FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2038919289> ?p ?o ?g. }

• W2038919289 endingPage "1142" @default.
• W2038919289 startingPage "1132" @default.
• W2038919289 abstract "Competitive binding experiments were performed using Y1-BS1 adrenal cells to provide information about the interaction of HDL apolipoproteins with scavenger receptor class B, type I (SR-BI). Exchangeable apolipoproteins apolipoprotein A-I (apoA-I), apoA-II, apoE-2, apoE-3, and apoE-4 as phospholipid complexes bind like HDL3 to SR-BI via their multiple amphipathic α-helices; the concentrations required to reduce the binding of HDL3 to SR-BI by 50% (IC50) were similar and in the range of 35–50 μg protein/ml. In the case of apoA-I, peptides corresponding to segments 1–85, 44–65, 44–87, 149–243, and 209–241 all had the same IC50 as each other (P = 0.86), showing that a specific amino acid sequence in apoA-I is not responsible for the interaction with SR-BI. The distribution of charged residues in the amphipathic α-helix affects the interaction, with class A and Y helices binding better than class G* helices. Synthetic α-helical peptides composed of either l or d amino acids can bind equally to the receptor. Association with phospholipid increases the amount of apolipoprotein binding to SR-BI without altering the affinity of binding. Lipid-free apolipoproteins compete only partially with the binding of HDL to SR-BI, whereas lipidated apolipoproteins compete fully.These results are consistent with the existence of more than one type of apolipoprotein binding site on SR-BI. Competitive binding experiments were performed using Y1-BS1 adrenal cells to provide information about the interaction of HDL apolipoproteins with scavenger receptor class B, type I (SR-BI). Exchangeable apolipoproteins apolipoprotein A-I (apoA-I), apoA-II, apoE-2, apoE-3, and apoE-4 as phospholipid complexes bind like HDL3 to SR-BI via their multiple amphipathic α-helices; the concentrations required to reduce the binding of HDL3 to SR-BI by 50% (IC50) were similar and in the range of 35–50 μg protein/ml. In the case of apoA-I, peptides corresponding to segments 1–85, 44–65, 44–87, 149–243, and 209–241 all had the same IC50 as each other (P = 0.86), showing that a specific amino acid sequence in apoA-I is not responsible for the interaction with SR-BI. The distribution of charged residues in the amphipathic α-helix affects the interaction, with class A and Y helices binding better than class G* helices. Synthetic α-helical peptides composed of either l or d amino acids can bind equally to the receptor. Association with phospholipid increases the amount of apolipoprotein binding to SR-BI without altering the affinity of binding. Lipid-free apolipoproteins compete only partially with the binding of HDL to SR-BI, whereas lipidated apolipoproteins compete fully. These results are consistent with the existence of more than one type of apolipoprotein binding site on SR-BI. The role of scavenger receptor class B, type I (SR-BI) in lipoprotein metabolism has been studied widely (1Krieger M. Charting the fate of the “good cholesterol”: identification and characterization of the high-density lipoprotein receptor SR-BI.Annu. Rev. Biochem. 1999; 68: 523-558Google Scholar). Initially identified for its ability to bind oxidized LDL (2Acton S.L. Scherer P.E. Lodish H.F. Krieger M. Expression cloning of SR-BI, a CD36-related class B scavenger receptor.J. Biol. Chem. 1994; 269: 21003-21009Google Scholar), SR-BI was subsequently shown to be the first molecularly defined receptor that can mediate the selective uptake of cholesteryl ester (CE) from HDL (3Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Google Scholar) and LDL (4Swarnakar S. Reyland M.E. Deng J. Azhar S. Williams D.L. Selective uptake of low density lipoprotein-cholesteryl ester is enhanced by inducible apolipoprotein E expression in cultured mouse adrenocortical cells.J. Biol. Chem. 1998; 273: 12140-12147Google Scholar, 5Stangl H. Hyatt M. Hobbs H.H. Transport of lipids from high and low density lipoproteins via scavenger receptor-BI.J. Biol. Chem. 1999; 274: 32692-32698Google Scholar) into cells. SR-BI resides in the plasma membrane of cells and is composed of a glycosylated extracellular domain and two membrane-spanning domains near the N- and C-terminal regions of the molecule (6Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.Proc. Natl. Acad. Sci. USA. 1997; 94: 12610-12615Google Scholar, 7Babitt J. Trigatti B. Rigotti A. Smart E.J. Anderson R.G. Xu S. Krieger M. Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.J. Biol. Chem. 1997; 272: 13242-13249Google Scholar). Comparison of SR-BI to a closely related scavenger receptor, CD36, shows that while both receptors can mediate binding of HDL, only SR-BI is efficient at promoting selective CE uptake, and this function is attributable directly to the extracellular domain of SR-BI (8Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. Comparison of class B scavenger receptors, CD36 and scavenger receptor BI (SR-BI), shows that both receptors mediate high density lipoprotein-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.J. Biol. Chem. 1999; 274: 41-47Google Scholar, 9Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.J. Biol. Chem. 1998; 273: 26338-26348Google Scholar). Also, mutational analysis of this region of the receptor revealed two arginine residues that may be important for efficient uptake of CE from HDL (10Gu X. Lawrence R. Krieger M. Dissociation of the high density lipoprotein and low density lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) using retrovirus library-based activity dissection.J. Biol. Chem. 2000; 275: 9120-9130Google Scholar). More recently, it was shown that the unique ability of SR-BI to mediate selective lipid uptake does not require accessory proteins or structural components of the cell membrane (11Liu B. Krieger M. Highly purified scavenger receptor class B, type I reconstituted into phosphatidylcholine/cholesterol liposomes mediates high affinity high density lipoprotein binding and selective lipid uptake.J. Biol. Chem. 2002; 277: 34125-34135Google Scholar), although a PDZ domain protein appears to be required for the receptor to properly translocate to the plasma membrane in hepatocytes in vivo (12Silver D.L. A carboxyl-terminal PDZ-interacting domain of scavenger receptor B, type I is essential for cell surface expression in liver.J. Biol. Chem. 2002; 277: 34042-34047Google Scholar). The most widely studied ligands for SR-BI are the serum lipoproteins, in particular HDL, due to its participation in reverse cholesterol transport (13Tall A.R. An overview of reverse cholesterol transport.Eur. Heart J. 1998; 19: A31-A35Google Scholar, 14Rothblat G.H. de La Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. Cell cholesterol efflux: integration of old and new observations provides new insights.J. Lipid Res. 1999; 40: 781-796Google Scholar). A range of ligands besides lipoproteins has been identified, including anionic phospholipids (15Rigotti A. Acton S.L. Krieger M. The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids.J. Biol. Chem. 1995; 270: 16221-16224Google Scholar), advanced glycation end products (16Ohgami N. Nagai R. Miyazaki A. Ikemoto M. Arai H. Horiuchi S. Nakayama H. Scavenger receptor class B type I-mediated reverse cholesterol transport is inhibited by advanced glycation end products.J. Biol. Chem. 2001; 276: 13348-13355Google Scholar), and apoptotic cells (17Murao K. Terpstra V. Green S.R. Kondratenko N. Steinberg D. Quehenberger O. Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes.J. Biol. Chem. 1997; 272: 17551-17557Google Scholar). Both the protein and lipid components of HDL can bind to SR-BI, but the selective uptake of lipids into the cell requires protein-protein interaction between the lipoprotein and the receptor since phospholipid vesicles and lipid emulsion particles devoid of apolipoproteins cannot mediate selective uptake (18Thuahnai S.T. Lund-Katz S. Williams D.L. Phillips M.C. Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor.J. Biol. Chem. 2001; 276: 43801-43808Google Scholar). Instead, apolipoprotein-free lipid particles seem to fuse with the cell membrane after interaction with SR-BI (18Thuahnai S.T. Lund-Katz S. Williams D.L. Phillips M.C. Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor.J. Biol. Chem. 2001; 276: 43801-43808Google Scholar). One model of SR-BI-mediated selective lipid uptake mechanism invokes formation of a hydrophobic channel between the bound lipoprotein particle and the cell membrane (19Rodrigueza W.V. Thuahnai S.T. Temel R.E. Lund-Katz S. Phillips M.C. Williams D.L. Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells.J. Biol. Chem. 1999; 274: 20344-20350Google Scholar), while another invokes a hemifusion process (9Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.J. Biol. Chem. 1998; 273: 26338-26348Google Scholar). Regardless of the mechanism, it is clear that there is a high correlation between lipoprotein binding and the selective uptake of lipids consistent with SR-BI-mediated lipid uptake involving the two sequential steps of lipoprotein particle binding and lipid uptake (8Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. Comparison of class B scavenger receptors, CD36 and scavenger receptor BI (SR-BI), shows that both receptors mediate high density lipoprotein-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.J. Biol. Chem. 1999; 274: 41-47Google Scholar, 9Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.J. Biol. Chem. 1998; 273: 26338-26348Google Scholar, 19Rodrigueza W.V. Thuahnai S.T. Temel R.E. Lund-Katz S. Phillips M.C. Williams D.L. Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells.J. Biol. Chem. 1999; 274: 20344-20350Google Scholar, 20de Beer M.C. Durbin D.M. Cai L. Mirocha N. Jonas A. Webb N.R. de Beer F.C. van Der Westhuyzen D.R. Apolipoprotein A-II modulates the binding and selective lipid uptake of reconstituted high density lipoprotein by scavenger receptor BI.J. Biol. Chem. 2001; 276: 15832-15839Google Scholar). With regard to efflux of cholesterol from cells, it is still controversial whether binding to the receptor is an absolute requirement for the process. There is a report that provides evidence that SR-BI-dependent efflux of cholesterol requires a productive binding of the extracellular lipid acceptor (21Liu T. Krieger M. Kan H.Y. Zannis V.I. The effects of mutations in helices 4 and 6 of ApoA-I on scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux suggest that formation of a productive complex between reconstituted high density lipoprotein and SR-BI is required for efficient lipid transport.J. Biol. Chem. 2002; 277: 21576-21584Google Scholar), while another study shows that binding of liposomal acceptors to SR-BI is not necessary (22M. de La Llera-Moya Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar). Clearly, a molecular understanding of the lipid selective uptake process requires that the interactions of apolipoproteins with SR-BI be characterized thoroughly. The initial study (23Xu S. Laccotripe M. Huang X. Rigotti A. Zannis V.I. Krieger M. Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake.J. Lipid Res. 1997; 38: 1289-1298Google Scholar) demonstrated that individual HDL apolipoproteins (apoA-I, apoA-II, and apoC-III), reconstituted into HDL particles, bind to SR-BI. Subsequent work with apoA-I has shown that its conformation is important for receptor interaction. Thus, conformational changes induced by adding different amounts of phospholipid to create HDL particles of different sizes alter the affinity of binding to SR-BI (24de Beer M.C. Durbin D.M. Cai L. Jonas A. de Beer F.C. van der Westhuyzen D.R. Apolipoprotein A-I conformation markedly influences HDL interaction with scavenger receptor BI.J. Lipid Res. 2001; 42: 309-313Google Scholar, 25Liadaki K.N. Liu T. Xu S. Ishida B.Y. Duchateaux P.N. Krieger J.P. Kane J. Krieger M. Zannis V.I. Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger receptor class B type I. Effect of lipid association and ApoA-I mutations on receptor binding.J. Biol. Chem. 2000; 275: 21262-21271Google Scholar). The apoA-I/SR-BI interaction is complex in that there is not a unique recognition site in the apolipoprotein because domains in both the N- and C-terminal regions of the molecule can mediate binding (26Williams D.L. de La Llera-Moya M. Thuahnai S.T. Lund-Katz S. Connelly M.A. Azhar S. Anantharamaiah G.M. Phillips M.C. Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic alpha-helix as a recognition motif.J. Biol. Chem. 2000; 275: 18897-18904Google Scholar). Apparently, N- and C-terminal regions can both bind because they contain an amphipathic α-helix that is a recognition motif for SR-BI (26Williams D.L. de La Llera-Moya M. Thuahnai S.T. Lund-Katz S. Connelly M.A. Azhar S. Anantharamaiah G.M. Phillips M.C. Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic alpha-helix as a recognition motif.J. Biol. Chem. 2000; 275: 18897-18904Google Scholar). The interactions of apoA-II and apoE with SR-BI are less well understood, and contradictory results have been published. Thus, it has been reported that the presence of apoA-II together with apoA-I in HDL particles can either increase (27Pilon A. Briand O. Lestavel S. Copin C. Majd Z. Fruchart J.C. Castro G. Clavey V. Apolipoprotein AII enrichment of HDL enhances their affinity for class B type I scavenger receptor but inhibits specific cholesteryl ester uptake.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1074-1081Google Scholar) or decrease (20de Beer M.C. Durbin D.M. Cai L. Mirocha N. Jonas A. Webb N.R. de Beer F.C. van Der Westhuyzen D.R. Apolipoprotein A-II modulates the binding and selective lipid uptake of reconstituted high density lipoprotein by scavenger receptor BI.J. Biol. Chem. 2001; 276: 15832-15839Google Scholar) binding to SR-BI. In the case of apoE, it has been claimed recently that apoE/phospholipid complexes can (18Thuahnai S.T. Lund-Katz S. Williams D.L. Phillips M.C. Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor.J. Biol. Chem. 2001; 276: 43801-43808Google Scholar, 28Li X. Kan H.Y. Lavrentiadou S. Krieger M. Zannis V. Reconstituted discoidal ApoE-phospholipid particles are ligands for the scavenger receptor BI. The amino-terminal 1–165 domain of ApoE suffices for receptor binding.J. Biol. Chem. 2002; 277: 21149-21157Google Scholar) and cannot (29Bultel-Brienne S. Lestavel S. Pilon A. Laffont I. Tailleux A. Fruchart J.C. Siest G. Clavey V. Lipid free apolipoprotein E binds to the class B Type I scavenger receptor I (SR-BI) and enhances cholesteryl ester uptake from lipoproteins.J. Biol. Chem. 2002; 277: 36092-36099Google Scholar) bind to SR-BI. Furthermore, contrary to the situation with apoA-I (25Liadaki K.N. Liu T. Xu S. Ishida B.Y. Duchateaux P.N. Krieger J.P. Kane J. Krieger M. Zannis V.I. Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger receptor class B type I. Effect of lipid association and ApoA-I mutations on receptor binding.J. Biol. Chem. 2000; 275: 21262-21271Google Scholar), it is reported that lipidation of apoE reduces its ability to bind to the receptor (29Bultel-Brienne S. Lestavel S. Pilon A. Laffont I. Tailleux A. Fruchart J.C. Siest G. Clavey V. Lipid free apolipoprotein E binds to the class B Type I scavenger receptor I (SR-BI) and enhances cholesteryl ester uptake from lipoproteins.J. Biol. Chem. 2002; 277: 36092-36099Google Scholar). The purpose of this study is to provide new quantitative information and to clarify some of the discrepancies concerning the binding of apolipoproteins to SR-BI. We find that apoA-I, apoA-II, and apoE in the lipid-free state lack the ability to fully compete for HDL binding to SR-BI. However, once these apolipoproteins are associated with phospholipids, they all demonstrate similar abilities to compete fully for the binding of HDL to SR-BI. Results using various domains of apoA-I and apoE and synthetic peptides show that the SR-BI/apolipoprotein interaction is not amino acid sequence specific, but that the class of amphipathic α-helix affects the binding. 