Cell-free extracts of the soil amoeba
Acanthamoeba palestinensis have been found to catalyze the incorporation of choline, L-serine and ethanolamine into phospholipids of intracellular membranes in the presence of Ca
2+ and the absence of other exogenous factors. Cell fractions enriched in endoplasmic reticulum and mitochondria contained the highest activity, while plasma membranes demonstrated no activity. Microsomal pellets were used to characterize the enzyme activity. Mg
2+ could not replace Ca
2+, but did not inhibit Ca
2+-dependent activity, while Mn
2+ inhibited activity. Choline incorporation was competitively inhibited by L-serine and less effectively by ethanolamine. L-Serine incorporation was competitively inhibited by ethanolamine and inhibited by choline only at low concentrations. Ethanolamine incorporation was competitively inhibited by L-serine, while choline had no inhibitory effects. The specificities of the competitive inhibition of incorporation of each alcoholic amine indicated that more than one enzyme or enzymatic site was involved and that incorporation occurred
via a direct base exchange mechanism.
Inactivation of incorporation enzymes occurred in an exponential fashion during incubation of microsomal membranes at 29° in the presence of Ca
2+. However, the measured rates of inactivation could not account for the low levels of incorporation experimentally observed with each of the three alcoholic amines. Quantitative analysis of the turnover of radioactive bases of prelabeled phospholipids predicted ongoing loss of the labeled bases from phospholipids that was not due to exchange, as well as the unavailability of a constantly increasing fraction of the labeled bases for such loss and/or exchange.
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