Macromolecular Accessibility of Fluorescent Taxoids Bound at a Paclitaxel Binding Site in the Microtubule Surface
2004; Elsevier BV; Volume: 280; Issue: 5 Linguagem: Inglês
10.1074/jbc.m407816200
ISSN1083-351X
AutoresJ. Fernando Dı́az, Isabel Barasoaı́n, André Arigony Souto, F. Amat‐Guerri, José M. Andreu,
Tópico(s)14-3-3 protein interactions
ResumoThe macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4′-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 ± 0.25 × 106 m-1 s-1 at 37 °C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism. The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4′-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 ± 0.25 × 106 m-1 s-1 at 37 °C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism. The paclitaxel (Taxol®, Bristol-Myers Squibb) binding site of microtubules (1Schiff P.B. Fant J. Horwitz S.B. Nature. 1979; 277: 665-667Crossref PubMed Scopus (3116) Google Scholar, 2Schiff P.B. Horwitz S.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1561-1565Crossref PubMed Scopus (1743) Google Scholar) is emerging as a ubiquitous target for substances extracted from many types of different organisms, including prokaryotes (epothilones from Sorangium cellulosum) (3Bollag D.M. McQueney P.A. Zhu J. Hensens O. Koupal L. Liesch J. Goetz M. Lazarides E. Woods C.M. Cancer Res. 1995; 55: 2325-2333PubMed Google Scholar) and eukaryotes, both vegetal (paclitaxel from yew) (4Wani M.C. Taylor H.L. Wall M.E. Coggon P. McPhail A.T. J. Am. Chem. Soc. 1971; 93: 2325-2327Crossref PubMed Scopus (3700) Google Scholar) and animal (discodermolide from sea sponges and eleutherobin from coral) (5Ter Haar E. Kowalski R.J. Hamel E. Lin C.M. Longley R.E. Gunasekera S.P. Rosenkranz H.S. Day B.W. Biochemistry. 1996; 35: 243-250Crossref PubMed Scopus (438) Google Scholar, 6Long B.H. Carboni J.M. Wasserman A.J. Cornell L.A. Casazza A.M. Jensen P.R. Lindel T. Fenical W. Fairchild C.R. Cancer Res. 1998; 58: 1111-1115PubMed Google Scholar). These compounds, called microtubule-stabilizing agents, bind to microtubules, stabilize them, and block their dynamics, thus disrupting cell division. Subsequently, because they block cell division they are useful as antitumor drugs. Paclitaxel and its derivatives are the main choices for the chemotherapy of ovarian cancer, metastatic breast cancer, head and neck cancer, and lung cancer (7Choy H. Crit. Rev. Oncol. Hematol. 2001; 37: 237-247Crossref PubMed Scopus (67) Google Scholar).The location of the paclitaxel binding site of microtubules has been the object of research for a decade. Early low resolution microtubule models placed the paclitaxel binding site in between the protofilaments of microtubules (8Andreu J.M. Bordas J. García de Díaz J.F. Ancos J. Gil R. Medrano F.J. Nogales E. Pantos E. Towns-Andrews E. J. Mol. Biol. 1992; 226: 169-184Crossref PubMed Scopus (134) Google Scholar). A compatible location was observed in the projection difference map of zincinduced tubulin sheets at 6.5 Å resolution (9Nogales E. Wolf S.G. Khan I.A. Luduen̈a R.F. Downing K.H. Nature. 1995; 375: 424-427Crossref PubMed Scopus (326) Google Scholar). This location of the binding site would have been compatible with the currently established facts that taxoids are able to access their binding site very rapidly (10Evangelio J.A. Abal M. Barasoain I. Souto A.A. Acun̈a A.U. Amat-Guerri F. Andreu J.M. Cell Motil. Cytoskeleton. 1998; 39: 73-90Crossref PubMed Scopus (68) Google Scholar, 11Díaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 12Díaz J.F. Barasoain I. Andreu J.M. J. Biol. Chem. 2003; 278: 8407-8419Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and that taxoids do not bind to unassembled tubulin (13Díaz J.F. Menéndez M. Andreu J.M. Biochemistry. 