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• W2000409657 abstract "Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparentK d(Pi) for the two phosphate sites at 23.3 and 1.9 mm, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt·MC1 complex at 14 s−1, yet AMP-PNP has no effect on the Mt·MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd·ADP·Pi intermediate. Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparentK d(Pi) for the two phosphate sites at 23.3 and 1.9 mm, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt·MC1 complex at 14 s−1, yet AMP-PNP has no effect on the Mt·MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd·ADP·Pi intermediate. Non-claret disjunctional protein (Ncd) 1The abbreviations used are: Ncd, non-claret disjunction; MC1, Ncd construct of Leu209–Lys700 amino acid residues; MC6, Ncd construct of Met333–Lys700 amino acid residues; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate; ATPγS, adenosine 5′-O-(3-thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; Mt·N, microtubule·Ncd complex; a.a., amino acids; Mgacetate, magnesium acetate; Kacetate, potassium acetate; DTT, dithiothreitol. 1The abbreviations used are: Ncd, non-claret disjunction; MC1, Ncd construct of Leu209–Lys700 amino acid residues; MC6, Ncd construct of Met333–Lys700 amino acid residues; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate; ATPγS, adenosine 5′-O-(3-thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; Mt·N, microtubule·Ncd complex; a.a., amino acids; Mgacetate, magnesium acetate; Kacetate, potassium acetate; DTT, dithiothreitol.is a member of the kinesin superfamily of molecular motors that translocate along microtubules by coupling ATP hydrolysis to force production (1Endow S.A. Henikoff A. Soler-Niedziela L. Nature. 1990; 345: 81-83Crossref PubMed Scopus (182) Google Scholar, 2McDonald H.B. Goldstein L.S.B. Cell. 1990; 61: 991-1000Abstract Full Text PDF PubMed Scopus (128) Google Scholar). Conventional kinesin, the founding member of this family, moves toward the plus end of neuronal microtubules, transporting membranous organelles from the cell body toward the synapse (3Bloom G.S. Endow S.A. Protein Profile. 1994; 1: 1059-1116PubMed Google Scholar, 4Vale R.D. Fletterick R.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 745-777Crossref PubMed Scopus (401) Google Scholar). Ncd, on the other hand, is responsible for the microtubule movements required for spindle organization and maintenance during oogenesis and the early cleavages of the Drosophila melanogaster embryo (5Endow S.A. Komma D.J. J. Cell Biol. 1997; 137: 1321-1336Crossref PubMed Scopus (110) Google Scholar, 6Matthies H.J.G. McDonald H.B. Goldstein L.S.B. Theurkauf W.E. J. Cell Biol. 1996; 134: 455-464Crossref PubMed Scopus (216) Google Scholar). Surprisingly, Ncd exhibits motility behavior that is quite different from that of kinesin even though the motor domains of kinesin and Ncd (∼320 amino acids) are remarkably similar both in amino acid sequence (∼40% identity) and three-dimensional structure (7Kull F.J. Sablin E.P. Lau R. Fletterick R.J. Vale R.D. Nature. 1996; 380: 550-555Crossref PubMed Scopus (579) Google Scholar, 8Sablin E.P. Kull F.J. Cooke R. Vale R.D. Fletterick R.J. Nature. 1996; 380: 555-559Crossref PubMed Scopus (324) Google Scholar, 9Sack S. Müller A. Marx M. Thormählen M. Mandelkow E.-M. Brady S.T. Mandelkow E. Biochemistry. 1997; 36: 16155-16165Crossref PubMed Scopus (181) Google Scholar). For example, Ncd drives movement to the minus end of microtubules, yet kinesin promotes plus end-directed movement (10Walker R.A. Salmon E.D. Endow S.A. Nature. 