Membrane integrity and phospholipid movement influence the base exchange reaction in rat liver microsomes.
Properties of Ca(2+)-stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the reaction were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent K(m) values for either aminoalcohol, maximal velocities of the reaction were significantly affected by Ca2+ concentration. No competition between these two soluble substrates used at equimolar concentrations close to their K(m) values was observed, suggesting the existence of two distinct phospholipid base exchange activities. The enzyme utilizing the electrically neutral serine was not sensitive to changes of membrane potential evoked by valinomycin in the presence of KCl. On the other hand, when positively charged ethanolamine served as a substrate, the enzyme activity was inhibited by 140 mM KCl and this effect was reversed by valinomycin. The rates of inhibition of phospholipid base exchange reactions by various thiol group modifying reagents were also found to differ. Cd2+ and lipophylic p-chloromercuribenzoic acid at micromolar concentrations were most effective. It can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membrane substrates. However, the influence of the -SH group modifying reagents on the protein-facilitated phospholipid motion across endoplasmic reticulum membranes can not be excluded, since an integral protein-mediated transverse movement of phospholipids within the membrane bilayer and Ca(2+)-mediated changes in configuration of the phospholipid polar head groups seem to be a regulatory step of the reaction. Indeed, when the membrane integrity was disordered by detergents or an organic solvent, 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 accessibility of phospholipid molecules.
Rakowska
M
M
Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Jasińska
R
R
Lenart
J
J
Komańska
I
I
Makowski
P
P
Dygas
A
A
Pikula
S
S
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry
D010714:Phosphatidylethanolamines; D013439:Sulfhydryl Reagents; D012694:Serine; D002118:Calcium