1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m303894200
ISSN1083-351X
AutoresAivar Lõokene, Liyan Zhang, Vello Tõugu, Gunilla Olivecrona,
Tópico(s)Enzyme Catalysis and Immobilization
ResumoLipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII), a component of plasma lipoproteins, for function in vivo. The hydrophobic fluorescent probe 1,1′-bis(anilino)-4,4′-bis(naphthalene)-8,8′-disulfonate (bis-ANS) was found to be a potent inhibitor of LPL. ApoCII prevented the inhibition by bis-ANS, and was also able to restore the activity of inhibited LPL in a competitive manner, but only with triacylglycerols with acyl chains longer than three carbons. Studies of fluorescence and surface plasmon resonance indicated that LPL has an exposed hydrophobic site for binding of bis-ANS. The high affinity interaction was characterized by an equilibrium constant Kd of 0.10–0.26 μm and by a relatively high on rate constant k ass = 2.0 × 104m–1 s–1 and a slow off-rate with a dissociation rate constant k diss = 1.2 × 10–4 s–1. The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII. Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII), a component of plasma lipoproteins, for function in vivo. The hydrophobic fluorescent probe 1,1′-bis(anilino)-4,4′-bis(naphthalene)-8,8′-disulfonate (bis-ANS) was found to be a potent inhibitor of LPL. ApoCII prevented the inhibition by bis-ANS, and was also able to restore the activity of inhibited LPL in a competitive manner, but only with triacylglycerols with acyl chains longer than three carbons. Studies of fluorescence and surface plasmon resonance indicated that LPL has an exposed hydrophobic site for binding of bis-ANS. The high affinity interaction was characterized by an equilibrium constant Kd of 0.10–0.26 μm and by a relatively high on rate constant k ass = 2.0 × 104m–1 s–1 and a slow off-rate with a dissociation rate constant k diss = 1.2 × 10–4 s–1. The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII. Lipoprotein lipase (LPL) 1The abbreviations used are: LPL, lipoprotein lipase; apoCII, apolipoprotein CII; ANS, 1-anilinonaphthalene-8-sulfonic acid; bis-ANS, 1,1′-bis(anilino)-4-,4′-bis(naphthalene)-8,8′-disulfonate; BSA, bovine serum albumin; T3, triiodothyronine.1The abbreviations used are: LPL, lipoprotein lipase; apoCII, apolipoprotein CII; ANS, 1-anilinonaphthalene-8-sulfonic acid; bis-ANS, 1,1′-bis(anilino)-4-,4′-bis(naphthalene)-8,8′-disulfonate; BSA, bovine serum albumin; T3, triiodothyronine. is one of the central proteins in blood lipid metabolism (for reviews, see Refs. 1Goldberg I.J. Merkel M. Front. Biosci. 2001; 6: 388-405Crossref PubMed Google Scholar, 2Wong H. Schotz M.C. J. Lipid Res. 2002; 43: 993-999Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 3Preiss-Landl K. Zimmermann R. Hammerle G. Zechner R. Curr. Opin. Lipidol. 2002; 13: 471-481Crossref PubMed Scopus (195) Google Scholar). The enzyme is bound to heparan sulfate proteoglycans at the vascular endothelium and hydrolyzes triacylglycerols and phospholipids in plasma lipoproteins so that lipolysis products can be taken up in adjacent cells for metabolic purposes. In addition and independent of catalysis, the LPL protein functions as a mediator for binding and uptake of lipoproteins by cells by bridging between the lipoproteins and heparan sulfate proteoglycans or receptors of the low density lipoprotein receptor family.LPL belongs to the family of mammalian triglyceride lipases together with pancreatic lipase, hepatic lipase, and endothelial lipase (2Wong H. Schotz M.C. J. Lipid Res. 2002; 43: 993-999Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Based on sequence homology with pancreatic lipase, models of the 55-kDa subunit of LPL have been created (4van Tilbeurgh H. Roussel A. Lalouel J.-M. Cambillau C. J. Biol. Chem. 1994; 269: 4626-4633Abstract Full Text PDF PubMed Google Scholar, 5Kobayashi Y. Nakajima T. Inoue I. Eur. J. Biochem. 