1990; 347: 780-782Crossref PubMed Scopus (294) Google Scholar, 11McDonald H.B. Stewart R.J. Goldstein L.S.B. Cell. 1990; 63: 1159-1165Abstract Full Text PDF PubMed Scopus (286) Google Scholar). In addition, the velocity of Ncd movement in microtubule gliding assays (100–230 nm/s) is slower than the movement reported for kinesin (500–800 nm/s) (12Vale R.D. Reese T.S. Sheetz M.P. Cell. 1985; 42: 39-50Abstract Full Text PDF PubMed Scopus (1410) Google Scholar, 13Yang J.T. Saxton W.M. Stewart R.J. Raff E.C. Goldstein L.S.B. Science. 1990; 249: 42-47Crossref PubMed Scopus (191) Google Scholar, 14Howard J. Hudspeth A.J. Vale R.D. Nature. 1989; 342: 154-158Crossref PubMed Scopus (740) Google Scholar, 15Stewart R.J. Thaler J.P. Goldstein L.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5209-5213Crossref PubMed Scopus (133) Google Scholar, 16Berliner E. Mahtani H.K. Karki S. Chu L.F. Cronan Jr., J.E. Gelles J. J. Biol. Chem. 1994; 269: 8610-8615Abstract Full Text PDF PubMed Google Scholar, 17Vale R.D. Funatsu T. Pierce D.W. Romberg L. Harada Y. Yanagida T. Nature. 1996; 380: 451-453Crossref PubMed Scopus (614) Google Scholar, 18Chandra R. Salmon E.D. Erickson H.P. Lockhart A. Endow S.A. J. Biol. Chem. 1993; 268: 9005-9013Abstract Full Text PDF PubMed Google Scholar, 19Allersma M.W. Gittes F. DeCastro M.J. Stewart R.J. Schmidt C.F. Biophys. J. 1998; 74: 1074-1085Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), and Ncd does not exhibit the highly processive movement along the microtubule lattice that is characteristic of kinesin (20Case R.B. Pierce D.W. Hom-Booher N.H. Hart C.L. Vale R.D. Cell. 1997; 90: 959-966Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Although the motility of Ncd and kinesin appear to be quite different, recent kinetic studies to compare Ncd and kinesin have indicated there are similarities in the ATPase mechanism. For both Ncd and kinesin, ADP release is the rate-limiting step in the absence of microtubules, and microtubules directly and dramatically activate the dissociation of ADP from both kinesin and Ncd (21Shimizu T. Sablin E. Vale R.D. Fletterick R. Pechatnikova E. Taylor E.W. Biochemistry. 1995; 34: 13259-13266Crossref PubMed Scopus (46) Google Scholar, 22Lockhart A. Cross R.A. McKillop D.F.A. FEBS Lett. 1995; 368: 531-535Crossref PubMed Scopus (36) Google Scholar, 23Pechatnikova E. Taylor E.W. J. Biol. Chem. 1997; 272: 30735-30740Crossref PubMed Scopus (34) Google Scholar). These preliminary studies have also shown that kinesin is a much faster ATPase than Ncd. Structural studies have also been pursued to compare the complexes produced by the interaction of either kinesin or Ncd with microtubules. These high resolution electron microscopic reconstructions have revealed important differences in the microtubule-motor complex that may provide insight into the mechanism of movement (24Arnal I. Metoz F. DeBonis S. Wade R.H. Curr. Biol. 1996; 6: 1265-1270Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Hirose K. Lockhart A. Cross R.A. Amos L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9539-9544Crossref PubMed Scopus (130) Google Scholar, 26Sosa 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, 27Hoenger A. Sack S. Thormählen M. Marx A. Müller J. Gross H. Mandelkow E. J. Cell Biol. 1998; 141: 419-430Crossref PubMed Scopus (118) Google Scholar, 28Hirose K. Cross R.A. Amos L.A. J. Mol. Biol. 1998; 278: 389-400Crossref PubMed Scopus (46) Google Scholar). For dimeric Ncd, both motor domains are clearly visible and are in direct contact with each other. However, one motor domain is bound to the microtubule with the second partner motor domain detached from the microtubule lattice and pointing toward the microtubule minus end (25Hirose K. Lockhart A. Cross R.A. Amos L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9539-9544Crossref PubMed Scopus (130) Google Scholar, 26Sosa 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, 27Hoenger A. Sack S. Thormählen M. Marx A. Müller J. Gross H. Mandelkow E. J. Cell Biol. 1998; 141: 419-430Crossref PubMed Scopus (118) Google Scholar). In contrast, dimeric kinesin under similar experimental conditions shows a different three-dimensional image reconstruction and diffraction pattern (24Arnal I. Metoz F. DeBonis S. Wade R.H. Curr. Biol. 1996; 6: 1265-1270Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Hirose K. Lockhart A. Cross R.A. Amos L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9539-9544Crossref PubMed Scopus (130) Google Scholar, 27Hoenger A. Sack S. Thormählen M. Marx A. Müller J. Gross H. Mandelkow E. J. Cell Biol. 1998; 141: 419-430Crossref PubMed Scopus (118) Google Scholar). The published data to date indicate that one motor domain is bound directly to the microtubule surface. However, the results for the partner motor domain are not as straightforward. It was originally proposed that the second head of kinesin was detached and only partially visible because of disorder (24Arnal I. Metoz F. DeBonis S. Wade R.H. Curr. Biol. 1996; 6: 1265-1270Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Hirose K. Lockhart A. Cross R.A. Amos L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9539-9544Crossref PubMed Scopus (130) Google Scholar). Furthermore, the authors proposed that the differences observed in the orientation of the second head of kinesin and Ncd reflected the difference in the mechanism of motility. Although Hoengeret al. (27Hoenger A. Sack S. Thormählen M. Marx A. Müller J. Gross H. Mandelkow E. J. Cell Biol. 1998; 141: 419-430Crossref PubMed Scopus (118) Google Scholar) recently reported similar experimental results, they argued quite convincingly that the second motor domain was also bound to the microtubule requiring separation of the motor domains at the neck helices. Regardless of the interpretation of the diffraction patterns or image reconstructions, the position of the second motor domain of kinesin is very different from the second motor domain of Ncd, illustrating by another criterion a fundamental difference in these two motors. Therefore, comparative studies of kinesin and Ncd are important in determining mechanistic and structural features that are both common and different for unidirectional motor proteins such as actomyosin, kinesin, dynein, DNA helicases, and RNA and DNA polymerases. We have pursued kinetic and equilibrium binding experiments using both monomeric and dimeric constructs of Ncd in the presence of various nucleotides and nucleotide analogs. These studies examine the microtubule affinity of the different Ncd-nucleotide intermediates. From these data we can infer which Ncd intermediates are bound to the microtubule and which intermediates are dissociated from the microtubule during the ATPase cycle. Such studies have been undertaken for kinesin and Ncd in the past, and it has been well established that the analogs resembling the ATP intermediate, such as AMP-PNP, are tightly bound to the microtubule and that the ADP intermediate is more weakly bound (23Pechatnikova E. Taylor E.W. J. Biol. Chem. 1997; 272: 30735-30740Crossref PubMed Scopus (34) Google Scholar, 29Crevel I.M.T.C. Lockhart A. Cross R.A. J. Mol. Biol. 1996; 257: 66-76Crossref PubMed Scopus (136) Google Scholar, 30Rosenfeld S.S. Rener B. Correia J.J. Mayo M.S. Cheung H.C. J. Biol. Chem. 1996; 271: 9473-9482Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, it has not been clearly established for Ncd which intermediate actually detaches from the microtubule during the ATPase cycle. We report here that in the absence of added nucleotide, in the presence of AMP-PNP, or in the presence of ADP, Ncd sediments with microtubules. Yet, in the presence of ADP plus phosphate, Ncd dissociates from microtubules as reported for kinesin (30Rosenfeld S.S. Rener B. Correia J.J. Mayo M.S. Cheung H.C. J. Biol. Chem. 1996; 271: 9473-9482Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and the