An evaluation of N-phenyl-1-naphthylamine as a probe of membrane energy state in Escherichia coli

Abstract

Colicin E1 and the uncoupler of oxidative phosphorylation, trifluoromethoxy-carbonylcyanidephenylhydrazone (FCCP), cause an increase in the fluorescence intensity of N-phenyl-1-naphthylamine bound to whole cells of Escherichia coli. It has been shown elsewhere that this fluorescence increase correlates well with de-energization. Addition of glucose causes a large cyanide-sensitive decrease of intensity, tentatively associated with energization, with the emission spectrum almost returning to the original trace with a peak at 417 nm. These data suggest that there may be a measurable competition between de-energization and energization of the cell membrane, and that the probe fluorescence intensity may be a general indicator of membrane energy level. The conclusions reached about cellular energy level from measurements of the probe fluorescence intensity correlate partly (a, b below, not c) with the energy level assayed physiologically through rates of active transport: (a) FCCP is found to be a poor inhibitor of proline transport if cells are first incubated with glucose, showing either competition between the processes of energization and de-energization or an increase in the envelope permeability barrier to FCCP caused by glucose addition. (b) Cyanide blocks the fluorescence decrease caused by glucose and inhibits proline and serine transport, consistent with the decrease in probe fluorescence intensity indicating an increase in membrane energization. However, (c) it appears that the amplitude of the fluorescence intensity decrease caused by glucose addition in the presence of FCCP and colicin E1 greatly exaggerates the extent of real membrane energization. Glucose added after uncoupler can cause only a small increase, and after colicin, a negligible increase in the proline transport rate, indicating that the magnitude of the fluorescence intensity decrease after glucose addition is not a true measure of membrane energization, but rather seems to amplify this energization greatly. Glucose addition does not cause a decrease in fluorescence intensity in cells treated with EDTA to remove lipopolysaccharide and an apparent barrier to the probe. The rotational relaxation time of the probe in intact cells appears to correlate somewhat better with the cellular energy level than does intensity.