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Title
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A dipeptide metalloendoprotease substrate completely blocks the response of cells in culture to cholera toxin
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Author
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Abstract
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| Prior exposure (15 min at 37 degrees C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mM N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH2), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH2, an inactive analogue of Cbz-Gly-Phe-NH2. The effect was reversible and dose- (IC50 as low as 0.2 mM depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH2 during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A(1) peptide of CT and NAD(+) activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH2-treated cells or when Cbz-Gly-Phe-NH2 was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gay-Phe-NH2 and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH2 on CT cytotoxicity, Several lines of evidence indicate that a perturbation of intracellular Ca2+ homeostasis by Cbz-Gly-Phe-NH2 is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of I-125-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH2 did not completely suppress the formation of the catalytically active A(1) fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH2 cannot be excluded. |
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Language
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English
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Source (journal)
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Journal of biological chemistry. - Baltimore, Md
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Publication
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Baltimore, Md
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2000
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ISSN
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0021-9258
[print]
1083-351X
[online]
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Volume/pages
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275
:39
(2000)
, p. 30240-30247
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ISI
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000089577900046
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Full text (Publisher's DOI)
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Full text (publisher's version - intranet only)
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