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Braun, V., Hantke, K., and Henning, U. (l975), FEBS Lett. 60, 26. Braun, V., Rotering, H., Ohmo, J. P., and Hagenmaier, H. (1976), Eur. J . Biochem. 70, 601. DeMartini, M., Inouye, S., and Inouye, M.( 1976). J . Bacteriol. 127, 564. Hantke, K., and Braun, V. (1973), Eur. J . Biochem. 34, 284. Harden, A., and Norris, D. (1971), J . Physiol. 42, 332. Hawkes, S., Meehan, T., and Bissell, M. (1976), Biochem. Biophys. Res. Commun. 68, 1226. Inouye, M. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 2396. Inouye, M., Shaw, J., and Shen, C. (1972), J . B i d . Chem. 247, 8 154. Inouye, S., Takeishi, K., Lee, N., DeMartini, M., Hirashima, A., and Inouye, M. (1976), J . Bacteriol. 127, 555. Lee, N., Chang, E., and Inouye, M. (1 977), Biochim. Biophys.
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Acta 465, 650. Lee, N., and Inouye, M. (1974), FEBS Lett. 39, 167. Nakaya, K . , Yabuta, M., Iinuma, F., Kinoshita, T., and Nakamura, Y. (l975), Biochem. Biophys. Res. Commun. 67, 760. Nazaki, Y., Schechter, N . M., Reynolds, J. A,, and Tanford, C. (l976), Biochemistry 15, 3884. Reynolds, J. A., and Tanford, C. ( I 970). Proc. Natl. Acad. Sci. U.S.A. 66, 1002. Riordan, V. F. ( I 973), Biochemistry 12, 391 5. Robinson, N C., and Tanford, C. (1975), Biochemistry 14, 369. Tu, S., and Grosso, L. (1976), Biochem. Biophys. Res. Commun. 72, 9. Weigele, M., DeBernards, S., Tengi, J., and Leinigruber, W. ( I 972), J . A m . Chem. Soc. 94, 5527. Yankulov Jr.. J. A. (l970), Biochemistry 9, 2433.
Interaction of Plasma “Arginine-Rich” Apolipoprotein with Dim yristoylphosphatidylcholine’ Robert I . Roth. Richard L. Jackson,* Henry J . Pownall,I and Antonio M. Gotto, Jr.*.§
ABSTRACT: Very low density lipoproteins isolated from the
plasma of cholesterol-fed rabbits contain abnormally high amounts of cholesterol, phospholipid, and an apoprotein referred to as the “arginine-rich” protein (ARP). I t is generally assumed that the major interaction between apolipoproteins and lipids is between the protein and the phospholipids. Therefore, we have studied in the present report the lipidbinding properties of A R P to dimyristoylphosphatidylcholine (DMPC) vesicles in order to determine the importance of this interaction for ARP. The interaction was studied by ultracentrifugal flotation, circular dichroism, and microcalorimetry. The binding studies were performed using low protein-to-lipid ratios so as to minimize protein-protein interaction and vesicle disintegration. The ARP-DMPC complexes were isolated by salt density ultracentrifugation in KBr and had an average DMPC to protein molar ratio of 625 to 1. The complexes were
v e r y low density lipoproteins (VLDL)’ of human plasma contain several apolipoproteins in variable amounts. (For a review, see Scanu 1972a,b; Scanu et al., 1975; Jackson et al., 1976.) These apoproteins have been designated apoB, apoC-I, apoC-11, apoC-I11 (Alaupovic, 1971) and a protein rich in arginine termed the “arginine-rich” protein (ARP) (Shore and
’
From the Marrs McLean Department of Biochemistr) and thc Dcpartrnent of Medicine, Baylor College of Medicine and The Methodist Hospital. Houston. Texas 77030. Receiced January 3, 1977. This material u as developed by the Atherosclerosis. Lipids and Lipoproteins section of the Kational Heart and Blood Vessel Research and Demonstration Center. Baglor College of Uedicine, a grant-supported research project of thc Aational Heart. Lung and Blood Institute. National Institutes of Health. Grant KO.H L 17269. Established Investigator of the American Heart Association. 3 Address all correspondence to this author at: Division of Atherosclcrosis and Lipoprotein Research, The Methodist Hospital, Houston. Texas 77030.