dissociation from microtubules is cooperative for dimeric MC1. The kinetic and equilibrium binding results described below indicate that the formation of the N·ADP·Piintermediate weakens the affinity of the Ncd motor domain for the microtubule. We propose that it is this intermediate that detaches from the microtubule during the ATPase cycle. [α-32P]ATP (>3,000 Ci/mmol) was purchased from NEN Life Science Products, PEI-cellulose F TLC plates (EM Science of Merck, 20 × 20 cm, plastic backed) from VWR Scientific (West Chester, PA), and taxol (Taxus brevifolia) from Calbiochem. AMP-PNP, ATP, GTP, DEAE-Sepharose FF, and SP-Sepharose were obtained from Pharmacia Biotech Inc. (Uppsala, Sweden). The following buffers were used for the experiments described: 25 mm Kacetate ATPase buffer for equilibrium binding experiments (20 mm HEPES, pH 7.2, with KOH, 0.1 mm EDTA, 0.1 mm EGTA, 2 mmMgacetate, 25 mm Kacetate, 1 mm DTT, 5% sucrose). 50 mm Kacetate ATPase buffer for kinetics experiments (20 mm HEPES, pH 7.2, with KOH, 0.1 mm EDTA, 0.1 mm EGTA, 5 mmMgacetate, 50 mm Kacetate, 1 mm DTT, 5% sucrose). Dimeric MC1 and monomeric MC6 were expressed from pET/MC1 and pET/MC6, respectively (18Chandra R. Salmon E.D. Erickson H.P. Lockhart A. Endow S.A. J. Biol. Chem. 1993; 268: 9005-9013Abstract Full Text PDF PubMed Google Scholar). These clones were generously provided by Dr. Sharyn Endow, Duke University Medical Center. MC1 protein expressed from the plasmid contains the N terminus 11 amino acids from bacteriophage T7 S10 protein (MASMTGGQQMG) followed by 2 linker residues (RD), and Leu209–Lys700 from MC1 as originally described (18Chandra R. Salmon E.D. Erickson H.P. Lockhart A. Endow S.A. J. Biol. Chem. 1993; 268: 9005-9013Abstract Full Text PDF PubMed Google Scholar). The MC6 protein expressed from the plasmid contains no T7 S10 protein amino acids and is composed of Ncd amino acids Met333–Lys700 with amino acid residue 699 changed from aspartic acid to glycine as originally described (18Chandra R. Salmon E.D. Erickson H.P. Lockhart A. Endow S.A. J. Biol. Chem. 1993; 268: 9005-9013Abstract Full Text PDF PubMed Google Scholar). Both constructs were sequenced to confirm that the Ncd insertions were unaltered. MC1 and MC6 were expressed separately in Escherichia coli strain BL21(DE3) and purified as described with minor modifications (18Chandra R. Salmon E.D. Erickson H.P. Lockhart A. Endow S.A. J. Biol. Chem. 1993; 268: 9005-9013Abstract Full Text PDF PubMed Google Scholar). Protein concentration of purified Ncd was determined using the Bradford method (IgG or ovalbumin as standard), by measuring absorbance at 280 nm, and by measuring absorbance at 259 nm to determine the concentration of the stoichiometrically bound ADP (εADP = 15,400 m−1cm−1). The calculated extinction coefficient of MC1 is 29,240 m−1 cm−1 at 280 nm, based on 26,740 m−1 cm−1 contributed by 2 tryptophans (εTrp = 5,690 m−1cm−1) and 12 tyrosines (εTyr = 1,280m−1 cm−1) plus one ADP bound per active site (εADP at A 280 nm = 2,500 m−1 cm−1). The extinction coefficient for MC6 is 27,960 m−1cm−1 at 280 nm, based on 25,460m−1 cm−1 contributed by 2 tryptophans and 11 tyrosines plus one ADP bound per active site. For dimeric MC1, there is good agreement of the Ncd motor domain concentration based on the A 259 determination, the Bradford determination, and the active site concentration (31Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1996; 35: 6321-6329Crossref PubMed Scopus (30) Google Scholar). TheA 280 measurement is significantly lower even when determined in the presence of 8 m urea to unfold the protein. In contrast, for monomeric MC6 the A 280measurement, the Bradford assay, and the active site determination are comparable, yet the A 259 measuring ADP concentration is significantly higher. Ncd concentrations reported are based on active site concentration. Microtubules were assembled from soluble tubulin (cold depolymerized and clarified by centrifugation) and stabilized with 20 μm taxol as described previously (32Gilbert S.P. Johnson K.A. Biochemistry. 