2002; 269: 4701-4710Crossref PubMed Scopus (62) Google Scholar, 6Razzaghi H. Day B.W. McClure R.J. Kamboh M.I. J. Mol. Graph. Model. 2001; 19: 487-494Crossref PubMed Scopus (16) Google Scholar). The active form of LPL is a noncovalent dimer of identical subunits (7Iverius P.-H. Östlund-Lindqvist A.M. J. Biol. Chem. 1976; 251: 7791-7795Abstract Full Text PDF PubMed Google Scholar, 8Osborne Jr., J.C. Bengtsson-Olivecrona G. Lee N.S. Olivecrona T. Biochemistry. 1985; 24: 5606-5611Crossref PubMed Scopus (121) Google Scholar) that are arranged in a head-to-tail fashion (5Kobayashi Y. Nakajima T. Inoue I. Eur. J. Biochem. 2002; 269: 4701-4710Crossref PubMed Scopus (62) Google Scholar, 9Wong H. Yang D. Hill J.S. Davis R.C. Nikazy J. Schotz M.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5594-5598Crossref PubMed Scopus (59) Google Scholar). LPL can engage in a number of interactions with heparin/heparan sulfate, receptors, lipid/water interfaces, the activator apolipoprotein CII (apoCII), and perhaps with other apolipoproteins. Fatty acids with long acyl chains have been shown to bind to LPL with high affinity (10Edwards K. Chan R.Y.S. Sawyer W.H. Biochemistry. 1994; 33: 13304-13311Crossref PubMed Scopus (23) Google Scholar) and to inhibit the catalytic activity of LPL as well as binding of LPL to lipid/water interfaces (lipoproteins, emulsion droplets) (11Bengtsson G. Olivecrona T. Eur. J. Biochem. 1980; 106: 557-562Crossref PubMed Scopus (106) Google Scholar) and to heparin (12Peterson J. Bihain B.E. Bengtsson-Olivecrona G. Deckelbaum R.J. Carpentier Y.A. Olivecrona T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 909-913Crossref PubMed Scopus (196) Google Scholar). Thus, fatty acids exert strong control over the activity of LPL. Their removal by uptake in tissues or by binding to albumin is essential to allow the action of the enzyme to proceed unabated.ApoCII, a 79-amino acid residue peptide belonging to the apoC family of exchangeable apolipoproteins, is a necessary activator for LPL in vivo (13LaRosa J.C. Levy R.I. Herbert P. Lux S.E. Fredrickson D.S. Biochem. Biophys. Res. Commun. 1970; 41: 57-62Crossref PubMed Scopus (450) Google Scholar, 14Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (157) Google Scholar). Deficiency of apoCII leads to massive accumulation of lipids in blood and symptoms similar to what is seen on LPL deficiency (15Breckenridge W.C. Little J.A. Steiner G. Chow A. Poapst M. N. Engl. J. Med. 1978; 298: 1265-1273Crossref PubMed Scopus (409) Google Scholar). Systematic mutagenesis of residues in the LPL binding domain of apoCII and studies of the three-dimensional structure of apoCII by NMR strongly suggest that the basis for the activation is formation of a complex between apoCII and LPL at the surface of a lipid droplet (16Shen Y. Lookene A. Nilsson S. Olivecrona G. J. Biol. Chem. 2002; 277: 4334-4342Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 17Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar). Although the region of apoCII responsible for the interaction with LPL has been localized to the C-terminal part of the peptide (18Kinnunen P.K.J. Jackson R.L. Smith L.C. Gotto Jr., A.M. Sparrow J.T. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4848-4851Crossref PubMed Scopus (128) Google Scholar), it is still unclear which part(s) of LPL interacts with apoCII. From studies of chimeras of LPL with the related hepatic lipase it has been concluded that apoCII may interact across the anti-parallel LPL dimer with binding sites both on the N-terminal and C-terminal folding domains of LPL (19Hill J.S. Yang D. Nikazy J. Curtiss L.K. Sparrow J.T. Wong H. J. Biol. Chem. 1998; 273: 30979-30984Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). By computer modeling of the molecular docking of a C-terminal fragment of apoCII, spanning residues 50–79, to the LPL monomer, the binding site(s) for apoCII was suggested to be located at the interface between the N-terminal and C-terminal folding domains of LPL and also engaging the lid region that covers the active site (6Razzaghi H. Day B.W. McClure R.J. Kamboh M.I. J. Mol. Graph. Model. 