1993; 32: 10067-10077Crossref PubMed Scopus (94) Google Scholar) (which suggests that the interprotofilament contact might be necessary for the existence of the binding site).The quest for the location of the binding site got a first answer from the atomic structure of tubulin. Tubulin had been an elusive protein for high resolution structural studies; its inhomogeneity and instability precluded the determination of its three-dimensional structure until the late nineties. In the brilliant study of Nogales et al. (14Nogales E. Wolf S.G. Downing K. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1778) Google Scholar), the structure of tubulin was solved by electron crystallography of paclitaxel-stabilized zinc-induced tubulin sheets, a polymer of tubulin whose topology is different from that of microtubules (15Larsson H. Wallin M. Edstrom A. Exp. Cell Res. 1976; 100: 104-110Crossref PubMed Scopus (136) Google Scholar). Although both microtubules and zinc sheets are composed of protofilaments that consist of tubulin dimers aligned head to tail, in the case of microtubules, protofilaments associate laterally and close to give a cylinder whose number of protofilaments ranges from 11 to 16 (8Andreu J.M. Bordas J. García de Díaz J.F. Ancos J. Gil R. Medrano F.J. Nogales E. Pantos E. Towns-Andrews E. J. Mol. Biol. 1992; 226: 169-184Crossref PubMed Scopus (134) Google Scholar, 16Mandelkow E.M. Schulteiss R. Rapp R. Müller M. Mandelkow E. J. Cell Biol. 1986; 102: 1067-1073Crossref PubMed Scopus (95) Google Scholar, 17Wade R.H. Chrétien D. Job D. J. Mol. Biol. 1990; 212: 775-786Crossref PubMed Scopus (93) Google Scholar). In the case of the zinc-induced sheets, protofilaments associate in an anti-parallel way, resulting in a flat sheet that is suitable for two-dimensional electron crystallography (9Nogales E. Wolf S.G. Khan I.A. Luduen̈a R.F. Downing K.H. Nature. 1995; 375: 424-427Crossref PubMed Scopus (326) Google Scholar).The protofilament structure directly determined from the zinc-induced sheets was fitted into electron microscopy density maps of microtubules (18Sosa H. Dias D.P. Hoenger A. Whittaker M. Wilson-Kubalek E. Sablin E. Fletterick R.J. Vale R.D. Milligan R.A. Cell. 1997; 90: 217-224Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 19Meurer-Grob P. Kasparian J. Wade R.H. Biochemistry. 2001; 40: 8000-8008Crossref PubMed Scopus (108) Google Scholar) to obtain pseudo-atomic resolution maps of microtubules (19Meurer-Grob P. Kasparian J. Wade R.H. Biochemistry. 2001; 40: 8000-8008Crossref PubMed Scopus (108) Google Scholar, 20Nogales E. Whittaker M. Milligan R.A. Downing K.H. Cell. 1999; 96: 79-88Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar, 21Chacón P. Wriggers W. J. Mol. Biol. 2002; 317: 375-384Crossref PubMed Scopus (291) Google Scholar). The constructed models are supported by the 8 Å three-dimensional reconstruction of microtubules from cryoelectron microscopy (22Li H. DeRosier D.J. Nicholson W.V. Nogales E. Downing K.H. Structure (Lond.). 2002; 10: 1317-1328Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Because the zinc-induced sheets employed included paclitaxel as stabilizer, the location of the paclitaxel binding in the microtubules was thus known. Surprisingly, the translation of the zinc sheet protofilaments into microtubules results in a paclitaxel binding site inside the lumen of the tube, hidden from the outer solvent (20Nogales E. Whittaker M. Milligan R.A. Downing K.H. Cell. 1999; 96: 79-88Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar).The kinetics of binding of several taxoids (including paclitaxel) to their site in the microtubules was subsequently determined (11Díaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 12Díaz J.F. Barasoain I. Andreu J.M. J. Biol. Chem. 2003; 278: 