The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl‐sn‐glycero‐3‐phosphoinositols (GPI) and diacyl‐sn‐glycero‐3‐phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl‐sn‐glycerophosphoinositols (diacyl‐GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [14C] arachidonoyl groups, diacyl‐GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl‐GPI in brain synaptosomes may be represented by two pools with half‐lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP‐dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A2 and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A2. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1‐acyl‐GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A2 in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl‐GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1‐acyl‐2‐[14C] arachidonoyl‐GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl‐GPI. The arachidonate released from incubation of labeled diacyl‐GPI may be the result of phospholipase A2 action which is not specific for diacyl‐GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl‐GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.

Incubation of synaptosomes together with 1-acyl-2-[14C]arachidonoyl-sn-glycerophosphoinositols (GPI) and sodium deoxycholate yielded diacylglycerols and free arachidonic acid. Diacylglycerol formation is attributed to hydrolysis by the diacyl-GPI-specific phospholipase C (EC 3.1.4.10), and this reaction requires sodium deoxycholate for optimal activity. The free arachidonic acid formed is attributed to hydrolysis of diacyl-GPI by phospholipase A (EC 3.1.1.5). Free fatty acid release was observed during incubation, even in the absence of bile salts, but this process was preferentially stimulated by sodium taurocholate. The release of fatty acids was not specific for diacyl-GPI, as similar release was obtained during incubation with other phosphoglycerides. In the presence of deoxycholate (2 mg/ml), the release of diacylglycerols was maximal at a diacyl-GPI concentration around 1.0 mM. However, the free fatty acid release was linear with respect to the substrate at least up to 1.4 mM. The rate of diacylglycerol release from diacyl-GPI was more rapid in the initial 30 min, whereas the free fatty acid release was linear with time up to 2 h. Under this incubation condition, calcium was found to stimulate both types of hydrolytic action, although the concentration needed to achieve this stimulation was rather high. This type of labeled precursor is potentially useful for studies of the different modes of diacyl-GPI degradation by enzymes in brain subcellular membranes.

The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [3H]guanosine for 1.5-7.5 h; 3-day-old rats were injected with [3H]guanosine and were killed 5-45 min later. [3H]tRNA was extracted, partially purified, and hydrolyzed to yield [3H]guanine and [3H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [3H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [3H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably betweren astrocyte and brain tRNAs. Thus, [3H]1-methylguanine represented up to 35% of the total [3H]methyl guanine radioactivity in astrocyte [3H]tRNA, while it became only negligibly labeled in brain [3H]tRNA. Conversely, brain [3H]tRNA contained more [3H]N2-methylguanine than did astrocyte [3H]tRNA. Approximately equal proportions of [3H]7-methylguanine were found in the [3H]tRNAs of both neural systems. The [3H]methylguanine composition of brain [3H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [3H]methylguanine composition of RPC-5 fractions containing, respectively, tRNAlys and tRNAphe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.

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