The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

Abstract

1. The distribution of Pi between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J.111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of Pi transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total Pi liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the Pi liberated was retained in the mitochondria. 2. Larger amounts of Pi appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this Pi were identified: (a) a slow efflux of Pi from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg2+, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and Pi and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and Pi, is unaffected by uncoupling agents and is activated by endogenous or added Mg2+. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the Pi liberated was recovered in the suspending medium, unless inhibitors of Pi-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg2+ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of Pi retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the Pi transporter provides the sole pathway for Pi efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg2+ on mersalyl inhibition of the Pi transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in Pi efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A.75, 4788–4792] are erroneous and should be discarded.