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. 2020 Mar 9;13:749–759. doi: 10.2147/IDR.S242304

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© 2020 Wang et al.

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Interaction of palmatine with QnrS and AAC(6′)-Ib-cr. Purification of recombinant proteins visualized using SDS-PAGE for (A) QnrS, (B) AAC(6′)-Ib-cr, (C) GyrA and (D) GyrB. Lane M, protein molecular mass marker; Lane 1, total cellular extracts before induction; Lane 2, total cellular extracts after IPTG induction; Lane 3, extracts after Ni-NTA column elution (E) Palmatine reduces the gyrase protective effect of QnrS. 20 μL reaction mixtures were analyzed in 1% TBE gels. Reaction mixtures contained 0.25 μg relaxed pBR322 plasmid DNA (lanes 1 to 6), 2.8 nM gyrase (lanes 2 to 6), 6 μM ciprofloxacin (lanes 3 to 6), 2 μM purified His₆-tag proteins (lane 4), 2 μM purified QnrS (lane 5) and 2 μM purified QnrS that were preincubated with 10 μM palmatine (lane 6). (F) Digital analysis of the most supercoiled isomer (yellow-dotted box). (G) Palmatine reduces acetylation of AAC(6′)-Ib-cr. 50 μM ciprofloxacin was incubated in the presence of 4 μM AAC(6ʹ)-Ib-cr protein or 4 μM AAC(6ʹ)-Ib-cr protein that was preincubated with 20 μM palmatine, an acetyl donor (acetyl-CoA) and an indicator (DTNB). Absorbance at 405 nm was measured as an index of acetylation (see text for details). 4 μM His₆-tag proteins were used for controls.