2001; 19: 487-494Crossref PubMed Scopus (16) Google Scholar). A different interaction site was recently reported by McIlhargey et al. (20McIlhargey T.T. Yang Y. Wong H. Hill J.S. J. Biol. Chem. 2003; 278: 23027-23035Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) based on experiments with cross-linking and mutagenesis. They proposed that LPL residues 65–68 and 73–79 are involved in the activation by apoCII. In the model of LPL these residues are part of a helix located in close proximity to the active site pocket.The molecular mechanism for the activation of LPL by apoCII is still unresolved. Both proteins bind independently to lipid droplets (21Bengtsson G. Olivecrona T. Eur. J. Biochem. 1980; 106: 549-555Crossref PubMed Scopus (96) Google Scholar, 22McLean L.R. Jackson R.L. Biochemistry. 1985; 24: 4196-4201Crossref PubMed Scopus (23) Google Scholar). Thus, apoCII is not needed for interfacial binding of LPL. ApoCII has effects on LPL activity only with triacylglycerols and phospholipids that contain acyl chains longer than seven carbons (23Rapp D. Olivecrona T. Eur. J. Biochem. 1978; 91: 379-385Crossref PubMed Scopus (50) Google Scholar, 24Shinomiya M. Jackson R.L. Biochem. Biophys. Res. Commun. 1983; 113: 811-816Crossref PubMed Scopus (7) Google Scholar, 25Shinomiya M. Jackson R.L. McLean L.R. J. Biol. Chem. 1984; 259: 8724-8728Abstract Full Text PDF PubMed Google Scholar). The level to which apoCII activates LPL in vitro depends not only on the substrate molecules but also on components like surfactants and other proteins that are present in the system (21Bengtsson G. Olivecrona T. Eur. J. Biochem. 1980; 106: 549-555Crossref PubMed Scopus (96) Google Scholar). The conditions when apoCII has effects on LPL activity, i.e. in the presence of emulsified lipids, are not easily compatible with biophysical techniques for studies of protein structure and interactions. Therefore, most information comes from indirect studies of enzyme kinetics and from studies of mutants of apoCII and LPL.In the present study we have used the aromatic hydrophobic probes 1,1′-bis(anilino)-4,4′-bis(naphthalene)-8,8′-disulfonate (bis-ANS), 1-anilinonaphthalene-8-sulfonic acid (ANS), and triiodothyronine (T3) to investigate the mechanism of LPL action. Bis-ANS and ANS are often used as sensitive probes to detect hydrophobic sites and conformational changes in proteins (26Primm T.P. Gilbert H.F. J. Biol. Chem. 2001; 276: 281-286Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 27Chatterjee S. Ghosh K. Dhar A. Roy S. Proteins Struct. Funct. Genet. 2002; 49: 554-559Crossref PubMed Scopus (8) Google Scholar, 28Carneiro F.A. Ferradosa A.S. Da Poian A.T. J. Biol. Chem. 2001; 276: 62-67Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). An additional advantage with these compounds is the possibility of studying irreversible inhibition after photoincorporation by irradiation with UV light (29Seale J.W. Brazil B.T. Horowitz P.M. Methods Enzymol. 1998; 290: 318-323Crossref PubMed Scopus (15) Google Scholar). We show that bis-ANS and ANS can be used to obtain specific information about the interaction of LPL with substrates and apoCII. Our results suggest that bis-ANS binds to an exposed hydrophobic site at or close to the binding site(s) for apoCII and for individual substrate molecules. A similar site is not present in pancreatic lipase or bile salt-stimulated lipase. We propose that apoCII binds close to the substrate binding site on LPL and that the activator may play an important role for the alignment of individual substrate molecules into the active site.EXPERIMENTAL PROCEDURESMaterials—LPL was purified from bovine milk as described (30Bengtsson-Olivecrona G. Olivecrona T. Methods Enzymol. 