. L.A.. White, H.M. and Pangburn. S.A. (1972) Biochemistry 11, 511-518 Jackson, R.L.. Baker. H.N., Taunton, O.D., Smith, L.C., Garner, C.W. and Gotto, A.M. (1973) J. Biol. Chem. 248, 2639-2644 Jonas, A. (1972) J. Biol. Chem. 247.7767-7772 Nelson, G.J. and’shorn. V. (1974) J. Biol. Chem. 249, 536-542 Scanu, A.M. and Ritter. M.C. (1973) Adv. Cfin. Chem. 16.111-151 Scanu. A. (1965) J. Lipid Res. 7, 295-306 Kruski. A.W. and Scanu, A.M. (1974) Chem. Phys. Lipids 13, 2748
19 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 44064412 20 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404427 21 Chervenka, C.H. (1970) A Manual of Methods for the Analytical Ultracentrifuge, Beckman Instruments, Inc., Palo Alto, CA. 22 Scanu, A., Toth, J., Edelstein, C., Koga, S. and Stiller, E. (1969) Biochemistry 8. 3309-3316 23 Liu, T.-Y. and Chang. Y.H. (1971) J. Biol. Chem. 246, 2842-2848 24 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-2’7t 25 Bartlett, G.F. (1959) J. Biol. Chem. 234, 466-468 26 Skipski, V.P.. Peterson, RF. and Barclay, M. (1962) J. Lipid Res. 3, 467-470 27 Zak, B., Dickenman, R.C., White. E.G., Burnett, H. and Cberney, P.J. (1954) Am. J. Clin. Patbol. 24, 1307-1315 28 Amen&, J.S. (1964) J. Lipid Res. 5, 270-272 29 Hazelwood. R.N. (1957) J. Am. Chem. Sot. 80, 2152-2156 30 Tanford. C. (1967) Physical Chemistry of Macromolecules John Wiley and Sons, New York 31 Baker, N.H.. Delahunty, T., Gotto, A.M. and Jackson, R.L. (1974) Proc. Nat]. Acad. Sci. U.S. 71, 3631-3634 32 Scanu, A.M. (1969) Biochim. Biophys. Acta 181, 268-274 33 Kruski, A.W. and Narayan, K.A. (1974) Anal. Biochem. 60.431-440 34 Scanu, A.M. and Kruski, A.W. (1975) International Encyclopedia &arm. Therapeutics, Chap. 2, Sect. 24, Pergamon Press, London 35 Scanu, A.M., ViteRo, L. and DeGanello, S. (1974) CRC Cnt. Rev. Biochem. March, 175-196