1,2-Dimyristoyl phosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) were purchased from Avanti Polar Lipids (Albaster, AL). Carrier-free 125I-Na (15 Ci/mg) was purchased from New England Nuclear (Boston, MA). Ham's F-10 and minimal essential media for tissue culture were purchased from Bio Whittaker (Walkersville, MD). Bovine serum albumin, horse serum, and fetal bovine serum were purchased from Sigma (St. Louis, MO). Cortrosyn, a synthetic analog of adrenocorticotropic hormone, was purchased from Organon (West Orange, NJ). Human HDL was isolated from normolipidemic donors by sequential ultracentrifugation (30Lund-Katz S. Weisgraber K.H. Mahley R.W. Phillips M.C. Conformation of apolipoprotein E in lipoproteins.J. Biol. Chem. 1993; 268: 23008-23015Google Scholar). ApoA-I and apoA-II were isolated from human HDL as described (31Weisweiler P. Isolation and quantitation of apolipoproteins A-I and A-II from human high-density lipoproteins by fast-protein liquid chromatography.Clin. Chim. Acta. 1987; 169: 249-254Google Scholar). Prior to use, the purified apolipoproteins were resolubilized in 6 M guanidine hydrochloride (GdnHCl) and dialyzed against Tris-buffered saline (TBS) (10 mM Tris, 150 mM NaCl, 0.1 mM EDTA; pH 7.4). Human apoE isoforms were expressed in Escherichia coli and purified according to the method of Morrow et al. (32Morrow J.A. Arnold K.S. Weisgraber K.H. Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.Protein Expr. Purif. 1999; 16: 224-230Google Scholar); apoE samples were solubilized in 6 M GdnHCl and 1% β-mercaptoethanol, and dialyzed extensively against 100 mM ammonium bicarbonate buffer prior to use. Synthetic peptides of human apoA-I were synthesized using an automated solid-phase peptide synthesizer as described (33Anantharamaiah G.M. Synthetic peptide analogs of apolipoproteins.Methods Enzymol. 1986; 128: 627-647Google Scholar). The peptides were blocked at the N- and C-terminal and corresponded to amino acid residues 1–43, 44–65, 44–87, and 209–241 of apoA-I with molecular masses of 4,824, 2,438, 5,087, and 3,749 Da, respectively. The amphipathic α-helical peptides L-18A (DWLKAFYDKVAEKLKEAF), D-18A (18A sequence but containing d amino acids), L-37pA (18A-proline-18A), D-37pA, and DL-37pA (D-18A-proline-L-18A; the proline separating the two 18A segments was the L form) have been described before (34Anantharamaiah G.M. Jones J.L. Brouillette C.G. Schmidt C.F. Chung B.H. Hughes T.A. Bhown A.S. Segrest J.P. Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine.J. Biol. Chem. 1985; 260: 10248-10255Google Scholar) and were prepared in a similar way; the molecular masses of the 18A peptides (which were N- and C-terminal blocked) and 37pA were 2,243 Da and 4,483 Da, respectively. Concentrations for the peptides were determined from the absorbance at 280 nm using the following molar absorbtivities: 8,250, 5,690, 11,380, 1,280, 13,940, and 6,970 M−1 cm−1 for apoA-I peptides 1–43, 44–65, 44–87, 209–241, the 18A peptide, and the 37pA peptide, respectively. As necessary, HDL and apolipoproteins were labeled with 125I using the iodine monochloride method (35Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar); the specific activities of the labeled products were in the range of 300 to 1,500 dpm/ng protein. Human apoA-I was subjected to fragmentation at three internal methionine residues using cyanogen bromide (CNBr), as described by Morrison et al. (36Morrison J. Fidge N.H. Tozuka M. Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.J. Biol. Chem. 1991; 266: 18780-18785Google Scholar); the peptides were purified by HPLC. Briefly, 30 mg of apoA-I was digested with 3 ml of CNBr in 70% trifluoroacetic acid (TFA) for 24 h. The reaction was terminated by the addition of deionized water, after which the reaction mixture was lyophilized and subjected to reversed-phase HPLC (VYDAC 22 mm inner diameter × 25 cm, particle size 10 μm) using an acetonitrile-water (containing 0.1% TFA) solvent system with a gradient of 0% to 60% acetonitrile for 90 min at a rate of 5 ml per min. Fractions at retention times 55.3 min, 57 min, 62 min, 65 min, and 68 min corresponding to fragments 1, 2, 3, 4, and 5, respectively, were collected. The samples were lyophilized and characterized using an analytical HPLC and mass spectral analysis using a PE-Sciox APT-III triple-quadrupole ion spray mass spectrometer (MS core facility at UAB-Birmingham). The fragments had retention times of 55.3 min, 57 min, 62 min, 65 min, and 68 min and corresponded to masses of 3,190 Da, 4,350 Da, 7,427 Da, 9,880 Da, and 10,700 Da, respectively. The samples of interest were the 9,880 Da fragment corresponding to the N-terminus and the 10,700 Da fragment corresponding to the C-terminus of apoA-I. The lyophilized samples were dissolved in TBS and the concentrations determined from the absorbance at 280 nm using the extinction coefficients 2.04 and 0.45 for 1 mg/ml solutions of the N- and C-terminal peptides, respectively. The method of Barenholz et al. (37Barenholz Y. Gibbes D. Litman B.J. Goll J. Thompson T.E. Carlson R.D. A simple method for the preparation of homogeneous phospholipid vesicles.Biochemistry. 1977; 16: 2806-2810Google Scholar) was adapted to prepare small unilamellar vesicles (SUVs). POPC was dissolved in chloroform-methanol (1:1, v/v) and dried under nitrogen onto the wall of a glass tube and placed in a vacuum oven to completely remove any remaining solvent. To form multilamellar vesicles (MLVs), the lipid was then rehydrated in TBS. This dispersion was sonicated on ice under nitrogen using a tapered titanium tip (Branson Sonifier 350) for 5 min followed by 1 min of cooling. The initially cloudy lipid mixture became translucent after this cycle was repeated 10 times. Thereafter, the sample was centrifuged in a Beckman 50 Ti rotor for 2 h at 4°C at 145,000 g to remove the titanium debris that was produced during the sonication, and to separate any remaining MLV from the small SUV layer. The top SUV layer was removed and used in competitive binding experiments. Discoidal reconstituted HDL (rHDL) complexes containing DMPC and apoA-I (2:1, w/w) were prepared by incubating the lipid-protein solution at the phase transition temperature of DMPC, 24°C. The appropriate amount of phospholipid dissolved in chloroform-methanol (1:1, v/v) was dried on the wall of a glass tube with nitrogen and allowed to dry completely in a vacuum oven. The dried lipid was then dissolved in TBS to form MLV. After equilibration in a 24°C water bath, the lipid and apoA-I solutions were mixed and further incubated in the same water bath for 30 min, at which time the initially turbid lipid-protein solution became clear. Under the experimental conditions used, there was essentially quantitative incorporation of protein into the lipid-protein complexes; formation of the complexes was assessed by analysis using nondenaturing 8–25% gradient electrophoresis gels (Amersham-Pharmacia). Similar discoidal complexes of apoA-II, apoE isoforms and the various peptides (38Mishra V.K. Palgunachari M.N. Datta G. Phillips M.C. Lund-Katz S. Adeyeye S.O. Segrest J.P. Anantharamaiah G.M. Studies of synthetic peptides of human apolipoprotein A-I containing tandem amphipathic alpha-helixes.Biochemistry. 1998; 37: 10313-10324Google Scholar) were prepared in the same fashion. Additionally, some rHDL particles were prepared by using DMPC-SUV and cycling the lipid-protein mixture across the phase transition temperature of DMPC. This was accomplished by cycling the lipid-protein solution at 10°C for 10 min followed by 10 min at 37°C several times. Results of experiments using rHDL prepared by direct incubation of apolipoprotein with either MLV or SUV were similar. Y1-BS1 murine adrenal cells were grown in Ham's F-10 medium (supplemented with 12.5% horse serum, 5% fetal bovine serum, and 50 μg/ml of gentamycin) in a humidified 5% CO2 incubator at 37°C as described before (19Rodrigueza W.V. Thuahnai S.T. Temel R.E. Lund-Katz S. Phillips M.C. Williams D.L. Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells.J. Biol. Chem. 1999; 274: 20344-20350Google Scholar). For experiments, Y1-BS1 adrenal cells were seeded in 12 or 24 well tissue culture flasks and allowed to grow for 2 to 3 days until ∼75% to 80% confluency was reached. The cells were then stimulated for 24 h with 100 nM cortrosyn (dissolved as a 100 mM stock solution in PBS) in serum-free Ham's F-10 medium to increase the expression of SR-BI protein. Immunoblot analysis confirmed the increased expression of SR-BI upon stimulation with hormone (data not shown). For competitive binding experiments, the monolayer was washed twice with serum-free Ham's F-10 medium (at 4°C) and then equilibrated at 4°C. Subsequently, 10 μg protein/ml of 125I-HDL3 plus increasing amounts of the unlabeled competitor in serum-free medium were incubated with the cell monolayer for 2 h at 4°C. Control wells were also included that contained only 125I-HDL3 and no competitor. After the incubation, the cell monolayer was washed three times with 2 ml of ice-cold PBS containing 0.1% BSA plus one additional wash with 2 ml of PBS alone, then 0.1 N NaOH was used to solubilize the cell monolayer. Aliquots of this lysate were taken to determine the cell-associated 125I in a γ counter, and the protein content was measured using a modified Lowry method (39Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). HDL binding and selective CE uptake were measured as described previously (19Rodrigueza W.V. Thuahnai S.T. Temel R.E. Lund-Katz S. Phillips M.C. Williams D.L. Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells.J. Biol. Chem. 1999; 274: 20344-20350Google Scholar). Control experiments were conducted to establish that incubation of 125I-HDL3 with unlabeled competitor did not lead to displacement of 125I-apolipoprotein from the HDL particle. We incubated 125I-HDL3 with a 10-fold excess of unlabeled apoA-I for 2 h at 4°C, and passed the mixture over a gel-filtration column (Pharmacia Superdex 200, 1.6 × 60 cm). Displaced 125I-apolipoprotein was not detected in the fractions that contained lipid-free apoA-I, and the specific activity of the 125I-HDL3 did not decrease by more than 5% (data not shown). To examine further the ability of apolipoproteins to exchange from human HDL3, the apolipoproteins on the lipoprotein particle were labeled by reductive methylation using [14C]formaldehyde. HDL3 labeled in this fashion was incubated with excess human VLDL (the VLDL-HDL phospholipid ratio was 10:1, v/v) at 37°C, and the VLDL was rapidly precipitated at different times by the addition of manganese phosphate. About 10% of the radiolabeled apolipoprotein transferred to VLDL within 1 min, but there was no further detectable transfer over a 24 h period. It follows that <10% of the labeled apoA-I on HDL3 particles is available for transfer to excess lipoprotein particles under the conditions of the compet" @default.
• W2038919289 created "2016-06-24" @default.
• W2038919289 creator A5005857840 @default.
• W2038919289 creator A5018727674 @default.
• W2038919289 creator A5044235202 @default.
• W2038919289 creator A5052965419 @default.
• W2038919289 creator A5061820301 @default.
• W2038919289 date "2003-06-01" @default.
• W2038919289 modified "2023-09-25" @default.
• W2038919289 title "A quantitative analysis of apolipoprotein binding to SR-BI: multiple binding sites for lipid-free and lipid-associated apolipoproteins" @default.
• W2038919289 cites W1161931977 @default.
• W2038919289 cites W1522459970 @default.
• W2038919289 cites W1558446072 @default.
• W2038919289 cites W1574380753 @default.
• W2038919289 cites W1598788587 @default.
• W2038919289 cites W1600384320 @default.
• W2038919289 cites W165264657 @default.
• W2038919289 cites W1863056418 @default.
• W2038919289 cites W1969913104 @default.
• W2038919289 cites W1971700135 @default.
• W2038919289 cites W1983038560 @default.
• W2038919289 cites W1985170971 @default.
• W2038919289 cites W1986963574 @default.
• W2038919289 cites W1996812601 @default.
• W2038919289 cites W1997678048 @default.
