The Incorporation of Phenylalanine and Uridine in Tetrahymena: A Pressure Study1

SYNOPSIS. The action of pressure was studied on the incorporation of labelled phenylalanine and uridine, and on the synthesis of water-soluble proteins in heat-synchronized Tetrahymena pyriformis. Incorporation of [¹⁴C] phenylalanine and [³H] uridine into TCA insoluble fractions was markedly decreased in pressuretreated Tetrahymena. Moreover, the incorporation was dependent upon the age of the cells. Water-soluble protein synthesis was unaffected. These results appear consistent with the proposal that pressure-induced division-delays occur by inhibiting the accumulation of “division proteins” which are essential for cell division.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Studies of Membrane Formation in Tetrahymena pyriformis. VIII. On the Origin of Membranes Surrounding Food Vacuoles*†

SYNOPSIS. A procedure was devised for the isolation of purified food vacuoles from Tetrahymena pyriformis fed particles of ferric oxide. Phospholipids extracted from vacuolar membranes were more similar in composition to the lipids of microsomes than to lipids of whole cells, cilia or post-microsomal supernatant. Fractionation of cells grown in the presence of [¹⁴C]palmitic acid or [³²P]inorganic phosphate also revealed similarities in the specific radioactivities of microsomes and vacuolar membranes. The data suggested that vacuolar membranes arise from a pool of cytoplasmic membranes.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

Lipid-mediated glycosylation of a white matter membrane glycoprotein with an apparent molecular weight of 24,000.

White matter membrane preparations from pig brain catalyze the transfer of [¹⁴C]mannose from exogenous [¹⁴C]mannosylphosphoryldolichol into an endogenous oligosaccharide lipid. Under the same incubation conditions label is also incorporated into endogenous membrane glycoproteins. The enzymatic labeling of both classes of endogenous acceptors is stimulated by the addition of Ca²⁺. Several enzymatic properties of the mannosyltransferase activity responsible for the transfer of mannose from mannosylphosphoryldolichol into the oligosaccharide lipid intermediate have been examined. The [Man-¹⁴C] oligosaccharide lipid synthesized by this in vitro system has the solubility, hydrolytic and chromatographic characteristics of a pyrophosphate-linked oligosaccharide derivative of dolichol. The free [Man-¹⁴C]oligosaccharide liberated from the carrier lipid by mild acid treatment is estimated to contain 8 glycose units. All of the [¹⁴C]mannosyl units in the [Man-¹⁴C]oligosaccharide derived from exogenous [¹⁴C]mannosylphosphoryldolichol are released as free [¹⁴C]mannose by an α-mannosi-dase. No [¹⁴C]mannose is released during incubation with a β-mannosidase. The presence of an N,N′-diacetylchitobiose unit at the reducing end of the lipid-bound [Man-¹⁴C]oligosaccharide is indicated by its susceptibility to digestion by endo-β-N-acetylglucosaminidase H. Pronase digestion of the enzymatically labeled [Man-¹⁴C]glycoprotein yields a single [Man-¹⁴C]gly-copeptide fraction on Bio-Gel P-6 that appears to be slightly larger than the free [Man-¹⁴C]oligosac-charide released from the carrier lipid by mild acid hydrolysis. The [Man-¹⁴C]glycopeptide is cleaved by endo-β-N-acetylglucosaminidase H, and the neutral [Man-¹⁴C]oligosaccharide product appears to be identical to the product formed when the lipid-bound [Man-¹⁴C]oligosaccharide is degraded by the endoglycosidase. The glycopeptide linkage in the [Man-¹⁴C]glycoprotein is stable to mild alkali treatment. These results are consistent with the dolichol-linked [Man-¹⁴C]oligosaccharide, mannosy-lated via exogenous [¹⁴C]mannosylphosphoryldoiichol, being subsequently transferred en bloc from dolichyl pyrophosphate to asparagine residues in endogenous membrane polypeptide acceptors. SDS-polyacrylamide gel electrophoresis of the [Man-¹⁴C]glycoprotein, labeled when white matter membranes are incubated with [¹⁴C]mannosylphosphoryldolichol. revealed a major labeled polypeptide with an apparent mol wt of 24,000. A minor labeled membrane glycoprotein is also seen, having an apparent mol wt of 105,000.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Mechanism of irreversible inactivation of phenylalanine-4- and tryptophan-5-hydroxylases by [4-36Cl, 2-14C]p-chlorophenylalanine: A revision

Intraperitoneal injection of [4-³⁶Cl, 2-¹⁴C]p-chlorophenylalanine (pCPA) (300 mg/kg) in rats revealed absence of chlorine in pure hepatic phenylalanine hydroxyase, while the carbon label appeared as 1–4 moles/mole of [¹⁴C]tyrosine in the inactivated phenylalanine and cerebral tryptophan-5-hydroxylase. Crystalline muscle aldolase and tyrosine hydroxylase also revealed the presence of [2-¹⁴C]tyrosine from [2-¹⁴C]pCPA without inactivating these enzymes. Injection of L-[(U)-¹⁴C] tyrosine led to its incorporation into the above enzymes, but to a different degree without altering the enzyme activity. Repeated injections ofp-chlorophenylacetic acid had no effect on phenylalanine or tryptophan-hydroxylase. Administration of pCPA did not change the levels of cerebral biopterins. Reexamination of the effect of cycloheximide on reversing enzymic inactivation by pCPA failed to confirm our earlier observation.     

