PHOSPHOLIPID TOPOLOGY OF THE INNER MITOCHONDRIAL-MEMBRANE OF RAT-LIVER

Mitoplasts from rat liver mitochondria were reacted with trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB), isethionyl acetimidate (IA), methyl acetimidate (MA) and methyl picolinimidate (MP) at 4 and 21 °C in order to elucidate the topology of phosphatidylethanolamine (PE). TNBS was found to penetrate the mitoplast membrane rapidly at 21 °C and more slowly at 4 °C. Penetration was not influenced by valinomycin but was inhibited by 4-acetamido-4‘-isothiocyanostilbene-2,2‘-disulfonic acid (SITS). Kinetic analysis of the TNBS reaction was required to elucidate the asymmetric arrangement of PE. The fast-reacting PE was considered to be located on the outer membrane surface. The slow-reacting PE may be located on the inner membrane surface. The fraction of PE which did not react with FDNB or MA is believed to be masked by tight binding to membrane proteins. The results suggest that three different pools of PE occur in the membrane, but their location cannot be firmly defined. Our interpretation is that 30-40% of the total membrane PE is located on the outer membrane surface, 45-60% is located on the inner membrane surface and 10-15% is buried within the membrane. IA labeled only 13% of the total PE at 4 °C at saturation of available sites. Perturbing the membrane by sonication, by treatment with lubrol and by freeze-thawing has little effect on the extent of labeling of PE by IA. Therefore, a large number of PE molecules are not available to react with IA due to either charge repulsion and/or tight binding to protein. MA, a penetrating probe, at 24 mM concentration labeled about 90% of the total PE molecules in mitoplasts in contrast to 20 mM MP which labeled only 50% of the PE molecules. The labeling of PE by MP was not influenced by disrupting the mitoplasts with lubrol. TNBS, FDNB, IA and MA have differential effects on the activities of glutamate dehydrogenase (GDH) (matrix enzyme) and ATPase (membrane enzyme). At saturating levels of probe, 20 mM MA at 4 °C inhibited GDH by 20% and inhibited ATPase by 16% whereas 20 mM IA inhibited GDH by only 8% and gave no inhibition of ATPase. TNBS at 2 mM concentration at 4 °C inhibited ATPase by 40% and inhibited GDH by 96%. FDNB at 2 mM concentration at 4 °C inhibited ATPase by 95% and inhibited GDH by 96%. © 1979, American Chemical Society. All rights reserved.