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stable for several days. The addition of DMPC to A R P induced an increase in the a helicity of the protein; the maximal change (from 45% to 65%) in a-helical content required 90 min with a t ! ,? of approximately 15 min. The enthalpy of association of A R P with D M P C was highly exothermic with a value AH = -614 kcal/mol of protein. The rate of heat release in this measurement was time dependent, requiring in excess of 20 min; however, the enthalpic changes were totally finished when the helical increase was only about one-half complete. Based on the kinetics of interaction, we suggest that the high enthalpy of binding may be associated with the increase in helicity of the protein; these two processes, though, are not sufficiently concomitant to account unequivocally for the heat release in terms of either protein-lipid interaction or protein structural changes.
Shore, 1972, 1973) or apoE (Utermann, 1975). Normal subjects, as well as those with hyperlipidemia, show individual quantitative differences in these VLDL proteins which may reflect variances in genetic, hormonal, and dietary factors. The “arginine-rich’’ protein of normal human VLDL, which was originally described by Shore and Shore (1 970) and characterized by Shelburne and Quarfordt (1974), comprises about 5- 15% of the total VLDL proteins. Shore et al. (1 974) found that the proportion of A R P is preferentially increased in VLDL I Abbreviations used are: VLDL, very low density lipoproteins: 4 R P . “arginine-rich“ protein: apoB. apoC-I, apoC-Il, opoC-Ill, and ARP. apoprotein constituents of VLDL: DMPC. dimyristoylphosphatidylcholine: CD, circular dichroism; DSC, differential scanning calorimetry: T,. gel liquid crystalline transition temperature: R,. Stokes radius: LP-X I and LP-X2, ~ H lipoproteins O isolated from plasma of obstructive jaundicc patients: Tris. tris(hydroxymethy1)aminomethane: EDTA. ethylencdiaminetetraacetic acid: DEAE. diethylaminoethyl.
-
"ARGININE-RICH"
PROTEIN-PHOSPHOLIPID
from cholesterol-fed rabbits; the amount of A R P increased from 10 to 15% of the total protein in normal VLDL to 50% in cholesterolemic rabbit VLDL. The induction of ARP by dietary cholesterol and its localization in cholesterol-rich particles raises several interesting questions concerning the role of the apoprotein in the transport of this lipid. In addition, ARP may also have a role in the regulation of cholesterol synthesis. The purpose of the present report is to describe the interaction of A R P with dimyristoylphosphatidylcholine (DMPC), and to determine the effects of binding upon the protein and lipid structures. The rationale for using phospholipid is based on previous findings (Scanu, 1972a,b) which suggested that the major interaction between the plasma apolipoproteins and lipids was one between the proteins and phospholipids. This concept is also consistent with recent studies on the intact lipoproteins as described in a recent review (Morrisett et al., 1977). Although VLDL does not contain significant amounts of DMPC, this phospholipid was chosen because its physical form and properties are well defined (Newman and Huang, 1975; Lentz et al., 1976), and its thermotropic transition at about 23 OC makes it particularly suitable for study. Materials and Methods Isolation and Purification of Rabbit "Arginine-Rich" Protein ( A R P ) . Blood was collected in 1% EDTA by cardiac puncture from 12-h-fasted New Zealand white rabbits that had been maintained on a diet of commercial rabbit chow plus 2% cholesterol (Sigma) for 2 months. The plasma cholesterol concentrations were typically greater than 1500 mg/ 100 mL. Very low density lipoproteins (VLDL) were isolated from the hypercholesterolemic plasma by ultracentrifugation at plasma density for 18 h at 55 000 rpm and 5 "C in a Beckman L2-65 using a 60 Ti rotor. Following separation of the top and bottom fractions by tube slicing, the VLDL top fraction was further purified by centrifugation using a SW 50.1 rotor at 45 000 rpm at 5 "C for 40 h. The VLDL obtained from this ultracentrifugation was analyzed by Geon-Pevikon block electrophoresis (Mahley et al., 1975) and shown to consist primarily of pmigrating material. The VLDL were delipidated by diethyl ether-ethanol (3: I ) as previously described for human