Acta, 1043 (1990) 119-121
Rat apolipoprotein B differs in solubility properties from human apolipoprotein B Vaijinath
I,*, David L. Stiers * and Diana M. Lee lq3
’ Lipoprotein and Atherosclerosis Research Program, ’ Electron Microscopy Laboratory, Oklahoma Medical Research Foundation and ’ Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (U.S.A.) (Received
Apolikprotein B (ApoB) in general is known as an insoluble protein in aqueous buffers without the initial aid of denaturing agents. However, following the total delipidization of rat plasma low-density lipoproteins, a considerable portion (28.2 f 3.0%) of rat ApoB was found directly soluble in an aqueous buffer, N-ethylmorpholine acetate (pH 7.3) as demonstrated by SDS-polyacrylamide gel electrophoresis, immunoblotting and electron microscopic analysis. On the other hand, this was not observed for human ApoB. This solubility difference may suggest some structural differences that exist between rat ApoB and human ApoB.
In the absence of the lipid moiety apolipoprotein B (ApoB), the major protein moiety of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL), has a strong tendency to undergo aggregation and is insoluble in aqueous buffers without the aid of denaturing agents. However, most of the studies on solubility and characterization of ApoB were done on human ApoB [l-4]. It has been generally assumed that ApoB from other species would be just as insoluble as human ApoB. The present study was undertaken to investigate whether ApoB of rat, an extensively used species for lipoprotein metabolism, would be similar to human ApoB in solubility properties. Blood samples were collected from ten fasted (16-18 h) male Sprague-Dawley rats (under ether anesthesia) by exsanguination of the abdominal aorta in tube containing 0.1 ml of 15% EDTA. Human plasma was collected by plasmapheresis from normolipidemic subjects after a 14 h fast. Immediately a preservative cocktail [l] and butylated hydroxytoluene (1% in 75% ethanol) were added at l/100 and l/200 dilution , respectively, to plasma.
* Present address: Nephrology Section (111 N), UCI-Long Beach Medical Program, Veterans Admimistration Medical Center, Long Beach, CA, U.S.A. Correspondence: D.M. Lee, Lipoprotein/Atherosclerosis Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, U.S.A. 0005-2760/90/$3.50
0 1990 Elsevier Science Publishers
Narrow density range of LDL (apparent density 1.032-1.043 g/ml) was isolated immediately from the fresh plasma by a single spin density gradient ultracentrifugation  and purified by reflotation [l]. The LDL samples were immediately delipidized and the ApoB pellet was washed with 5 ml of 0.1 M N-ethylmorpholine acetate (NEMA) buffer (pH 7.3) containing preservatives [l]. All steps were carried out under N,. NEMA solution was collected for further analysis of ApoB. The insoluble ApoB was then totally dissolved in 6 M guanidine . HCl buffer and dialyzed against 6 M urea buffer, both containing preservatives [l]. Protein concentrations in LDL, in NEMA solutions and in 6 M urea solutions were determined by the Lowry method  using human serum albumin as standard. The presence of ApoB in NEMA solution was analyzed by 3.3% SDS-polyacrylamide gel electrophoresis (PAGE) according to the method of Fairbanks et al.  as modified earlier . To calculate the percent of soluble ApoB in NEMA solution, samples were analyzed by separate SDS-PAGE gels with 3.3% gel concentration on top 2 cm and 10% gel concentration on the lower 6 cm. Thus, lower molecular weight apolipoproteins, mainly apolipoprotein E (and trace amounts of apolipoprotein C), exhibited a sharp band in the lower 10% gel while the ApoB band appeared in the upper 3.3% gel (figure not shown). Densitometric scanning was performed on these gels by using Gilford spectrophotometer. ApoB concentration soluble in NEMA was obtained by subtracting the protein portion Division)
Fig. 1. Analyses of ApoB on 3.3% SDS-PAGE in NEMA solution obtained from delipidized rat or human LDL. (1) Rat ApoB (after removal of soluble portion) in 6 M urea. 0.02 M Tris, 0.02% EDTA, 0.13% r-amino caproic acid (pH 7.3) (6 M urea buffer). (2) NEMA solution of delipidized rat LDL. (3) Human ApoB in 6 M urea buffer. (4) NEMA solution of delipidized human LDL. (5) Calibration protein standards. Each sample, containing about 15 pg of protein (except sample 4 with little measurable protein), was applied onto each gel.