1993; 32: 4677-4684Crossref PubMed Scopus (77) Google Scholar). Steady state ATPase measurements were determined by following the hydrolysis of [α-32P]ATP to form [α-32P]ADP·Pi as described previously (32Gilbert S.P. Johnson K.A. Biochemistry. 1993; 32: 4677-4684Crossref PubMed Scopus (77) Google Scholar). All experiments were conducted on a Beckman Optima XLA analytical ultracentrifuge equipped with absorbance optics and an An60Ti rotor. Temperature was calibrated as described previously (33Correia J.J. Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1995; 34: 4898-4907Crossref PubMed Scopus (51) Google Scholar) with the following modifications. All experiments were conducted in 20 mm HEPES, pH 7.2, with KOH, 5 mm Mgacetate, 0.1 mm EGTA, 0.1 mm EDTA, 50 mm Kacetate, 0.1 mmDTT, 5% sucrose including 50–100 μm MgATP, MgADP, or MgAMP-PNP as noted. The density of each buffer was measured in either a Mettler Paar DMA 02D or an Anton Parr DMA 5000 precision density meter and used accordingly. The average density value was 1.0199 at 24.7 °C. Buffer viscosity (average value 1.0858 at 24.7 °C) was measured in a Cannon-Manning semi-micro viscometer. The partial specific volume, υ, of each construct (MC1 = 0.7255; MC6 = 0.7266) was calculated by the method of Cohen and Edsall and included the effect of a single bound nucleotide molecule (34Perkins S.J. Eur. J. Biochem. 1986; 157: 169-180Crossref PubMed Scopus (543) Google Scholar, 35Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge1992: 90-125Google Scholar). Velocity data were analyzed using DCDT (36Stafford III, W.F. Anal. Biochem. 1992; 203: 295-301Crossref PubMed Scopus (520) Google Scholar) as described (33Correia J.J. Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1995; 34: 4898-4907Crossref PubMed Scopus (51) Google Scholar). Velocity data were also analyzed with the newest version of SVEDBERG (version 5.01) which utilizes a modified Fujita-MacCosham function and allows direct fitting of the data to molecular weight (37Philo J.S. Biophys. J. 1997; 72: 435-444Abstract Full Text PDF PubMed Scopus (214) Google Scholar). It had previously been shown that the molecular weight of samples that aggregate during sedimentation equilibrium can be reasonably estimated by these methods (30Rosenfeld S.S. Rener B. Correia J.J. Mayo M.S. Cheung H.C. J. Biol. Chem. 1996; 271: 9473-9482Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Equilibrium experiments were performed at 16,000 rpm for MC1 and 22,000, 30,000 and 32,000 rpm for MC6 in charcoal-filled epon six channel centerpieces. Data sets were fit individually or jointly with NONLIN (38Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar) to an appropriate association scheme. The sequence-derived molecular weights, including one bound nucleotide, correspond to the monomer, M1, in an association scheme. For fits to an association scheme, M1 was held at the correct value. For MC1 the molecular weight of an MC1·ADP complex is 57,789 or 115,578 for a dimer, and for MC6 the molecular weight of the MC6·ADP complex is 42,025. The best fit of sedimentation equilibrium data for MC6 is a singular monomeric species. For MC1 at room temperature, the continuous loss of material from the gradient and the presence of irreversible aggregates in the presence of AMP-PNP complicated estimates of aK d for dimerization (see “Results”). At 3.6 °C, the best fit of sedimentation equilibrium data is either a single dimeric species or a monomer-dimer model with aK d < 1.5 nm. Equilibrium binding experiments were performed at 25 °C in ATPase buffer at 25 mm Kacetate with all concentrations reported as final