134
Biochimica et Biophysica Acta, 5 7 6 ( 1 9 7 9 ) 1 3 4 - - 1 4 0 © Elsevier/North-Holland Biomedical Press
BBA 38067
ISOLATION AND PARTIAL CHARACTERIZATION OF A GUINEA PIG SERUM APOLIPOPROTEIN COMIGRATING WITH APO-E ON SODIUM DODECYL SULFATE-POLYACRYLAMIDE ELECTROPHEROGRAMS *
MARTHA
M E N G , L U K E G U O ** a n d R . O S T W A L D
Department of Nutritional Sciences, University of California, Berkeley, CA 94 720 (U.S.A.) (Received April 26th, 1978)
Key words: Arginine-rich; Cholesterol; Apolipoprotein; (Guinea pig serum)
Summary Study of guinea pig plasma lipoproteins has shown that they contain a polypeptide that comigrates with the arginine-rich polypeptide (apo-E) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This comigrating polypeptide differs from apo-E in its amino acid composition, immunological crossreactivities, electrophoretic mobility in urea polyacrylamide gel, and elution volume from Sephadex gel columns. It is present in very low density lipoproteins and low density lipoproteins from both control and cholesterol-fed guinea pigs. Introduction Dietary cholesterol has been shown to induce substantial changes in the plasma lipoproteins of a number of species including the guinea pig [1--5]. Among the most striking changes in the guinea pig is the very large increase of an apolipoprotein rich in arginine with molecular weight of approx. 34 000 (apo-E) [6]. This apoprotein is present in minute quantity in lipoproteins d ~ 1.006 g/ml and d 1.01--1.05 g/ml of control guinea pigs, but constitutes up to 50% in d 1.07-..1.20 g/ml of guinea pigs fed a 1% cholesterol-containing diet. We are now reporting that there is an apolipoprotein present in the plasma lipoproteins of both control and cholesterol-fed guinea pigs that has an electrophoretic mobility on sodium dodecyl sulfate (SDS)-polyacrylamide gels very * We are ad opting the n o m e n c l a t t t r e apo-E now c o m m o n l y used in the literatwre for an arginine-rich apol i p o p r o t e i n described in o t h e r s p e c i e s t h a t is very similar to t ha t found in guinea pigs. In previous c o m m u n i c a t i o n s we have referred to it as arginine-rich p ol ype pt i de , ARP [1,6]. ** Present address: Cardiovascular Research Institute, University of California, San Franciscol Calif. 94143, U.S.A. Abbreviations: VLDL, very low density lipoprotein, d ~ 1.006 g/ml: LDL, low density liPoprotein, d 1.01--1.05 g/ml: HDL 1 , high density lipoprotein, d 1.07--1.09 g/ml~ HDL2, d 1.09--1.20 g/ml.
135 similar to apo-E, but a different elution volume on Sephadex gel, a different electrophoretic mobility on polyacrylamide gel in the presence of urea, a different amino acid composition, and different immunological cross-reactivities. Materials and Methods Guinea pigs (from Simonson Lab., Gilroy, Calif.) were fed either Purina guinea pig chow supplemented with 5% cottonseed oil. or the same fatcontaining diet supplemented with 1% cholesterol. The animals were autopsied when the erythrocyte count of the cholesterol-fed guinea pigs dropped below 2.5 • 10 ~ cells/mra ~. Blood was collected in 0.1% sodium-EDTA from animals fasted overnight (15--17 h}. Lipoproteins were isolated by standard flotation techniques [ 7 ]. Four classes of lipoprotein, VLDL (d < 1.006 g/ml), LDL (d 1.01--1.05 g/ml), HDL, (d 1.07--1.09 g/ml), and HDL2 (d 1.09--1.20 g/ml) were isolated as reported previously [6]. After dialysis and lyophilization, lipoprotein fractions were delipidated at a concentration of 1--3 mg protein/ml in a solution containing 0.15 M NaC1, 10 -3 M EDTA, pH 7.4, by extractions with diethylether/ethanol (3 : 2, v/v and 3 : 1, v/v), similar to the procedure of Shore and Shore [8]. Recoveries of protein after delipidation were greater than 95% and the apoprotein contained less than 1% residual phospholipids. Each sample of apolipoprotein, dissolved in 0.05 M Tris-HCl (pH 8.8) containing 6 M urea, 10 -3 M EDTA and 0.01% NAN3, was applied to a column (1.25 × 100 cm) of Sephadex G-150. The column was equilibrated a n d eluted with the same solution, at a flow rate of 5 ml/h. The effluent was monitored for absorbance at 280 nm. After determination of protein content in all fractions [9] the material from each peak was pooled, dialyzed against distilled water, and freeze-dried. Samples of each peak materials were dissolved in appropriate buffers and subjected to electrophoresis in SDS- and urea-polyacrylamide gels. SDS (0.1%) gel electrophoresis on 10% polyacrylamide [10] was carried out at 8 mA/tube. Gels were stained with 0.05% Coomassie Brilliant Blue in 25% isopropanol/10% acetic acid overnight and destained in 7.5% acetic acid. Molecular weight markers used to estimate the molecular weights of the apoproteins were bovine serum albumin, fraction V, Mr = 68 000 (Armour Pharmaceutical); ovalbumin, Mr = 43 000 (Sigma Chem. Co.); bovine heart lactate dehydrogenase, Mr = 36 000 (Sigma Chem. Co.); bovine pancreas a~hymotrypsinogen, Mr = 25 700 (Sigma Chem. Co.); and egg white lysozyme. Mr = 14 300 (Mann). The Tris buffer (pH 8.6)/8 M urea system on 7.5% acrylamide gel [11] was used as modified by Shore and Shore [12]. Gels were fixed with 12.5% (w/v) trichloroacetic acid for 1 h, stained by immersion in. a solution of 0.1% Coomassie Brilliant Blue in 12.5% trichloroacetic acid overnight, and destained in 10% trichloroacetic acid. Amino acid analyses were carried out essentially as described by Moore et al. [13] using a Beckman Model 121 amino acid analyzer. Double immunodiffussion was carried out as described by Ouchterlony [14]. Preparation of antisera has been reported previously [ 1].
