期刊
ANALYTICAL BIOCHEMISTRY
卷 398, 期 2, 页码 203-211出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.12.020
关键词
Flow cytometry; BCECF; Fluorescence; High throughput; Chemical library; Yeast; Vacuoles; pH; V-ATPase; Pumps; V-ATPase inhibitors; Disulfiram; Concanamycin A
资金
- National Science Foundation (NSF) [MCB-0728833]
- National Institutes of Health (NIH) [1U54MH084690, CA118100]
Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise. V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC50 = 26 mu M), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved. (C) 2009 Elsevier Inc. All rights reserved.
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