8407-8419Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The association rate constant of paclitaxel to stabilized microtubules assembled from pure tubulin is very high (3.6 × 106 m-1 s-1), being an order of magnitude slower for MAP 1The abbreviations used are: MAP, microtubule-associated protein; AB, microtubular protein assembly buffer, GAB, glycerol assembly buffer; Mes, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; paclitaxel, 4,10-diacetoxy-2a-(benzoyloxy)-5b,20-epoxy-1,7b-dihydroxy-9-oxotax-11-en-13a-yl(2R,3S)-3-[(phenylcarbonyl)-amino]-2-hydroxy-3-phenylpropionate); docetaxel, 4-acetoxy-2a-(benzoyloxy)-5b,20-epoxy-1,7b,10b-trihydroxy-9-oxotax-11-en-13a-yl(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropionate). 1The abbreviations used are: MAP, microtubule-associated protein; AB, microtubular protein assembly buffer, GAB, glycerol assembly buffer; Mes, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; paclitaxel, 4,10-diacetoxy-2a-(benzoyloxy)-5b,20-epoxy-1,7b-dihydroxy-9-oxotax-11-en-13a-yl(2R,3S)-3-[(phenylcarbonyl)-amino]-2-hydroxy-3-phenylpropionate); docetaxel, 4-acetoxy-2a-(benzoyloxy)-5b,20-epoxy-1,7b,10b-trihydroxy-9-oxotax-11-en-13a-yl(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropionate).-stabilized microtubules and for native cytoskeletons of PtK2 cells (12Díaz J.F. Barasoain I. Andreu J.M. J. Biol. Chem. 2003; 278: 8407-8419Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Because a binding site hidden in the microtubule lumen should severely restrict accessibility of the ligands to their target, these results were apparently contradictory with the observed location of the binding site. These kinetic constants are incompatible with any kind of passive diffusion through a pore on the microtubule surface as big as 25 Å diameter (12Díaz J.F. Barasoain I. Andreu J.M. J. Biol. Chem. 2003; 278: 8407-8419Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The binding reaction of taxoids to microtubules is diffusion-controlled, indicating that the observed binding step is not restricted, and so it corresponds to the binding of the ligand to an exposed site. Because binding is slowed down by a factor of 10 by the presence of MAPs that bind to the microtubule outer surface, the kinetic evidence points to a primary binding site accessible on the microtubule surface.To reconcile the kinetic data with the structural and biochemical evidence of a paclitaxel binding site placed in the microtubule lumen (20Nogales E. Whittaker M. Milligan R.A. Downing K.H. Cell. 1999; 96: 79-88Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar, 23Rao S. Horwitz S.B. Ringel I. J. Natl. Cancer Inst. 1992; 84: 785-788Crossref PubMed Scopus (153) Google Scholar, 24Rao S. Krauss N.E. Heerding J.M. Swindell C.S. Ringel I. Orr G.A. Horwi S.B. J. Biol. Chem. 1994; 269: 3132-3134Abstract Full Text PDF PubMed Google Scholar, 25Rao S. Orr G.A. Chaudhary A.G. Kingston D.G. Horwitz S.B. J. Biol. Chem. 1995; 270: 20235-20238Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 26Giannakakou P. Sackett D.L. Kang Y.K. Zhan Z. Buters J.T. Fojo T. Poruchynsky M.S. J. Biol. Chem. 1997; 272: 17118-17125Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 27Gonzalez-Garay M.L. Chang L. Blade K. Menick D.R. Cabral F. J. Biol. Chem. 1999; 274: 23875-23882Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 28Rao S. He L. Chatkravarty S. Ojima I. Orr G.A. Horwitz S.B. J. Biol. Chem. 1999; 274: 37990-37994Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), an external initial binding site together with a hypothetical transport mechanism toward the internal binding site have been proposed (12Díaz J.F. Barasoain I. Andreu J.M. J. Biol. Chem. 2003; 278: 8407-8419Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In this