of apolipoprotein E from the total protein in NEMA solution. Thus, by delipidizing a known volume of LDL, the amounts of soluble ApoB and insoluble ApoB could be calculated. Immunoblotting of ApoB in NEMA solution was carried out according to Towbin et al. [lo] and immunoblotted as described previously [ll] using anti-rat ApoB antisera raised in rabbits . Morphological study was carried out as described earlier  on proteins present in NEMA solution after dialysis against 1% ammonium acetate buffer (pH 7.3) and dilution made at 1 : 500. Analysis of ApoB on 3.3% SDS-PAGE in both NEMA solution and the remaining insoluble ApoB residue totally solubilized in 6 M urea buffer obtained from delipidized rat LDL showed a distinct band corresponding to ApoB-100 (Fig. 1). On the other hand, under the same conditions, NEMA solution obtained from delipidized human plasma LDL did not show ApoB band (Fig. 1). These results indicate that, unlike human plasma ApoB, rat ApoB is partially soluble in aqueous buffer without the initial use of detergents or denaturing agents. The presence of rat ApoB in NEMA solution was consistently observed in three batches of different preparations. ApoB accounted for 47.2 k 5.1% (n = 3) of total soluble protein in NEMA solution. The mass of insoluble ApoB was estimated by protein determination after complete solubilization in 6 M urea. These protein analyses indicated that 28.2 + 3.0% (n = 3) of the total rat ApoB was directly soluble in NEMA. To demonstrate that the protein band in NEMA solution which comigrated with rat ApoB in urea buffer
on SDS-PAGE (Fig. 1) was indeed ApoB, immunoblotting and electron microscopic studies were carried out. As shown in Fig. 2, immunoblot of NEMA solution showed the presence of ApoB doublet band and ApoB48 as observed with ApoB in 6 M urea buffer, confirming the presence of ApoB in NEMA solution. Further evidence was provided by the globular nature of ApoB particles observed in the electron micrographs of negatively stained particles in NEMA solution (Fig. 3). The average diameter of the particles was 10.39 + 1.69 nm (n = 250), which is closely comparable to human ApoB particle size . From these three different lines of evidence, viz.. SDS-PAGE analysis, immunoblotting and electron microscopy, it was demonstrated for the first time that rat ApoB, unlike human ApoB or ApoB of any other species reported, is soluble in considerable proportion in aqueous buffer without the initial use of any detergents or denaturing agents. Although human ApoB was made to solubilize in aqueous buffer, the ApoB had to undergo the following steps: (1) dissolving in 6 M guanidine . HCl, (2) extensive dialysis against 6 M urea and (3) dialysis against aqueous buffer [1,2]. These results suggest that there may be important structural differences between rat ApoB and human ApoB. The differences could be either in the amino acid sequence
Fig. 2. Immunoblotting of rat ApoB. (1) Rat ApoB solublhzed in 6 M urea buffer. (2) Concentrated NEMA solution obtained from delipidized rat LDL. Both samples were immunoblotted with anti-rat ApoB .
Fig. 3. Electron micrographs with 1% ammonium acetate,
of negatively stained ApoB in NEMA solution from delipidized rat LDL. NEMA solution was dialyzed and diluted 0.05% EDTA, 0.13% e-aminocaproic acid (pH 7.3) to a suitable protein concentration before application to a grid. Average particle size was 10.39 f 1.69 nm (n = 250).
or in the post-translational modification or both. Studies with [i4C]-labeled methylamine on reduced and carboxymethylated rat ApoB revealed that rat ApoB also contained the thiolester-linked fatty acids , similar to that present in human ApoB . Although it contained oleic acid, in addition to palmitic acid and stearic acid  as found in human ApoB [14,15], we cannot envision that this difference in fatty acid composition could enhance the water-solubility of rat ApoB to such an extent. Thus, we propose that differences in amino acid sequence or in carbohydrate, or both, may exist between rat ApoB and human ApoB. These structural differences in ApoB may have an important bearing on the differences in lipid metabolism between the two species. We believe that the presence of soluble ApoB in rat plasma is conceptually important. Since the soluble ApoB from rat plasma could be obtained without the initial treatment with denaturing agents or detergents, we hope that this finding could stimulate interest for further structural and metabolic studies of rat ApoB. We wish to express our appreciation to Tina Mok for excellent technical assistance and to Joan Pilcher for typing this manuscript. This investigation was supported in part by grant HL23181 from the National Heart, Lung and Blood Institute of the National Institutes of Health and by the resources of the Oklahoma Medical Research Foundation.
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