Reaction of amino-phospholipids of the inner mitochondrial membrane with fluorodinitrobenzene and trinitrobenzenesulfonate.
Mitoplasts from rat liver mitochondria and ETPH particles from beef heart mitochondria were reacted with TNBS and FDNB in sucrose-mannitol-bicarbonate buffer pH 8.2 (BUFFER A) and in sodium chloride-bicarbonate buffer pH 8.5 (buffer B). Mitoplasts and ETPH particles are more stable in buffer A and very little hydrolysis of phospholipids occurs during the reaction period. In this buffer TNBS reacts to a lesser extent with phosphatidylethanolamine (PE) than does FDNB. The data suggests that with mitoplasts 65% of the total PE is localized on the outer surface of the membrane. With mitoplasts the labeling of membrane proteins is much more extensive with FDNB and suggests that 66% of the proteins are localized on the inner membrane surface. Thus a dual type of asymmetry occurs in the mitoplast membrane with more PE localized on the outer surface and more protein localized on the inner surface. In buffer B, extensive degradation of the dinitrophenylated and trinitrophenylated PE and LPE occurs to yield DNP-GPE and TNP-GPE respectively. DNP-GPE and TNP-GPE are degraded by a phosphodiesterase to DNP-ethanolamine and TNP-ethanolamine. When ETPH particles are labeled with TNBS and FDNB, washed, and incubated in buffer A and buffer B, a resynthesis of TNP-PE and DNP-PE occurs in buffer A by acylation of TNP-LPE whereas DNP-PE continues to be formed, primarily from DNP-GPE. These studies provide evidence for an asymmetric arrangement of PE in the inner mitochondrial membrane and demonstrate the presence of membrane-bound phospholipases which act on dinitrophenylated and trinitrophenylated amino-phospholipids. A membrane bound phosphodiesterase is also present which degrades dinitrophenylated or trinitrophenylated GPE. The degradative reactions prevail in bicarbonate-NaCl buffer B whereas acylation reactions prevail in sucrose-mannitol buffer A.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004139:Dinitrofluorobenzene / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D008931:Mitochondria, Muscle / Q000378:metabolism; D009206:Myocardium; D009578:Nitrobenzenes / Q000378:metabolism*; D009928:Organ Specificity; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D014302:Trinitrobenzenesulfonic Acid / Q000378:metabolism*
D009578:Nitrobenzenes; D010743:Phospholipids; D014302:Trinitrobenzenesulfonic Acid; D004139:Dinitrofluorobenzene