values after mixing. In the binding stoichiometry experiment (Fig. 6), 2 μm MC1 was incubated with taxol-stabilized microtubules (0–8 μm tubulin; 40 μm taxol) in a 220-μl reaction. For the experiments performed at constant tubulin concentration (Figs. 7 and 10), 2 μm MC1 or MC6 were incubated with taxol-stabilized microtubules (4 μmtubulin) for 15 min to preform the M·N complex. This complex was then incubated with one of the following nucleotides for an additional 30 min to allow the system to reach equilibrium: MgAMP-PNP, MgADP, or MgADP + potassium phosphate, pH 7.2. The 220-μl reaction mixture was then added to Ultra-Clear centrifuge tubes (Beckman Instruments, Palo, Alto, CA) and centrifuged in a Beckman Airfuge at 30 pounds/square inch (100,000 × g) for 30 min to separate free Ncd in the supernatant from Ncd which sediments with the microtubules. 110 μl of the supernatant were removed and combined with 28 μl of 5× Laemmli sample buffer. The remaining supernatant was removed and discarded. ATPase buffer (100 μl) was added to the tube without disturbing the pellet and then gently removed to wash away any residual supernatant. Subsequently, the pellet was resuspended in 55 μl of 5× sample buffer plus 220 μl of ATPase buffer (the original sample volume). The supernatant and pellet from each sample were boiled for 5 min, loaded at equal volume on an 8% acrylamide, 2 m urea SDS gel, and electrophoresed. To determine the concentrations of Ncd and tubulin in the supernatant and pellet, the Coomassie Blue-stained gels were analyzed using an Envisions ENV24Pro digital scanner (Envisions Solutions Technology, Burlingame, CA) and quantified using NIH Image version 1.60. MC1 and MC6 showed a linear increase in Coomassie Blue-staining intensity as a function of protein concentration loaded in the range of 0.025 to 12 μm protein.Figure 7Binding of MC1 dimer to microtubules in the presence of either AMP-PNP, ADP, ADP + Pi, and ADP + Kacetate. The figure shows images of Coomassie Blue-stained SDS gels following sedimentation of Mt·MC1 (2 μm MC1; 4 μm tubulin) complex. The supernatant (S) and pellet (P) for each reaction were loaded consecutively with the concentration of nucleotide added indicated above each supernatant/pellet pair. A, reaction in the presence of increasing concentrations of MgAMP-PNP (0–20 mm). 0 mmAMP-PNP lanes are the sample with microtubules in the absence of MC1. B, reaction in the presence of increasing concentrations of MgADP (0–20 mm).C, reaction in the presence of 2 mm MgADP and increasing concentrations of potassium phosphate buffer, pH 7.2 (0–22 mm). D, reaction in the presence of 2 mm MgADP and increasing concentrations of Kacetate (10–18 mm). Note that only the ADP + Pi experimental conditions (C) resulted in a significant amount of MC1 partitioning to the supernatant.View Large Image Figure ViewerDownload (PPT)Figure 1MC1 and MC6 protein preparations.Coomassie-stained 8% acrylamide, 2 m urea gel showing the representative preparations of MC1 and MC6 as described under “Experimental Procedures.” MW, molecular mass markers in kDa; MC1, purified MC1 after DEAE chromatography migrating at ∼56 kDa; MC6, purified MC6 after SP-Sepharose chromatography migrating at ∼42 kDa.View Large Image Figure ViewerDownload (PPT)Figure 10Binding of MC6 monomer in the presence of AMP-PNP, ADP, or ADP + Pi. The figure shows images of Coomassie Blue-stained SDS gels following sedimentation of the Mt·MC6 complex (2 μm MC6; 4 μm tubulin).A, reaction in the presence of increasing concentrations of MgAMP-PNP (1–10 mm). B, reaction in the presence of increasing concentrations of MgADP (0.5–4 mm).C, reaction in the presence of 0.5 mm MgADP and increasing concentrations of potassium phosphate (0–20 mm).View Large Image Figure ViewerDownload (PPT) The microtubule-Ncd equilibrium binding data were fit to one