model, taxoids would bind to a putative binding site located in one of the two different types of pores existent in the B-lattice of the microtubules (pore type I, closer to the site in the lumen), being later transported to the luminal site. In this hypothetical mechanism, binding to the external and luminal sites should be mutually exclusive because only 1.0 taxoid molecules bind/tubulin dimer. This could be accomplished if both sites share an element that switches between the two alternative binding sites.The purpose of this study is to obtain direct evidence of the existence of an external taxoid binding site. To do so, the accessibility of microtubule-bound taxoids to a large macromolecule (an antibody) has been probed, employing fluorescein derivatives of paclitaxel (Flutax-1, Flutax-2, and Hexaflutax) and polyclonal and monoclonal antibodies against the fluorescein moiety. Because it is impossible for a large molecule like an antibody to fit through any realistic pore in the microtubule wall, taxoid molecules bound to the luminal site of the microtubules will be protected from antibodies. Taxoid molecules bound to a site on the surface would react with the antibody, so providing evidence of an exposed taxoid binding site.In this study, the existence of a specific ternary complex of microtubules, Hexaflutax, and 4-4-20 (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy, providing direct evidence of the existence of an external binding site for the ligand. Further kinetic evidence is given by the kinetics of binding of the antibody to microtubule-bound Hexaflutax, which is fast, indicating that the fluorescein groups of the ligand are homogeneously exposed to the outer solvent. From these results it can be concluded that these fluorescent taxoids bind to an outer site on the microtubules which is shared with paclitaxel.MATERIALS AND METHODSTubulin, Taxoids, and Antibodies—Purified calf brain tubulin and chemicals were as described previously (29Díaz J.F. Andreu J.M. Biochemistry. 1993; 32: 2747-2755Crossref PubMed Scopus (406) Google Scholar). For glycerol-induced assembly, tubulin was equilibrated directly in 10 mm phosphate, 1 mm EGTA, 0.1 mm GTP, 3.4 m glycerol, pH 6.8, buffer. All tubulin samples were clarified by centrifugation at 50,000 rpm, 4 °C, for 10 min using TL100.2 or TL100.4 rotors in Beckman Optima TLX centrifuges. After centrifugation 6 mm MgCl2 and up to 1 mm GTP were added to the solution, final pH 6.5 (glycerol assembly buffer, GAB). Microtubule protein, containing tubulin and MAPs, was prepared in microtubular protein assembly buffer (AB) (100 mm Mes, 1 mm EGTA, 1 mM MgSO4, 2 mm 2-mercaptoethanol, 1 mm GTP, pH 6.5), as described previously (30de Pereda J.M. Wallin M. Billger M. Andreu J.M. Cell Motil. Cytoskeleton. 1995; 30: 153-163Crossref PubMed Scopus (10) Google Scholar). Stabilized mildly cross-linked microtubules were prepared as described previously (11Díaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 12Díaz J.F. Barasoain I. Andreu J.M. J. Biol. Chem. 2003; 278: 8407-8419Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 31Andreu J.M. Barasoain I. Biochemistry. 2001; 40: 11975-11984Crossref PubMed Scopus (66) Google Scholar). Flutax-1 (7-O-[N-(fluorescein-4′-carbonyl)-l-alanyl]paclitaxel) and Flutax-2 (7-O-[N-(2,7-difluorofluorescein-4′-carbonyl)-l-alanyl]paclitaxel) were synthesized as described previously (11Díaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 32Souto A.A. Acun̈a A.U. Andreu J.M. Barasoain I. Abal M.Y Amat-Guerri F. Angew. Chem. Int. Ed. Eng. 1995; 34: 2710-2712Crossref Scopus (56) Google Scholar). 