VLDL (Brown et al., 1969) and the lipid-free proteins dissolved in a buffer containing 8 M guanidine hydrochloride, 10 mM Tris-HCI, 1 mM EDTA, 1 mM NaN3, pH 7.6. The soluble proteins were subjected to chromatography at room temperature on a column of Sephadex G-200 (2.6 X 260 cm) equilibrated with the same buffer. The fraction corresponding to ARP, as determined by polyacrylamide gel electrophoresis, was dialyzed overnight against 8 M urea, 10 mM Tris-HCI, 1 mM EDTA, 1 m M NaN3, pH 8.2, and was then applied to a column ) 1.6 X 60 cm) of DEAE-cellulose equilibrated with the same dialysis buffer. ARP was eluted from the column with a continuous gradient from 0 to 0.25 M NaCl in the urea buffer. A R P was then dialyzed against 0.15 M NaCI, 10 mM Tris-HCI, 1 mM EDTA, 1 mM NaN3, pH 7.6 (referred to hereafter as the standard buffer), concentrated to 0.23 mg/mL by ultrafiltration using an Amicon cell equipped with a UM-IO membrane, and stored at 4 "C. Although great care was taken in these studies to avoid conditions which might give rise to aggregated states of ARP, we were unable to show that all of the apoprotein was ir, a monomeric form. The purity of the isolated A R P was demonstrated by polyacrylamide electrophoresis in sodium dodecyl sulfate. Synthesis of [ I4C]D M P C and Preparation of Vesicles. [ I4C]DMPC was synthesized from glycerol phosphorylcholine (Sigma) and [ 14C]myri~tic anhydride (Amersham/Searle) as described by Robles and van den Berg (1969). Bilamellar
INTERACTION
vesicles were prepared by dissolving 10 mg of DMPC in 1 mL of benzene and then drying the phospholipid by lyophilization; 1 mL of standard buffer was then added and the lipid was subjected to sonication for 30 min at 30 "C. Titanium particles were removed by slow speed centrifugation. Sonically irradiated preparations of DMPC contained about 90% small bilamellar vesicles and about 10% multilamellar structures as judged by chromatography on Sepharose 4B. Rechromatography of the small bilayer vesicles yielded a preparation which contained 5-1096 large vesicles; this finding suggested that an equilibrium between small quantities of multilamellar structures and the bilamellar vesicles cannot be avoided. DMPC was not degraded by sonication as demonstrated by thin-layer chromatography on silica gel in a solvent system of CHCI3/ MeOH/H20,65:25:4. All experiments were performed at least twice using well-sonicated but nonchromatographed vesicles as well as chromatographically isolated vesicles with no significantly different results. Preparation of ARP-DMPC Complexes. To 1 .O mL of ARP (-0.20 mg/mL in standard buffer) was added 0.075 mL of DMPC (-40 mg/mL in standard buffer) at 23 "C. After vortexing for 10 s, the mixture was placed directly into a Cary 61 spectropolarimeter cuvette which was maintained at 26 "C and the C D spectrum recorded as described below. After 5 h , the mixture was removed and the ARP-DMPC complex was separated from residual free lipid and free protein by gradient ultracentrifugation in KBr. The gradient solutions consisted of the appropriate quantity of KBr in the standard buffer and were prepared in 5 mL nitrocellulose tubes with a Buchler peristaltic pump and gradient maker. The samples were immediately centrifuged in an SW 50.1 rotor at 45 000 rpm and 25 "C for 60 h. The content of each tube was fractionated with an ISCO fractionator. Each fraction was analyzed for absorbance at 280 nm, for phospholipid I4C radioactivity, and for KBr density (Bausch and Lomb refractometer). Circular Dichroism. Circular dichroism was measured at 26 "C on a Cary 61 spectropolarimeter using cells of I-mm path length as described previously (Morrisett et al., 1973). The a-helical content was estimated from the [ f l ] 2 2 2 by the relation YOa helix = ( 0 2 2 2 3000)/(36 000 3000) (Morrisett et al., 1973). For experiments where changes in [ f l ] 2 2 2 were observed over time, the spectropolarimeter monitored the ellipticity at 222 nm for -30 s at each time interval. Protein concentrations were determined by amino acid analysis. Gel Permeation Chromatography of ARP-DMPC Complexes. Chromatography of complexes were performed on columns (1.6 X 30 cm) of Sepharose 4B (Pharmacia) equilibrated with standard buffer at 23 "C and eluted at an approximate flow rate of 12 mL/h under a hydrostatic pressure of
