Table 1.

Isolation of RepA–ClpA–ClpP and ClpA–ClpP complexes

Experiment	Additions	Amount retained, pmol
		RepA	ClpA
1	RepA, ClpA, ClpP, ATP[γ-S]	6.3 ± 0.2	8.3 ± 0.3
2	RepA, ClpA, ClpP	<0.5	<0.5
3	RepA, ClpA, ATP[γ-S]	<0.5	<0.5
4	RepA, ClpP, ATP[γ-S]	<0.5	—
5	RepA, ClpA, ClpP, ATP[γ-S] followed by treatment with 1 M NaCl	<0.5	<0.5
6	ClpA, ClpP, ATP[γ-S]	—	6.0 ± 0.2
7	ClpA, ClpP	—	<0.5
8	ClpA, ATP[γ-S]	—	<0.5
9	ClpA, ClpP, ATP[γ-S], followed by treatment with 1 M NaCl	—	<0.5

Complexes were formed by incubating 28 pmol of [³H]ClpA, 9 pmol ClpP, and 18 pmol of [¹⁴C]RepA in 20 μl of buffer A containing 1 mM ATP[γ-S] for 20 min at 23°C. Reaction volumes were adjusted to 100 μl with buffer A lacking Mg²⁺ and containing 0.1 mg/ml BSA and 0.005% Triton X-100. Samples were centrifuged at 2040 × g for 6 min through Ultrafree-MC 300,000 NMWL filters (Millipore). The amount of [¹⁴C]RepA and [³H]ClpA was quantitated by measuring radioactivity in the retentates and filtrates. In the presence of ATP[γ-S], 0.9 pmol of ClpA was nonspecifically bound and has been subtracted from all ClpA values. Similarly, 1.5 pmol of RepA was nonspecifically bound and has been subtracted from all RepA values. ClpP alone was not retained as demonstrated by the recovery of 95% of ClpP in the filtrate as detected by Bio-Rad Protein assay. Results are means (±SEM) of three to seven independent experiments.