1991; 197: 345-356Crossref PubMed Scopus (100) Google Scholar). Bis-ANS and ANS were from Molecular Probes. Mutants of apoCII were expressed and purified as described in a previous study (16Shen Y. Lookene A. Nilsson S. Olivecrona G. J. Biol. Chem. 2002; 277: 4334-4342Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The apoCII fragment spanning residues 50–79 was synthesized for a previous study (31Lycksell P.-O. Ohman A. Bengtsson-Olivecrona G. Johansson L.B. Wijmenga S.S. Wernic D. Gräslund A. Eur. J. Biochem. 1992; 205: 223-231Crossref PubMed Scopus (15) Google Scholar). Pancreatic lipase and colipase were purified from porcine pancreas as previously described (32Garner Jr., C.W. Smith L.C. J. Biol. Chem. 1972; 247: 561-565Abstract Full Text PDF PubMed Google Scholar, 33Chapus C. Desnuelle P. Foglizzo E. Eur. J. Biochem. 1981; 115: 99-105Crossref PubMed Scopus (66) Google Scholar). Bile salt-stimulated lipase purified from human milk was a kind gift from Dr. Lars Bläckberg, Umeå University. Tributyrin, tripropionin, triacetin, tricaprylin p-nitrophenyl butyrate, bovine serum albumin (fraction V), and gum arabic were obtained from Sigma. 125I-LPL was prepared by the lactoperoxidase method and purified on heparin-Sepharose (34Olivecrona G. Lookene A. Methods Enzymol. 1997; 286: 102-116Crossref PubMed Scopus (22) Google Scholar). Inactive, monomeric LPL was prepared by treatment with 1 m guanidinium chloride as previously described (8Osborne Jr., J.C. Bengtsson-Olivecrona G. Lee N.S. Olivecrona T. Biochemistry. 1985; 24: 5606-5611Crossref PubMed Scopus (121) Google Scholar, 34Olivecrona G. Lookene A. Methods Enzymol. 1997; 286: 102-116Crossref PubMed Scopus (22) Google Scholar). The concentration of bis-ANS was determined by absorbance at 394 nm using A 394 = 16000 cm–1m–1 (35Shi L. Palleros D.R. Fink A.L. Biochemistry. 1994; 33: 7536-7546Crossref PubMed Scopus (128) Google Scholar).Conditions for Pretreatment of Lipases by Bis-ANS—Incubations with LPL were made under conditions when the enzyme was as stable as possible. Depending on the purpose for the experiment this was done either at 10 °C in 20 mm Tris-Cl, 0.5 m NaCl, pH 7.4, or at 25 °C in 20 mm Tris-Cl, pH 8.5, containing 4 mm sodium deoxycholate. Stock solutions of bis-ANS (5 μm to10 mm) were made in methanol. Pancreatic lipase and bile-stimulated lipase were incubated with bis-ANS at 25 °C in 20 mm Tris-Cl, pH 7.4, in the presence of 0.5 or 0.15 m NaCl, respectively.Enzyme Assays—Most of the activity measurements with triacylglycerols as substrates were performed by continuous titration of fatty acids released using a pH-stat (Methrome, Herisau, Switzerland) at 25 °C. For these experiments stock solutions of the substrates were prepared by sonication of the triacylglycerols in gum arabic/water solution (MSE Soniprep). The enzymatic activity was recorded at pH 8.5 in 0.15 m NaCl containing 30 mm triacylglycerol and 0.2% gum arabic. In several experiments the reaction medium also contained bovine serum albumin as specified in the figure legends to Figs. 1 and 3.Fig. 3Effect of apoCII on reactivation of LPL previously inhibited by bis-ANS. A, effects of different substrates on reactivation. The substrates used were tributyrin (▾), tricaprylin (○), tripropionin (•), and triacetin (▿). The activity measurements were performed in solutions that contained 0.7 mg of BSA/ml, 15 IU heparin/ml, and 0.15 m NaCl. LPL (10 nm) was mixed with substrates (30 mm), and the reaction proceeded for 10 min before the addition of bis-ANS (8 μm final concentration). After 10 min, apoCII was added to the incubation mixtures from a stock solution in 6 m guanidine chloride (0.55 mm). B, reactivation by mutants/fragment of apoCII. Conditions were the same as in panel A but with the variants of apoCII mutants Y63F (▾) or Q70E (○) and the insertion mutant 63A (•), peptide spanning residues 50–79 from the C terminus of apoCII (▿). C, effects of apoCII at different concentrations of bis-ANS. Emulsified tricaprylin was used as substrate (30 mm). Reactivation was measured in the absence (•) as well as in the presence of 18 μm (○) or 30 μm (▾) bis-ANS. In all panels 100% corresponds to the LPL activity measured in the absence of apoCII and bis-ANS.