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. Author manuscript; available in PMC: 2013 Feb 17.
Published in final edited form as: Nat Commun. 2012 Mar 27;3:754. doi: 10.1038/ncomms1760

Figure 7.

Figure 7

Mechanism studies of K560 inactivation. Values are mean±SEM, sample sizes n are indicated. (a–b) Role of oligomerization. The oligomerization state (a) and activity (b) of K560 immediately after Ni purification (0 hr), and after a 24 hr ice incubation (24 hr). (a) Size exclusion chromatography using Superdex-200. K560 content was verified by Coomassie (not shown) and bead assay (b). Peak positions 1 and 2 were well correlated with the dimeric and monomeric forms of Eg5-513-GFP analyzed using the same column43. Relative abundance of peak 1 vs. 2 was 4.73±0.86 at 0 hr, and 4.4±0.43 at 24 hr (mean±data range, n=2 each). (b) Motor activity in peak 1 elute, analyzed by bead assay (n~30) at 1mM ATP. When compared at the same motor concentration per bead, ~5-fold less K560 dilution was required to reach the same bead binding fraction at the 0th hr,. (c) Role of nucleotide-bound state. The fraction of K560 competent for microtubule-binding at 4mM AMPPNP, before and after an 1.5hr incubation on ice, assayed by immunoblot against kinesin heavy chain (AKIN01). Cross reactivity of the polyclonal antibody AKIN01 with tubulin (‘Tub’) is used for loading reference. Input reference lanes are as indicated (‘Inputs’). Ni-purified K560 was dialyzed against 5mM EDTA to deplete bound nucleotide, and then supplemented with 10mM Mg2+ (‘Dialyzed’) or 10mM Mg2+/10 mM ADP (‘Dialyzed+ADP’). Controls used un-dialyzed K560 undergoing identical ice incubation (‘Un-Dialyzed’). The effect of CK2 during incubation is quantified later in text. We hypothesize that an additional freeze-thaw cycle may contribute to the increased magnitude of inactivation observed in (c) vs. (b). (d) Role of head-head interaction. Co-sedimentation of K339 monomer with microtubules at 4 mM AMPPNP, assayed by immunoblot against kinesin heavy chain (AKIN01). Cross reactivity of AKIN01 with tubulin (‘Tub’) was used for loading reference. Shown are microtubule pulldowns using Ni-purified K339 immediately after thawing (‘Before’), or after incubation with or without CK2 (1.5:1 kinase:monomer) for 1.5 hr on ice (‘After’). Input reference lanes are as indicated (‘Inputs’). The lane marked as ‘Other’(in ‘After’ immunoblot, d) was not part of the current study.