MEMBRANE INTEGRITY AND PHOSPHOLIPID MOVEMENT INFLUENCE THE BASE-EXCHANGE REACTION IN RAT-LIVER MICROSOMES

Properties of Ca2+ -stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the react ion were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent Km values for either aminoalcohol, maxi mal velocities of the reaction were significantly affected by Ca2+ con centration. No competition between these two soluble substrates used a t equimolar concentrations close to their Km values was observed, sugg esting the existence of two distinct phospholipid base exchange activi ties. The enzyme utilizing the electrically neutral serine was not sen sitive to changes of membrane potential evoked by valinomycin in the p resence of KCL. On the other hand, when positively charged ethanolamin e served as a substrate, the enzyme activity was inhibited by 140 mM K CI and this effect was reversed by valinomycin. The rates of inhibitio n of phospholipid base exchange reactions by various thiol group modif ying reagents were also found to differ. Cd2+ and lipophylic p-chlorom ercuribenzoic acid at micromolar concentrations were most effective. I t can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membran e substrates. However, the influence of the -SH group modifying reagen ts on the protein-facilitated phospholipid motion across endoplasmic r eticulum membranes can not be excluded, since an integral protein-faci litated transverse movement of phospholipids within the membrane bilay er and Ca2+-mediated changes in configuration of the phospholipid pola r head groups seem to be a regulatory step of the reaction. Indeed, wh en the membrane integrity was disordered by detergents or an organic s olvent, the reaction was inhibited, although not due to the transport of its water-soluble substrates is affected, but due to modulation of physical state of the membrane bilayer and, in consequence, the access ibility of phospholipid molecules.