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The activity of LPL against p-nitrophenyl butyrate was measured at pH 7.4 at room temperature in 0.1 m phosphate buffer containing 15 IU heparin/ml and 0.15 m NaCl. The concentration of substrate ranged between 0.1 and 0.5 mm. The release of p-nitrophenol was continuously monitored at 400 nm.Sucrose Density Gradient Centrifugation—For determination of the aggregation state of LPL·bis-ANS complexes, sedimentation was performed at 10 °C in linear gradients of sucrose (5–20%). The gradients (3.6 ml) were made in 20 mm Tris, 1.5 m NaCl, 1 mg/ml BSA, pH 7.4. Samples of LPL in the same buffer (0.5 μm, 200 μl total volume) were preincubated with bis-ANS in a 2- and 20-fold molar excess for 1 h and then applied on top of the gradients, which also contained the corresponding concentrations of bis-ANS. Centrifugation was for 18 h in a Beckman Coulter SW 60 rotor, and fractions of 0.24 ml were collected from the bottom of the tubes. In this case, activity measurements were made using [3H]triolein-labeled Intralipid (10%) as substrate (kindly prepared by The Upjohn Co., and the assay contained in addition to the long chain triacylglycerols 1 mm BSA and 5% rat serum as a source of apoCII to promote regain of activity of the inhibited enzyme (16Shen Y. Lookene A. Nilsson S. Olivecrona G. J. Biol. Chem. 2002; 277: 4334-4342Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Heparin-Sepharose Chromatography—This was carried out at 4 °C on a small column (2 ml of gel) equilibrated with 20 mm Tris-Cl, pH 7.4, 0.15 m NaCl, and 10% glycerol. After application of the sample and washing with buffer without NaCl, LPL was eluted by a linear gradient of NaCl from 0 to 1.5 m. The salt gradient was determined by conductivity using known concentrations of NaCl made up in the same buffer as a standard.Measurements of Fluorescence and Circular Dichroism—Fluorescence measurements were performed using a Spex FluoroMax-2 fluorometer. The experiments were done either at 25 °C in 20 mm Tris-Cl, pH 8.5, containing 4 mm sodium deoxycholate or at 10 °C in 20 mm Tris-Cl, pH 8.5, containing 15 IU heparin/ml and 0.15 m NaCl. Stock solutions of bis-ANS were prepared in methanol. LPL concentrations varied between 30 and 100 nm. Circular dichroism (CD) measurements were carried out using a Jasco J-700 spectropolarimeter. The experiments were performed at 10 °C in 20 mm phosphate buffer, pH 7.4, containing 0.5 m NaCl.Binding Studies on BIAcore—The binding studies were performed on a BIAcore 3000 instrument using sensorchips CM5. Most of the experimental details concerning immobilization of heparin and kinetic analyses of data are described in previous studies (36Lookene A. Chevreuil O. Ostergaard P. Olivecrona G. Biochemistry. 1996; 35: 12155-12163Crossref PubMed Scopus (113) Google Scholar, 37Lookene A. Savonen R. Olivecrona G. Biochemistry. 1997; 36: 5267-5275Crossref PubMed Scopus (67) Google Scholar). The binding experiments were carried out at 25 °C in 10 mm Hepes buffer, pH 7.4, containing 0.15 m NaCl. To estimate how many bis-ANS molecules that bound per LPL, we used the established relationship that for proteins a surface density of 1 ng/mm2 increases the response by about 1000 units. We assumed that this relationship was valid also for bis-ANS (38Davis T.M. Wilson W.D. Anal. Biochem. 2000; 284: 348-353Crossref PubMed Scopus (162) Google Scholar).Photoincorporation of Bis-ANS—This was carried out according to Seale et al. (29Seale J.W. Brazil B.T. Horowitz P.M. Methods Enzymol. 