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.
In the present study the relationship between lipid sole pathway for PS formation. Sundler et al. [3] reported that peroxidation, changes in the redox state of membrane and in isolated rat hepatocytes, at physiological concentrations of phosphatidylethanolamine (PE) synthesis via base exchange ethanolamine (25 ~tM), 8-9% of the total PE synthesis could reaction in rat liver microsomes was investigated. It was found be attributed to Ca2+-dependent incorporation of ethanolthat PE synthesis is enhanced in the presence of antioxidants, amine via the BE reaction. The reaction does not result in a butylated hydroxytoluene (BHT), or unsaturated free fatty acids, net increase of the total PE content, but is rather responsible Prooxidants, tert-butyl hydroxyperoxide (BHP), ferrous ions for significant remodelling of preexisting membrane phosphocombined with ascorbate or NADPH (via cytochrome P450-lipids. The most abundant molecular species of PS and PE are dependent proteins), increased the amount of lipid peroxidation known to contain long-chain polyunsaturated fatty acids: araproducts in the membrane, and in consequence inhibited the chidonic (20:4), docosatetraenoic (22:4)and docosahexaenoic reaction. The effect of BHP was fully reversed by reduced (22:6) in the sn-2 position of their glycerol moiety [4]. glutathione and dithiothreitol (DTT), whereas the effect of other compounds could be reversed only by BHT. In contrast, a Furthermore, Ellingson and Seenaiah [5] have documented reversal of the inhibitory effect of cadmium ions on base that stearoyl-polyunsaturated molecular species of PE and exchange activity was observed in the presence of DTT, but PC are preferentially converted to PS by the phospholipid not BHT. Therefore, both the -SH/-S-S-ratio in the membrane, BE reaction in rat liver microsomes. affected by BlIP and cadmium ions, and the lipid hydroxyper-PE, kept in a bilayer configuration by interactions with oxides (rather than aldehydes), generated by ferrous ions and other membrane phospholipid molecules, is able to induce ascorbate or NADPH, are equally responsible for the inactivalocal nonbilayer structures by creating hexagonal phases [6]. tion of the ethanolamine base exchange enzyme in rat liverThis property of PE may be responsible for regulation of the microsomes. This may suggest that the synthesis of PE via the activity of many membranous enzymes, for example cytobase exchange reaction may be considered an element of the chrome P450 [7]. The latter class of enzymes participates in superfine cellular machinery involved in the repair of damage to unsaturated fatty acid chains of phospholipids caused by reactive hydroxylation processes of different hydrophobic molecules, oxygen species under oxidative stress, like fatty and bile acids, phospholipids, steroid hormones and/or xenobiotics [8]. Moreover, the PE hexagonal phase
The procedures for purification and reconstitution of rat brain microsomal membrane protein that causes fusion of liposomes at acidic pH are described. A 1,860-fold purification was achieved, starting from the detergent-solubilized microsomal membranes. The fusion process was assayed spectrofluorimetrically by monitoring the formation of terbium-dipicolinic acid complex (Wilschut, J. et al. 1980. Biochemistry 19:6011-6021) evoked by the protein after mixing of two populations of liposomes. The fusogenic activity of the protein inserted into the membrane of Tb3+-containing vesicles was found to be strongly dependent on phospholipid composition and was higher in vesicles enriched with exogenous phosphatidylserine, phosphatidylglycerol and phosphatidylethanolamine than in those prepared with an excess of phosphatidylcholine. The vesicles enriched in negatively charged phospholipids were bound to Concanavalin A coupled to Sepharose-4B and could be released from this column only in the presence of a high concentration of alpha-methylmannopyranoside and detergent, indicating a glycoprotein nature of the fusogenic protein. Furthermore, these data show that protein inserted into membrane has its oligosaccharide chains exposed to the environment.
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