title: Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.
abstract: SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes.
authors.affiliation: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Ying-Hsin
authors.initials: YH
authors.lastname: Hsieh
authors.affiliation: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Ying-Ju
authors.initials: YJ
authors.lastname: Huang
authors.affiliation: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Jin-Shan
authors.initials: JS
authors.lastname: Jin
authors.affiliation: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Liyan
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Hsiuchin
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Chun
authors.initials: C
authors.lastname: Jiang
authors.affiliation: Department of Chemistry, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Binghe
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States. Electronic address: biopct@gsu.edu.
authors.forename: Phang C
authors.initials: PC
authors.lastname: Tai
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D004927:Escherichia coli Infections / Q000382:microbiology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D026901:Membrane Transport Proteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012395:Rose Bengal / Q000494:pharmacology*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014981:Xenopus
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D012395:Rose Bengal; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins
keywords: Channel activity; Oocytes; Proteo-liposomes; Rose Bengal inhibition; SecA ATPase