136 Results and Discussion Gel filtration of the soluble components of control apo-LDL yielded four major fractions (Fig. 1). The first fraction did not contain protein and gave no bands on SDS- or urea-polyacrylamide gels and was not studied further. SDSpolyacrylamide gel electrophoresis showed that fractions I and II each contained as major component a polypeptide with an apparent molecular weight of 34 000 that we have previously described (arginine-rich polypeptide, ARP or apo-E) [1,6]. In addition, these two fractions contained minute amounts of A-I (Mr = 25 000) and some high molecular weight components. Fraction III contained apo-C, Mr = 8000--11 000. The polypeptide eluted as fraction II yielded a rather broad band on SDS-polyacrylamide gels with a mobility similar to apo-E, i.e. with a molecular weight range of 31 000--36 000 (mean 34 000). Urea-polyacrylamide gel analysis showed that apo-E from fraction I contained three very closely spaced multiple bands with a mean relative mobility (RE) of 0.22, whereas the protein from fraction II yielded a broad diffuse band with a mean RF of 0.44. Cholesterol LDL and control VLDL were examined by the same methods.
009
"gT
SDS-PAGE
I
~
]]]:
M.W.
008
~34,000 ~ 25, 0 0 0
007
g
I1,000
0 006 (D O~
K
I.d 0 0 5 ~j, Z
Biochimica et Biophysica Acta, 5 7 6 ( 1 9 7 9 ) 1 3 4 - - 1 4 0 © Elsevier/North-Holland Biomedical Press
BBA 38067
ISOLATION AND PARTIAL CHARACTERIZATION OF A GUINEA PIG SERUM APOLIPOPROTEIN COMIGRATING WITH APO-E ON SODIUM DODECYL SULFATE-POLYACRYLAMIDE ELECTROPHEROGRAMS *
MARTHA
M E N G , L U K E G U O ** a n d R . O S T W A L D
Department of Nutritional Sciences, University of California, Berkeley, CA 94 720 (U.S.A.) (Received April 26th, 1978)
Key words: Arginine-rich; Cholesterol; Apolipoprotein; (Guinea pig serum)
Summary Study of guinea pig plasma lipoproteins has shown that they contain a polypeptide that comigrates with the arginine-rich polypeptide (apo-E) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This comigrating polypeptide differs from apo-E in its amino acid composition, immunological crossreactivities, electrophoretic mobility in urea polyacrylamide gel, and elution volume from Sephadex gel columns. It is present in very low density lipoproteins and low density lipoproteins from both control and cholesterol-fed guinea pigs. Introduction Dietary cholesterol has been shown to induce substantial changes in the plasma lipoproteins of a number of species including the guinea pig [1--5]. Among the most striking changes in the guinea pig is the very large increase of an apolipoprotein rich in arginine with molecular weight of approx. 34 000 (apo-E) [6]. This apoprotein is present in minute quantity in lipoproteins d ~ 1.006 g/ml and d 1.01--1.05 g/ml of control guinea pigs, but constitutes up to 50% in d 1.07-..1.20 g/ml of guinea pigs fed a 1% cholesterol-containing diet. We are now reporting that there is an apolipoprotein present in the plasma lipoproteins of both control and cholesterol-fed guinea pigs that has an electrophoretic mobility on sodium dodecyl sulfate (SDS)-polyacrylamide gels very * We are ad opting the n o m e n c l a t t t r e apo-E now c o m m o n l y used in the literatwre for an arginine-rich apol i p o p r o t e i n described in o t h e r s p e c i e s t h a t is very similar to t ha t found in guinea pigs. In previous c o m m u n i c a t i o n s we have referred to it as arginine-rich p ol ype pt i de , ARP [1,6]. ** Present address: Cardiovascular Research Institute, University of California, San Franciscol Calif. 94143, U.S.A. Abbreviations: VLDL, very low density lipoprotein, d ~ 1.006 g/ml: LDL, low density liPoprotein, d 1.01--1.05 g/ml: HDL 1 , high density lipoprotein, d 1.07--1.09 g/ml~ HDL2, d 1.09--1.20 g/ml.
