Incorporation of 32P-labelled intermediates into the phospholipids of cell-free preparations of rat brain

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Abstract
In suitably "reinforced" water dispersions of rat brain anaerobic labeling of lipid P from inorganic P32 was increased by addition of coenzyme A (CoA), glutathione, or cytidine triphosphate (CTP) to the incubation medium. Labeling was not significantly affected by the addition of [alpha] -giycerophosphate ([alpha]-GP) (0.001 [image]), glycerol, choline, serine, ethanol-amine or inositol. In phosphorylating mitochondria prepared from isotonic-sucrose homogenates of rat brain, labeling of lipid P from inorganic P32 was increased by addition of CTP, but not by addition of CoA or glutathione. Labeling was decreased by addition of [alpha]-GP (0.005[image]), but not by addition of [beta]-GP or glycerol. Examination of the hydrolysis products of labeled phospholipids showed that the increase in labeling produced by the addition of CoA to the anaerobic system (hypotonic dispersion) was almost exclusively the result of an increase in labeling of GP (presumably from phosphatidic acid) and a hydrolysis product of diphosphoinositide (IP), whereas that produced by addition of CTP to either system was largely due to an increase in the labeling of IP. In both systems P32 from radioactive adenosine triphosphate (ATP32), phosphorylcholine P32Ch) and [alpha]-GP32 was incorporated into lipid P without prior breakdown into inorganic P32. Incorporation of p32Ch into lipid P of both systems was almost entirely into lecithin and was greatly stimulated by addition of CTP, but not by addition of CoA. The requirement for CTP could not be replaced by uridine, inosine or guanosine triphosphates. Incorporation of [alpha] -GP32 into lipid P of both systems was stimulated by addition of CoA, but not by addition of CTP. Radioactivity of hydrolysis products derived from labeled phospholipids was largely in GP and IP. Labeling of GP, but not that of IP, was increased by addition of CoA. Labeling of lipid P from both P32Ch and [alpha]-GP32 was inhibited in the hypotonic dispersion by conditions that inhibited glycolysis, and in mitochondria by conditions that inhibited oxidative phosphorylation. The results are discussed in relation to the biosynthesis of phospholipids in brain.