• W2038919289 cites W2001995220 @default.
• W2038919289 cites W2008676611 @default.
• W2038919289 cites W2017120858 @default.
• W2038919289 cites W2023426559 @default.
• W2038919289 cites W2024131238 @default.
• W2038919289 cites W2027323531 @default.
• W2038919289 cites W2030479102 @default.
• W2038919289 cites W2031423487 @default.
• W2038919289 cites W2031696510 @default.
• W2038919289 cites W2034968148 @default.
• W2038919289 cites W2040571306 @default.
• W2038919289 cites W2043832254 @default.
• W2038919289 cites W2059685472 @default.
• W2038919289 cites W2061216302 @default.
• W2038919289 cites W2066298745 @default.
• W2038919289 cites W2071043133 @default.
• W2038919289 cites W2074631079 @default.
• W2038919289 cites W2076490603 @default.
• W2038919289 cites W2081695716 @default.
• W2038919289 cites W2088800437 @default.
• W2038919289 cites W2090760822 @default.
• W2038919289 cites W2092270878 @default.
• W2038919289 cites W2116446227 @default.
• W2038919289 cites W2127016165 @default.
• W2038919289 cites W2145532583 @default.
• W2038919289 cites W2156818917 @default.
• W2038919289 cites W2170417134 @default.
• W2038919289 cites W2181847367 @default.
• W2038919289 cites W2242726375 @default.
• W2038919289 cites W2336481777 @default.
• W2038919289 cites W98152484 @default.
• W2038919289 doi "https://doi.org/10.1194/jlr.m200429-jlr200" @default.
• W2038919289 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12671027" @default.
• W2038919289 hasPublicationYear "2003" @default.
• W2038919289 type Work @default.
• W2038919289 sameAs 2038919289 @default.
• W2038919289 citedByCount "65" @default.
• W2038919289 countsByYear W20389192892012 @default.
• W2038919289 countsByYear W20389192892013 @default.
• W2038919289 countsByYear W20389192892014 @default.
• W2038919289 countsByYear W20389192892015 @default.
• W2038919289 countsByYear W20389192892016 @default.
• W2038919289 countsByYear W20389192892017 @default.
• W2038919289 countsByYear W20389192892018 @default.
• W2038919289 countsByYear W20389192892019 @default.
• W2038919289 countsByYear W20389192892020 @default.
• W2038919289 countsByYear W20389192892021 @default.
• W2038919289 countsByYear W20389192892022 @default.
• W2038919289 countsByYear W20389192892023 @default.
• W2038919289 crossrefType "journal-article" @default.
• W2038919289 hasAuthorship W2038919289A5005857840 @default.
• W2038919289 hasAuthorship W2038919289A5018727674 @default.
• W2038919289 hasAuthorship W2038919289A5044235202 @default.
• W2038919289 hasAuthorship W2038919289A5052965419 @default.
• W2038919289 hasAuthorship W2038919289A5061820301 @default.
• W2038919289 hasBestOaLocation W20389192891 @default.
• W2038919289 hasConcept C185592680 @default.
• W2038919289 hasConcept C2778163477 @default.
• W2038919289 hasConcept C55493867 @default.
• W2038919289 hasConcept C62746215 @default.
• W2038919289 hasConceptScore W2038919289C185592680 @default.
• W2038919289 hasConceptScore W2038919289C2778163477 @default.
• W2038919289 hasConceptScore W2038919289C55493867 @default.
• W2038919289 hasConceptScore W2038919289C62746215 @default.
• W2038919289 hasIssue "6" @default.
• W2038919289 hasLocation W20389192891 @default.
• W2038919289 hasLocation W20389192892 @default.
• W2038919289 hasOpenAccess W2038919289 @default.
• W2038919289 hasPrimaryLocation W20389192891 @default.
• W2038919289 hasRelatedWork W1531601525 @default.
• W2038919289 hasRelatedWork W1990781990 @default.
• W2038919289 hasRelatedWork W2384464875 @default.
• W2038919289 hasRelatedWork W2606230654 @default.

• next