1998; 290: 318-323Crossref PubMed Scopus (15) Google Scholar). Briefly, samples of LPL in 20 mm Tris-Cl, 0.5 m NaCl, pH 7.4, or in 20 mm Tris-Cl, pH 8.5, 4 mm deoxycholate in the presence of various concentrations of bis-ANS were irradiated at 4 °C by UV light (250 nm). For comparison, samples of LPL without bis-ANS, were given the same treatment. After irradiation, unbound bis-ANS was removed by extensive dialysis against 0.05% SDS or against the deoxycholate-containing buffer, depending on the purpose for the experiment. For determination of the amount of bis-ANS bound by spectrophotometry, the LPL·/bis-ANS complexes were precipitated in 50% ice-cold acetone. Control experiments showed that free bis-ANS was not precipitated by this treatment.Fragmentation of LPL by Proteinases or CNBr—Limited proteolysis of complexes of LPL (0.3 mg/ml) and bis-ANS with trypsin or chymotrypsin was done at room temperature in 20 mm Tris-Cl, 4 mm deoxycholate, pH 8.5. After 15 min with the proteinases (1.5 μg/ml), SDS was added, and the samples were heated to 95 °C before analyses by SDS-PAGE (39Bengtsson-Olivecrona G. Olivecrona T. Jörnvall H. Eur. J. Biochem. 1986; 161: 281-288Crossref PubMed Scopus (41) Google Scholar, 40Lookene A. Bengtsson-Olivecrona G. Eur. J. Biochem. 1993; 213: 185-194Crossref PubMed Scopus (44) Google Scholar). Treatment with CNBr was carried out as previously described (41Nykjær A. Nielsen M. Lookene A. Meyer N. Roigaard H. Etzerodt M. Beisiegel U. Olivecrona G. Gliemann J. J. Biol. Chem. 1994; 269: 31747-31755Abstract Full Text PDF PubMed Google Scholar).Determination of Kinetic Parameters—The kinetic parameters Ki and Ar , characterizing the inhibitory efficiency (explained below under "Results"), were obtained from the equation, A=Ar+B×KiKi+C(Eq. 1) where A is the measured activity, Ar is a calculated constant describing the activity of the LPL·bis complex, C is the concentration of bis-ANS, Ki is the inhibition constant, and B is a proportional constant.Fluorescence titration curves were analyzed by the equation, F=a×CKd+C+b×C(Eq. 2) where F is fluorescence intensity, a and b are proportional constants, and C is the concentration of bis-ANS. The program SigmaPlot (SPSS Inc., Chicago, IL) was used for the determination of the kinetic parameters.RESULTSEffects of Bis-ANS on the Activity of LPL against Different Lipid Substrates—Preliminary experiments showed that bis-ANS inhibited LPL both with water-soluble and with emulsified substrates. The efficiency of the inhibition was dependent on the substrate and the assay system used. There was no detectable inhibition of LPL when the assays were run in the presence of high concentrations of BSA and apoCII. In contrast, in the presence of low concentrations of BSA ( 75 μm), binding of LPL to the emulsion particles was decreased. Because we were focused mainly on the effects of the high affinity binding of bis-ANS to LPL, we did not further investigate the effects at high concentrations of the inhibitor.Table IIIEffects of bis-ANS on binding of LPL to emulsion droplets of tributyrinConcentration of bis-ANSDistribution of LPLμmfree/bound02.0 ± 0.30.42.2 ± 0.23.03.0 ± 0.28.02.4 ± 0.122.02.8 ± 0.242.03.2 ± 0.175.07.9 ± 0.3 Open table in a new tab Another possible explanation for the inhibition was that bis-ANS caused dissociation of active LPL dimers to inactive monomers. Ultracentrifugation in sucrose density gradients in the presence of bis-ANS at a molar excess of 2:1 or 20:1 over LPL demonstrated that the remaining LPL activity (measured against triolein) sedimented corresponding to dimeric enzyme (Fig. 2A). LPL monomers have lower affinity than LPL dimers for heparin, and separation can therefore also be achieved by chromatography on heparin-Sepharose (42Bengtsson-Olivecrona G. Olivecrona T. Biochem. J. 1985; 226: 409-413Crossref PubMed Scopus (44) Google Scholar, 43Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Crossref PubMed Scopus (109) Google Scholar). We therefore investigated binding of complexes of bis-ANS and LPL to heparin-Sepharose (Fig. 2B). The peak of absorbance at 390 n
Referência(s)