of the following equations by a nonlinear least squares method using KaleidaGraph software (Synergy Software, Reading, PA). The binding stoichiometry of MC1 and microtubules (Fig. 6 B) was determined by plotting the data as the MC1 dimer concentration sedimenting with microtubules as a function of total microtubules (expressed as α,β-tubulin heterodimer concentration). The analysis assumes a single binding site on the microtubule for each MC1 dimer consistent with the image reconstruction data on the mode of interaction between Ncd and microtubules (25Hirose K. Lockhart A. Cross R.A. Amos L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9539-9544Crossref PubMed Scopus (130) Google Scholar, 26Sosa 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, 28Hirose K. Cross R.A. Amos L.A. J. Mol. Biol. 1998; 278: 389-400Crossref PubMed Scopus (46) Google Scholar). The microtubule pelleting experiments cannot differentiate between both motor domains bound to the microtubule or a single motor domain bound with the partner domain detached; however, both motor domains bound to the microtubule is considered to be unlikely. Therefore, the data were fit to the quadratic Equation 1. [Mt·N]=((N0+Mt0+Kd)−((N0+Mt0+Kd)2−4N0Mt0)1/2)/2Equation 1 where Mt·Nis the concentration of MC1 dimer bound to the microtubule; N 0 is the total MC1 dimer concentration; Mt 0 is total tubulin concentration, and K d is the dissociation constant. The Mt·Ncomplex was also plotted as a function of free microtubules not in complex with MC1 and fit to a hyperbola shown in Equation 2. [Mt·N]=[N][Mf]/(Kd+Mf)Equation 2 where N is the maximum concentration of MC1 dimer that sediments with microtubules; M f is the concentration of free tubulin not in complex with Ncd, andK d is the the apparent dissociation constant. Both methods of fitting the data provide approximately equivalent constants for K d and binding stoichiometry although Equation 1provides a better fit to the data. The MC1·ADP·Pi equilibrium binding results shown in Fig. 8 A were initially fit to the Hill equation to estimate the degree of cooperativity in ligand (Pi) binding (see Equation 3). Ys=[Pi]n/(Kh+[Pi]n)Equation 3 where Y s is the fraction of MC1 partitioning to the supernatant; Pi is the concentration of potassium phosphate; K h is the product of theK d values of the cooperative sites; and nis the Hill coefficient which provides an estimate of the number of cooperative sites. The data were also fit to a model assuming two stepwise binding reactions for phosphate with the association constantsK 1 and K 2. The reactions have been simplified showing only the binding of ligand Pi, although it is assumed that phosphate binds the N·ADP intermediate rather than free N (see Equations 4 and 5). N+Pi⇄N·PiK1=[N·Pi]/[N][Pi]Equation 4 N·Pi+Pi⇄Pi·N·PiK2=[Pi·N·Pi]/[N·Pi][Pi]Equation 5 Fractional partitioning (Y s) to the supernatant assumes that both motor domains are detached from the microtubule as N·ADP·Pi intermediate and can be defined as shown in Equation 6. Ys=[Pi·N·Pi]/[N]+[N·Pi]+[Pi·N·Pi]Equation 6 and using Equations Equation 4, Equation 5, Equation 6, fractional partitioning to the supernatant is as shown in Equation 7. Ys=K1K2[Pi]2/(1+K1[Pi]+K1K2[Pi]2)Equation 7 Data in Fig. 8 B were plotted as the fraction of MC1 partitioning to the supernatant as a function of MgADP concentration and fit to the function shown in Equation 8. Ys=[N][ADP]/(Kd(ADP)+[ADP])+IEquation 8 where is N is the maximum MC1 dimer partitioning to the supernatant; K d(ADP) is the apparent dissociation constant for ADP; and I is the concentration of MC1 partitioning to the supernatant extrapolated to 0 μmADP, 10 mm potassium phosphate. Stopped-flow experiments were performed with a KinTek Stopped-flow Instrument (model SF-2001, KinTek" @default.
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