Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl- symport.
We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53-59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomyces and Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30-120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.
Ohkuma
S
S
Laboratory of Biochemistry, Department of Molecular and Cell Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa, Ishikawa 920-0934, Japan. ohkuma@kenroku.kanazawa-u.ac.jp
Sato
T
T
Okamoto
M
M
Matsuya
H
H
Arai
K
K
Kataoka
T
T
Nagai
K
K
Wasserman
H H
HH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017920:Antiporters / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D011353:Prodigiosin / Q000031:analogs & derivatives / Q000494:pharmacology*; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014475:Uncoupling Agents / Q000494:pharmacology*; D025262:Vacuolar Proton-Translocating ATPases
D017920:Antiporters; D002712:Chlorides; D011510:Proteolipids; D014475:Uncoupling Agents; C114260:hydrogen-chloride symporter; C098402:metacycloprodigiosin; C020485:proteoliposomes; C077331:prodigiosin 25C; D006859:Hydrogen; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D011353:Prodigiosin