135 similar to apo-E, but a different elution volume on Sephadex gel, a different electrophoretic mobility on polyacrylamide gel in the presence of urea, a different amino acid composition, and different immunological cross-reactivities. Materials and Methods Guinea pigs (from Simonson Lab., Gilroy, Calif.) were fed either Purina guinea pig chow supplemented with 5% cottonseed oil. or the same fatcontaining diet supplemented with 1% cholesterol. The animals were autopsied when the erythrocyte count of the cholesterol-fed guinea pigs dropped below 2.5 • 10 ~ cells/mra ~. Blood was collected in 0.1% sodium-EDTA from animals fasted overnight (15--17 h}. Lipoproteins were isolated by standard flotation techniques [ 7 ]. Four classes of lipoprotein, VLDL (d < 1.006 g/ml), LDL (d 1.01--1.05 g/ml), HDL, (d 1.07--1.09 g/ml), and HDL2 (d 1.09--1.20 g/ml) were isolated as reported previously [6]. After dialysis and lyophilization, lipoprotein fractions were delipidated at a concentration of 1--3 mg protein/ml in a solution containing 0.15 M NaC1, 10 -3 M EDTA, pH 7.4, by extractions with diethylether/ethanol (3 : 2, v/v and 3 : 1, v/v), similar to the procedure of Shore and Shore [8]. Recoveries of protein after delipidation were greater than 95% and the apoprotein contained less than 1% residual phospholipids. Each sample of apolipoprotein, dissolved in 0.05 M Tris-HCl (pH 8.8) containing 6 M urea, 10 -3 M EDTA and 0.01% NAN3, was applied to a column (1.25 × 100 cm) of Sephadex G-150. The column was equilibrated a n d eluted with the same solution, at a flow rate of 5 ml/h. The effluent was monitored for absorbance at 280 nm. After determination of protein content in all fractions [9] the material from each peak was pooled, dialyzed against distilled water, and freeze-dried. Samples of each peak materials were dissolved in appropriate buffers and subjected to electrophoresis in SDS- and urea-polyacrylamide gels. SDS (0.1%) gel electrophoresis on 10% polyacrylamide [10] was carried out at 8 mA/tube. Gels were stained with 0.05% Coomassie Brilliant Blue in 25% isopropanol/10% acetic acid overnight and destained in 7.5% acetic acid. Molecular weight markers used to estimate the molecular weights of the apoproteins were bovine serum albumin, fraction V, Mr = 68 000 (Armour Pharmaceutical); ovalbumin, Mr = 43 000 (Sigma Chem. Co.); bovine heart lactate dehydrogenase, Mr = 36 000 (Sigma Chem. Co.); bovine pancreas a~hymotrypsinogen, Mr = 25 700 (Sigma Chem. Co.); and egg white lysozyme. Mr = 14 300 (Mann). The Tris buffer (pH 8.6)/8 M urea system on 7.5% acrylamide gel [11] was used as modified by Shore and Shore [12]. Gels were fixed with 12.5% (w/v) trichloroacetic acid for 1 h, stained by immersion in. a solution of 0.1% Coomassie Brilliant Blue in 12.5% trichloroacetic acid overnight, and destained in 10% trichloroacetic acid. Amino acid analyses were carried out essentially as described by Moore et al. [13] using a Beckman Model 121 amino acid analyzer. Double immunodiffussion was carried out as described by Ouchterlony [14]. Preparation of antisera has been reported previously [ 1].
136 Results and Discussion Gel filtration of the soluble components of control apo-LDL yielded four major fractions (Fig. 1). The first fraction did not contain protein and gave no bands on SDS- or urea-polyacrylamide gels and was not studied further. SDSpolyacrylamide gel electrophoresis showed that fractions I and II each contained as major component a polypeptide with an apparent molecular weight of 34 000 that we have previously described (arginine-rich polypeptide, ARP or apo-E) [1,6]. In addition, these two fractions contained minute amounts of A-I (Mr = 25 000) and some high molecular weight components. Fraction III contained apo-C, Mr = 8000--11 000. The polypeptide eluted as fraction II yielded a rather broad band on SDS-polyacrylamide gels with a mobility similar to apo-E, i.e. with a molecular weight range of 31 000--36 000 (mean 34 000). Urea-polyacrylamide gel analysis showed that apo-E from fraction I contained three very closely spaced multiple bands with a mean relative mobility (RE) of 0.22, whereas the protein from fraction II yielded a broad diffuse band with a mean RF of 0.44. Cholesterol LDL and control VLDL were examined by the same methods.
009
"gT
SDS-PAGE
I
~
]]]:
M.W.
008
~34,000 ~ 25, 0 0 0
007
g
I1,000
0 006 (D O~
K
I.d 0 0 5 ~j, Z
