Untitled

Abstract

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.

An antagonist of ATP‐regulated potassium channels, the guanidine derivative U‐37883A, stimulates the synthesis of phosphatidylserine in rat liver endoplasmic reticulum membranes

“…We have tested the effect of antidiabetic sulfonylureas and U‐37883A on PS synthesis in ER membranes in vitro. All the antidiabetic sulfonylureas applied, glibenclamide, glipizide, HB985, and HB699, even at 100 μM concentration, were unable to affect significantly PS synthesis at 1 mM CaCl 2 concentration, equal to EC 50 value of the PLBE reaction [16]. In contrast, stimulation of PS synthesis by 100 μM U‐37883A was observed at 1 mM CaCl 2 , as well as at 50 μM CaCl 2 (Fig.…”

“…Adult male Wistar rats weighing 150–180 g were killed after being starved for 16 h. Liver ER membranes were prepared according to Rakowska et al [16], resuspended at a protein concentration of 10–20 mg/ml in a buffer containing 250 mM sucrose, 40 mM HEPES, pH 7.4, and stored at −72°C.…”

Positive feedback between ethanolamine‐specific phospholipid base exchange and cytochrome P450 activities in rat liver microsomes. The effect of clofibric acid

“…Such fluctuations create a pool of phospholipid substrate which is used exclusively for the synthesis of PS via a calcium‐dependent serine‐specific PLBE reaction [7]. The pool of newly synthesised PS via this pathway can be subsequently metabolised to PE by PS decarboxylase in mitochondria [42]or via the ethanolamine‐specific PLBE reaction in ER membranes [25]. PS decarboxylase activity determined in the solubilised system (Table 2) when the efficiency of the reaction was not limited by PS transport, was significantly decreased (by 21%) in starved rats in comparison to animals fed ad libitum and only slightly increased after CLA treatment.…”

“…The PLBE activity was measured in an assay system containing 0.25 mg of ER membrane protein, 40 mM HEPES, pH 7.4, 1 mM CaCl 2 and 50 μM [2‐ 14 C]ethanolamine (specific activity 1.5 mCi/mmol), as described previously [25]. Ethanolamine kinase (EC 2.7.1.82) activity was determined according to Weinhold and Rethy [26].…”

Serine base‐exchange in rat liver nuclei

“…The assay of the serine base‐exchange reaction was performed according to the method used for rat liver microsomes [22] with modification. The incubation medium contained 0.5 mg protein of the indicated fraction, 100 mM sucrose, 50 mM HEPES, pH 7.4, 1 mM CaCl 2 , 0.8 mM MgCl 2 and 50 μM L ‐[U‐ 14 C]serine (specific activity 9 mCi/mmol), in a total volume of 0.25 ml.…”