Table 1.

Summary of ATPase measurement, MT-gliding assays, and single-molecule experiments of the homodimeric and heterodimeric kinesin constructs

	Construct	ATPase assay		Gliding assay	Beads assay
		kcat (s−1⋅head−1)	Km(MT), μM	Velocity, nm/s	Stall force, pN
Homodimer	WT	28.3  ± 2.5	0.4  ± 0.2	679  ± 59	6.3  ± 0.9
	WT	27.8  ± 1.7	0.5  ± 0.2	683  ± 42	6.0  ± 0.3
	L11/L11	11.1  ± 1.2	1.1  ± 0.3	179  ± 23	1.0  ± 0.2
	L12/L12	0.8	ND	0	0
	L8/L8	20.8  ± 3.1	1.2  ± 0.6	514  ± 31	4.0  ± 0.5
	L13/L13	19.8  ± 2.0	0.3  ± 0.2	5  ± 1	0
Heterodimer	WT/L11	20.2  ± 1.7	1.0  ± 0.3	202  ± 29	1.8  ± 0.3
	WT/L12	16.6  ± 2.2	2.0  ± 0.4	101  ± 25	0.8  ± 0.2
	WT/L8	22.7  ± 1.4	0.5  ± 0.2	554  ± 29	6.0  ± 0.7
	WT/L13	24.1  ± 0.7	0.2  ± 0.1	8  ± 1	0

The values of k_cat, K_m(MT), the gliding velocity, and the stall force are shown as the mean ± SD. The stall force was determined from the level of the plateau of the traces as shown in Fig. 3. ND, not determined.

^*WT kinesin homodimers with His-tags (WT-His/WT-His).

^† WT kinesin homodimers prepared with two different tags (WT/WT-His). The stall force of WT/WT-His was measured by using 1.0-μm beads.

^‡ The k_cat and K_m(MT) of L12/L12 could not be accurately measured because of low affinity to MTs. The indicated value is the ATPase rate with 50 μM tubulin.

^§ In the gliding assay, the L12/L12 homodimers did not retain MTs. In the beads assay, attachment signals were observed only when they exist in excess, but no movement was detected.

^¶ These constructs did not show processive behavior at a single-molecule level, although the beads moved continuously when more than one molecule was bound.