7-Hexaflutax (7-O-{N-[6-(fluorescein-4′-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) was synthesized by the reaction of 5 mg of 7-O-[l-alanyl]paclitaxel with a 50% excess of 6-(fluorescein-4′-carboxamido)-n-hexanoic acid succinimidyl ester (Molecular Probes, Eugene, OR) using basically the same procedure. The structures of the ligands are shown in Fig. 1. 7-Hexaflutax was purified by preparative TLC on silica gel with chloroform/hexane/methanol (1:1: 0.2, v/v/v) as eluent. Yield, 4.13 mg, 55%; 1H RMN (400 MHz, CDCl3, room temperature) (see numbering (32Souto A.A. Acun̈a A.U. Andreu J.M. Barasoain I. Abal M.Y Amat-Guerri F. Angew. Chem. Int. Ed. Eng. 1995; 34: 2710-2712Crossref Scopus (56) Google Scholar)): 8.89 (m, 3H, H-5′F, o-H-Ph(a)), 8.25 (s, 1H, H-3′F), 7.67 (d, 2H, o-H-Ph(c)), 7.52–7.05 (m, 12H, H-6′F, m-, p-H-Ph(a), o-, m-, p-H-Ph(b), m-, p-H-Ph(c)), 6.70 (d, 2H, H-2F, H-7F), 6.49 (s, 2H, H-4F, H-5F), 6.42 (d, 2H, H-1F, H-8F), 6.13 (s, 1H, H-10), 5.95 (t, 1H, H-13), 5.34–5.48 (m, 3H, H-2, H-7, H-3′), 4.85 (d, 1H, H-5), 4.54 (d, 1H, H-2′), 4.12 (q, 1H, CH-Ala), 3.99 (m, 2H, H-20), 3.70 (d, 1H, H-3), 3.42 (q, 2H, CH2NH), 3.26 (t, 2H, CH2CO), 2.26 (m, 1H, H-6a), 2.15 (s, 3H, 4-CH3CO), 2.08 (m, 2H, H-14), 1.97 (s, 3H, 10-CH3CO), 1.79 (s, 3H, H-18), 1.60 (m, 1H, H-6b), 1.59 (s, 3H, H-19), 1.40–1.50 (m, 4H, 2×CH2), 1.08 (d, 3H, CH3-Ala), 0.97 (s, 3H, H-17), 0.93 (s, 3H, H-16) ppm. Electrospray ionization mass spectrometry (positive mode): 1,396.3 [MH+]. Hexaflutax purity was 97.7% (high performance liquid chromatography (10Evangelio J.A. Abal M. Barasoain I. Souto A.A. Acun̈a A.U. Amat-Guerri F. Andreu J.M. Cell Motil. Cytoskeleton. 1998; 39: 73-90Crossref PubMed Scopus (68) Google Scholar)). Taxoids were dissolved in dimethyl sulfoxide, and their concentrations were measured spectrophotometrically as described previously (10Evangelio J.A. Abal M. Barasoain I. Souto A.A. Acun̈a A.U. Amat-Guerri F. Andreu J.M. Cell Motil. Cytoskeleton. 1998; 39: 73-90Crossref PubMed Scopus (68) Google Scholar, 12Díaz J.F. Barasoain I. Andreu J.M. J. Biol. Chem. 2003; 278: 8407-8419Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 29Díaz J.F. Andreu J.M. Biochemistry. 1993; 32: 2747-2755Crossref PubMed Scopus (406) Google Scholar) except for the Hexaflutax concentration, which was measured in 0.5% SDS, 50 mm sodium phosphate buffer, pH 7.0, employing an extinction coefficient of ϵ458 24,200 ± 600 m-1. The solubility of Hexaflutax in GAB in the absence of tubulin was found to be of the order of 10 μm. Docetaxel (Taxotere®); was kindly provided by Rhône-Poulenc Rorer (Antony, France). Antifluorescein polyclonal rabbit IgG and 4-4-20 antifluorescein monoclonal mouse IgG (33Kranz D.M. Voss Jr., E.W. Mol. Immunol. 1981; 18: 889-898Crossref PubMed Scopus (75) Google Scholar) were gifts from Prof. Edward W. Voss Jr. (University of Illinois at Urbana-Champaign). They were concentrated by precipitation with 50% ammonium sulfate, centrifuged for 25 min at 19,000 rpm in a SS-34 rotor employing a Sorvall RC-5B centrifuge, and the pellets were resuspended in 10 mm phosphate and 150 mm NaCl, pH 7.0. The remaining ammonium sulfate was eliminated with a gel filtration chromatography (HiTrap desalting, Amersham Biosciences) in the same buffer, and finally the antibodies were concentrated using Centricon 10 concentrators (Amicon, Bedford, MA) and kept at -80 °C until used. Prior to the measurements the concentration of sites in the polyclonal antibody was carefully titrated with fluorescein, Flutax-1, Flutax-2, and Hexaflutax, 20% of the total IgG was found to be active against fluorescein. The IgG fraction of a nonimmune rabbit serum was purified as described previously (34Arevalo M.A. Nieto J.M. Andreu D. Andreu J.M. J. Mol. Biol. 1990; 214: 105-120Crossref PubMed Scopus (48) Google Scholar) and concentrated as the other IgGs. P11E12 (anti-β-tubulin N-terminal) and P12E11 (anti-α-tubulin C-terminal) mouse monoclonal IgGs were obtained as described previously (35De Inés, C. (1995) Interaction of Cellular Microtubules with Specific Antibodies and New Antitumoral Compounds, Ph.D. thesis, pp. 59–92, Universidad Complutense de MadridGoogle Scholar, 36Chan M.F. Radeke M.J. de Inés C. Barasoain I. Kohlstaedt L.A. Feinstein S.C. Biochemistry. 