Presence of Base-Exchange Activity in Rat Brain Nerve Endings: Dependence on Soluble Substrate Concentrations and Effect of Cations

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Abstract
The calcium-dependent, energy-independent incorporations of 14C-labeled bases, choline, ethanolamine, and serine, into their corresponding membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, were compared in microsomes and in subcellular fractions prepared from a lysed crude mitochondrial (P2) pellet of whole rat brain. When activities were measured in the presence of an extracellular (1.25 mM) concentration of Ca2+, recovered activities were highest in the microsomal fraction, although substantial activity remained associated with the P2 homogenate even after repeated washing of the pellet. When this washed P2 homogenate was subfractionated, enrichment of all three exchange activities was obtained only in a fraction that was fivefold enriched over the homogenate and sevenfold enriched over the microsomal fraction in Na+, K+-ATPase, a plasma membrane marker. This strongly suggests that the base-exchange enzymes are normal constituents of synaptosomal plasma membranes. The three exchange activities were measured in synaptosomes prepared from whole rat brain in the presence of various substrate (base) concentrations, and kinetic constants were calculated. The Vmax values for choline, ethanolamine, and serine exchange were, respectively, 1.27 .+-. 0.09, 1.60 .+-. 0.17, and 0.56 .+-. 0.06 nmol/mg of protein/h; the respective Km (apparent) values were 241 .+-. 29, 65 .+-. 18, and 77 .+-. 22 .mu.M. Endogenous levels of the three bases, choline, ethanolamine, and serine, in whole (microwaved) rat brains were 20 .+-. 8, 78 .+-. 28, and 639 .+-. 106 nmol/g, respectively. That ethanolamine and serine incorporations had lower Km values than choline incorporation suggests that these bases are preferentially incorporated into their respective phospholipids. Moreover, that endogenous choline levels are well below those needed for enzyme saturation suggests that the incorporation of choline into phosphatidylcholine via this pathway may be sensitive to transitory increases in free choline concentration (such as occur intrasynaptically after acetylcholine is released and hydrolyzed). Various polyvalent cations (Mg2+, Ba2+, Ni2+, Co2+, and La3+) inhibited synaptosomal base-exchange activity with a rank order of potency similar to their ability to block calcium channels.