Characterization of the inhibitory mechanism of 1-methyl-4- phenylpyridinium and 4-phenylpyridine analogs in inner membrane preparations

M. R. Gluck, M. J. Krueger, R. R. Ramsay, S. O. Sablin, T. P. Singer*, W. J. Nicklas

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

91 Citations (Scopus)

Abstract

We have investigated the mechanism of the inhibition of membrane-bound NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) and a series of its 4'-alkyl-substituted analogs of increasing hydrophobicity, as well as their neutral, desmethyl congeners. Comparison of hydrophobicity, as measured by partition coefficients, with the IC50 for the inhibition of NADH oxidase activity in mitochondrial inner membrane preparations shows a negative correlation, but the cationic inhibitors are more effective than the neutral analogs with similar hydrophobicity. The presence of 10 μM tetraphenylboron (TPB-) potentiates the inhibitory power of positively charged analogs up to 4'-pentyl-MPP+, while the neutral inhibitors are unaffected by TPB-. Without TPB-, the more hydrophilic analogs give incomplete inhibition, but the inclusion of TPB- permits the attainment of complete inhibition, accompanied by the appearance of sigmoidal titration curves. These data support the hypothesis that MPP+ analogs, like rotenone, are bound at two sites on the enzyme and occupancy of both is required for complete inhibition. TPB-, by forming ion pairs with the cationic analogs, facilitates their equilibration to both sites in membrane preparations. When present in molar excess over the MPP+ analog, TPB- partially reverses the inhibition by decreasing its concentration in the more hydrophilic binding site. The effect of temperature and of pH on the IC50 values for inhibition support the concept of dual binding sites, and the pH dependence of the inhibition reveals the participation of two ionized protein groups in the binding, one of which may be a thiol group.

Original languageEnglish
Pages (from-to)3167-3174
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number5
Publication statusPublished - 1 Jan 1994

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