The fluorescence intensity of the lipophilic probe N‐phenyl‐1‐naphthylamine responds to the oxidation‐reduction state of the respiratory chain in everted membrane vesicles of Escherichia coli

Abstract

N-Phenyl-l-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to metabolic changes in everted inner membrane vesicles from this organism. NPN was bound to the vesicles to produce a steady-state level of fluorescence intensity. Addition of substrate or ATP did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of substrate through the respiratory chain, there was an increase in the fluorescence of NPN. Reoxidation of the components of the respiratory chain by the addition of oxygen, ferricyanide, fumarate or nitrate decreased fluorescence to the steady-state level until the oxidant had been completely reduced. The fluorescence changes were insensitive to the state of energization of the membrane. It is proposed that NPN responds to the state of reduction of components of the respiratory chain either directly by reacting with a component of the chain or indirectly through an effect transmitted to the membrane by a change in the conformation of respiratory chain components.

The fluorescence intensity of the lipophilic probe N‐phenyl‐1‐naphthylamine responds to the oxidation‐reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles

“…Addition of KCN prior to substrate resulted in an immediate increase in the fluorescence of NPN when ascorbate was added ( fig.2, curve 2). This was accompanied by an increase in the aerobic steady-state level of reduction of cytochrome o (not shown but see fig.4 of [7]). The inhibitor blocked reoxidation of cytochrome o by H202 and prevented quenching of NPN fluorescence by HzOz.…”

“…It is sensitive to uncouplers, and responds to membrane energization by ATP hydrolysis or substrate oxidation through the respiratory chain. The second component, which can also be demonstrated in inverted inner membrane vesicles, is insensitive to uncouplers but responds to the state of reduction of components of the respiratory chain either directly by reacting with a component of the chain or indirectly through an effect transmitted to the membrane by a change in the conformation of respiratory chain components [7]. Since membrane vesicles contain all of the components of the respiratory chain, i.e.…”