1998; 37: 17692-17703Crossref PubMed Scopus (81) Google Scholar, 37Modig C. Olsson P.E. Barasoain I. de Inés C. Andreu J.M. Roach M.C. Luduen̈a R.F. Wallin M. Cell Motil. Cytoskeleton. 1999; 42: 315-330Crossref PubMed Scopus (11) Google Scholar). MOPC 21-purified mouse IgG was from Sigma.Cosedimentation Assay of Ternary Complex Formation—10 μm GTP-tubulin in PEDTA 7 GTP buffer (10 mm sodium phosphate, 1 mm EDTA, 7 mm MgCl2, 1 mm GTP, pH 6.7) in the presence of 12 μm Hexaflutax was incubated at 37 °C for 30 min, in the presence or absence of 12 μm 4-4-20 monoclonal antibody and 100 μm docetaxel; alternatively, 4 μm paclitaxel binding sites in preassembled cross-linked microtubules (4.5 μm) in GAB were incubated for 1 min at 25 °C with the desired reac-tives. The samples were immediately centrifuged for 20 min at 50,000 rpm in a TLA 100 rotor, employing a Beckman Optima TLX ultracen-trifuge. The supernatants were taken and the pellets carefully resuspended in 10 mm sodium phosphate, 1% SDS, pH 7.0, buffer. An aliquot of supernatants and pellets was diluted 1/5 in 10 mm sodium phosphate, 1% SDS, pH 7.0, buffer and incubated for 15 min at 80 °C to dissociate the antibody-fluorescein complex. The fluorescence of the samples was measured in a Shimadzu RF-540 spectrofluorometer employing λexc 495, λems 520 nm, 5 nm excitation and emission slits, to quantify the amount of ligand in supernatant and pellets. The supernatants and pellets were also diluted 1/2 in electrophoresis sample buffer, and the presence of antibodies in them was assayed with a nonreducing SDS-PAGE in 7.5% acrylamide gel.Electron Microscopy of the Ternary Complex—A mixture of 4 μm sites in cross-linked microtubules, 2 μm Hexaflutax, and 2 μm 4-4-20 antibody was adsorbed for 1 min to a copper grid covered with formvar and carbon. The grids were blotted and then incubated over a drop of GAM-10, a nanogold-labeled goat anti-mouse antibody (British Biocell International, Cardiff, UK), for 5 min. The grids were then washed with 5 drops of GAB and stained for 2 min with 2% uranyl acetate in water. The grids were observed in a JEOL JEM-1200-EXII microscope.Fluorescence Spectroscopy and Anisotropy Measurements—Corrected fluorescence spectra were acquired with a photon counting Fluorolog-3–221 instrument (Jobin Yvon-Spex, Longiumeau, France), employing 1 nm excitation and 5 nm emission bandwidths, at 25 °C. Fluorometric concentration measurements were made with a Shimadzu RF-540 spectrofluorometer. Anisotropy values were collected in the Fluorolog T-format mode with vertically polarized excitation and corrected for the sensitivity of each channel with horizontally polarized excitation (38Lackowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic, New York1999: 291-318Crossref Google Scholar).Binding of Taxoids to Microtubules—Binding constants of Hexaflutax to microtubules were obtained using anisotropy titration measurements. 