Asymmetric distribution of phosphatidylethanolamine in the endoplasmic reticulum demonstrated using...

At pH 7.4 approximately one third of the phosphatidylethanolamine (PE) of rat liver microsomes is labelled by trinitrobenzenesulphonic acid (TNBS). The same fraction of the PE was labelled, when a fixed concentration of microsomes were incubated with concentrations of TNBS from 1.5 mM to 12 mM, or when the TNBS concentration was fixed at 3.0 mM and the microsomal protein varied between 1.2 and 12.0 mg. Microsomes incubated with TNBS remain closed indicated by retention of mannose-6-phosphatase latency, retention of labelled vesicular contents and by the appearance of the vesicles in the electron microscope. When the microsomal vesicles were opened by alkaline pH or after passage through the French pressure cell the % of PE labelled increased up to 90% of the total. The small % remaining unlabelled may be due to some vesicles remaining closed or to steric hindrance by the relatively bulky label on both phospholipid and protein. Phospholipase C hydrolyses approximately one third of the PE in closed microsomal vesicles. After treatment of microsomes with phospholipase C the % PE available for labelling by TNBS decreased and was inversely proportional to the % PE hydrolysed. These results suggest that the same pool of PE is available for either hydrolysis by phospholipase C or for labelling by TNBS, and that this pool is that of the outer leaflet of the microsomal membrane bilayer.