Differential inhibition of respiration and its dependent H + extrusion by fluorescamine in rat liver mitochondria

Abstract

Rat liver mitochondria were treated with varying amounts of fluorescamine ranging from 0 to 30 nmol/mg of protein. The biochemical activities of the modified mitochondria were analyzed. It was found that the respiration rate in the absence of ADP was not significantly affected, but that the state 3 respiration rate and the accompanying P O ratio decreased as the labeling extent increased. It was also observed that the treatment inhibited the stimulation of respiration induced by the presence of uncouplers. However, the modification has no effect on the discharging rate of proton gradient by uncouplers. The intrinsic activities of NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome oxidase of the inner membrane were not affected by the modification. Measurement of the respiration-dependent proton extrusion (in the presence of valinomycin and potassium ion) with secondary ion movements inhibited, showed that the initial extrusion rate was reduced progressively. However, the observed amounts of proton extruded (ΔH +) and Δμ H + were not affected. The observed reduction of the oxygen consumption rate was much less than that of the proton extrusion rate with increased labeling. These results suggest that some fluorescamine titratable primary amino groups may be involved in the controlling of the proton extrusion process. The implications on the mechanism of coupling in respirationdependent proton extrusion are discussed.