The cytotoxic action of diphtheria toxin and its degradation in intact vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase

Toshiyuki Umata, Yoshinori Moriyama, Masamitsu Futai, Eisuke Mekada

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Abstract

The role of vacuolar-type H+-ATPaSe (V-ATPase) in the cytotoxic action of diphtheria toxin (DT) was studied by using bafilomycin A1, a specific inhibitor of V-ATPase. Studies with acridine orange showed that the acidification of intracellular acidic compartments was inhibited strongly when Vero cells were treated with 500 nM bafilomycin A1, indicating that bafilomycin effectively inhibits V-ATPase when it is added to the culture medium. The toxicity of DT to Vero cells, which was determined by the inhibition of protein synthesis by DT, was inhibited partially by bafilomycin at 10 nM and inhibited completely at 500 nM. Therefore, V-ATPase is involved in the expression of the toxicity of DT. Studies using 125I-labeled DT showed that bafilomycin inhibited the degradation of internalized DT, indicating that V-ATPase is also involved in this step. Subcellular fractionation revealed that 125I-DT accumulated mainly in the endosome fraction, and not in the lysosome fraction, when the cells were incubated with 125I-DT in the presence of bafilomycin. Under the cell fractionation conditions similar to those used for the DT-treated cells, we determined the location of 125I-labeled epidermal growth factor in the degradation pathway. The result suggests that bafilomycin A1 does not inhibit the transport of epidermal growth factor to lysosome.

Original languageEnglish
Pages (from-to)21940-21945
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number35
Publication statusPublished - Dec 15 1990
Externally publishedYes

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Vacuolar Proton-Translocating ATPases
Diphtheria Toxin
Vero Cells
Degradation
Fractionation
Lysosomes
Epidermal Growth Factor
Toxicity
Cell Fractionation
bafilomycin A1
Acridine Orange
Acidification
Endosomes
Culture Media
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

The cytotoxic action of diphtheria toxin and its degradation in intact vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. / Umata, Toshiyuki; Moriyama, Yoshinori; Futai, Masamitsu; Mekada, Eisuke.

In: Journal of Biological Chemistry, Vol. 265, No. 35, 15.12.1990, p. 21940-21945.

Research output: Contribution to journalArticle

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abstract = "The role of vacuolar-type H+-ATPaSe (V-ATPase) in the cytotoxic action of diphtheria toxin (DT) was studied by using bafilomycin A1, a specific inhibitor of V-ATPase. Studies with acridine orange showed that the acidification of intracellular acidic compartments was inhibited strongly when Vero cells were treated with 500 nM bafilomycin A1, indicating that bafilomycin effectively inhibits V-ATPase when it is added to the culture medium. The toxicity of DT to Vero cells, which was determined by the inhibition of protein synthesis by DT, was inhibited partially by bafilomycin at 10 nM and inhibited completely at 500 nM. Therefore, V-ATPase is involved in the expression of the toxicity of DT. Studies using 125I-labeled DT showed that bafilomycin inhibited the degradation of internalized DT, indicating that V-ATPase is also involved in this step. Subcellular fractionation revealed that 125I-DT accumulated mainly in the endosome fraction, and not in the lysosome fraction, when the cells were incubated with 125I-DT in the presence of bafilomycin. Under the cell fractionation conditions similar to those used for the DT-treated cells, we determined the location of 125I-labeled epidermal growth factor in the degradation pathway. The result suggests that bafilomycin A1 does not inhibit the transport of epidermal growth factor to lysosome.",
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