200 nm Hexaflutax in GAB was incubated for 30 min with increasing concentrations of binding sites in stabilized cross-linked microtubules (from 0 to 10 μm) at the desired temperature, and the anisotropy of the solution was measured in a POLARSTAR BMG plate reader in the polarization mode, employing the 480-P excitation filter and the 520-P emission filters (38Lackowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic, New York1999: 291-318Crossref Google Scholar). Alternatively, the binding constants of Hexaflutax and the 4-4-20-Hexaflutax complex were measured using a centrifugation assay. Samples of 1 μm taxoid binding sites in cross-linked microtubules were incubated for 30 min at 25 °C with different concentrations of Hexaflutax in the presence and absence of antibody. The samples were centrifuged for 20 min at 50,000 rpm in a TL100 rotor employing a Beckman TLX ultracentrifuge. The pellets and supernatants were diluted 1/5 in 10 mm sodium phosphate, pH 7.0, 1% SDS and warmed up at 95 °C for 3 min to dissociate the Hexaflutax-antibody complex and their fluorescence measured employing a Shimadzu RF-540 spectrofluorometer (excitation wavelength 495 nm, emission wavelength 527 nm, 5 nm excitation and emission slits). The concentration of ligand in the samples was calculated using Hexaflutax spectrophotometric concentration standards.Kinetics of Binding of Antibodies to Microtubules—The fast kinetics of binding of the 4-4-20 antibody to Hexaflutax bound to stabilized microtubules or free in solution was measured by the change of intensity of fluorescence using a Bio-Logic SF300S stopped flow device equipped with a fluorescence detection system with an excitation wavelength of 484 nm and a filter with a cutoff of 520 nm in the emission pathway. The fast kinetics of binding of the anti-fluorescein polyclonal IgG to Flutax-1, Flutax-2 bound to stabilized microtubules, was measured by the change of intensity of fluorescence using a High-Tech Scientific SS-51 stopped flow device equipped with a fluorescence detection system using an excitation wavelength of 492 nm and a filter with a cutoff of 530 nm in the emission pathway. The slow kinetics of binding of 4-4-20 antibody to Hexaflutax bound to MAPs containing microtubules was measured with a photon counting instrument Fluorolog-3-221 (Jobin Yvon-Spex, Longiumeau, France) with an excitation wavelength of 495 nm (0.1 nm band pass to prevent photolysis) and an emission wavelength 525 nm (5 nm band pass). The fitting of the kinetic curves was done with a nonlinear least squares fitting program based on the Marquardt algorithm (39Bevington P.R. Data Reduction and Error Analysis for the Physical Sciences. McGraw-Hill Book Co., New York1969: 235-240Google Scholar); additionally the FITSIM package (40Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (667) Google Scholar) was employed for the data shown in Fig. 4B.Fig. 4A, kinetics of binding of 4-4-20 IgG to free Hexaflutax at 37 °C. In the stopped flow device a 2 μm solution of 4-4-20 IgG (solid line) or GAB (dotted line) was mixed with 200 nm Hexaflutax (final concentrations 1 or 0 μm 4-4-20 IgG and 100 nm Hexaflutax); green line, fitting of the data to a monoexponential curve. Upper inset, residues between the experimental and the fitting curve (green line). Lower inset, dependence of the observed kinetic rate on the concentration of 4-4-20 IgG. B, kinetics of binding of 4-4-20 IgG to Hexaflutax bound to paclitaxel binding sites in cross-linked microtubules in GAB at 37 °C. In the stopped flow device a 2 μm solution of 4-4-20 IgG (solid line) or GAB (dotted line) was mixed with a solution of 200 nm Hexaflutax and 2 μm sites (final concentrations 1 or 0 μm 4-4-20 IgG) and 100 nm Hexaflutax bound to 1 μm sites (cyan line), fitting the data to the sum of three exponentials (green, red, and blue lines). Data were corrected for the observed photobleaching (3%). Upper inset, residues between the experimental and the fitting curves (cyan line residues to the three-exponential fitting, orange line residues to a two-exponential fitting). Lower inset, dependence of the observed kinetic rate of the second phase (red line) on the concentration of 4-4-20 IgG.View Large Image
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