TABLE 3.

Effect of prior copurification with vesicles on the fraction of membrane-associating H-MinD^a

Reaction	Nucleotide		% H-MinD in pellet
	Step 1	Step 2	Step 1	Step 2
1	ATP	ATP	66	76
2	ATP	ATPγS	66	58
3	ATP	ADP	66	21
4	ATPγS	ATP	51	77
5	ATPγS	ATPγS	51	62
6	ATPγS	ADP	51	23

^aIn step 1, H-MinD (5 μM) was incubated in RB with phospholipid vesicles (1.0 mg/ml), MgCl₂ (1 mM), and either ATP or ATPγS (100 μM) for 10 min at 30°C. Mixtures were sedimented for 1 min (16,000 × g). The pellet fractions were resuspended in the original volume of RB containing 1 mM MgCl₂ and incubated for 5 min at 30°C. In step 2, the suspensions were split into aliquots to which ATP, ATPγS, or ADP was added to 500 μM. Mixtures were incubated for another 10 min and subjected to centrifugation for 25 min at 16,000 × g. As indicated, 66% (ATP) or 51% (ATPγS) of H-MinD fractionated with the vesicles in the first step. Consequently, the total concentration of H-MinD in the reactions during the second step was 3.3 μM (reactions 1 to 3) or 2.6 μM (reactions 4 to 6).