PARTICULATE AND SOLUBILIZED FUCOSYL TRANSFERASES FROM MOUSE BRAIN

The transfer of [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to endogenous and exogenous acceptors by particulate and solubilized preparations from mouse brain is described. Suspensions of brain microsomes incorporated [¹⁴C]fucose into a heterogenous group of glycoprotein products, which have a distribution on gel electrophoresis similar to those synthesized in vivo. Fucosyl transferase, extracted from brain microsomes by Triton X-100, transferred [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to terminal galactose residues exposed by mild acid hydrolysis of porcine plasma glycoprotein. Comparison of the specific activities of the solubilized fucosyl transferase from a number of organs showed that, in the presence of the exogenous acceptor which was used, the transferase of brain was more active than the transferases from all other organs tested, with the exception of kidney. Examination of subcellular fractions of brain, with endogenous and exogenous acceptors, showed that activity was limited to fractions containing microsomal membranes, whereas synaptosomal and other fractions were virtually inactive.     

Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse-labeling experiments indicated that [¹⁴C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t₅ (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [¹⁴C]phenylalanine was observed when the isolated forespores at t₅ were incubated with the corresponding mother-cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t₅ was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the ¹⁴C-labeled cytoplasmic protein had a molecular weight of about 20,000, and that a protein having the same molecular weight was present in the t₅ forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the ¹⁴C-labeled membranous-soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.     

A RELATIONSHIP OF N-ACETYLASPARTATE BIOSYNTHESIS TO NEURONAL PROTEIN SYNTHESIS12

A detailed time study of the incorporation of label from sodium-[1-¹⁴C]acetate, [1-¹⁴C]ethanol, and [2-¹⁴C]glucose into the aspartyl moiety of N-acetylaspartic acid (NAA) was conducted. As expected the specific activity of aspartate increased rapidly with time and peaked within 15-20 min after which it fell sharply; but significantly, that of the aspartyl moiety of NAA rose very slowly even after the specific activity of aspartate had fallen to less than 1 per cent of the peak values. A rat brain microsomal free supernatant preparation was shown enzymatically to incorporate label from sodium-[1-¹⁴C]acetate into the t-RNA fraction from which was isolated N-[1-¹⁴C]acetylaspartic acid. From these observations we were inclined to speculate that NAA-t-RNA may serve as an initiator of neuronal protein synthesis.     

Some effects of aluminium on rat brain protein synthesis

1. Infant rats and rabbits received intraperitonal aluminium (Al) chloride (5, 10 or 20 mg Al/kg body weight) every third day from one to four weeks of age.2. When the polysomal fraction was tested in a protein synthesizing system, a significant increase in the incorporation of [¹⁴C] leucine, [¹⁴C] phenylalanine, or [³⁵S] methionine into proteins in vitro was observed at the higher doses in rats but not rabbits.3. The incorporation of [³⁵S]methionine into brain ferritin was measured using polysomal mRNA or mRNA “stored” in the ribonucleoprotein (RNP) particle fraction.4. The results suggest that Al exposure causes the mobilization of ferritin mRNA from the latter fraction to the polysomal fraction for increased ferritin synthesis.     

The effects of individual amino acids on the incorporation of labelled amino acids into proteins by brain homogenates

Abstract— The effects of phenylalanine and other amino acids on incorporation of several different ¹⁴C-labelled amino acids into cerebral protein were studied in brain homogenates. Excess of some amino acids had a varied effect with different ¹⁴C-labelled amino acids. Of the unlabelled-labelled amino acid combinations tested the maximal inhibition was obtained with the following: (1) phenylalanine, which inhibited the incorporation of [¹⁴C]tyrosine, and (2) leucine, which inhibited incorporation of [¹⁴C]isoleucine. In both cases the inhibition occurred principally in proteins that were recovered in the 800 g and 13,000 g sediments. Only a small degree of inhibition occurred in proteins that sedimented at 100,000 g, and no inhibition occurred in proteins of the 100,000 g supernatant.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

Cell-free biosynthesis of arphamenine A

Arphamenine A was synthesized in a cell-free system obtained from the arphamenine-producing strain, Chromobacterium violaceum BMG361-CF4. L-[14C]-phenylalanine was converted to beta-phenylpyruvic acid by phenylalanine amino-transferase obtained from the 10,000 x g supernatant (S10 fraction). [14C]-Benzylmalic acid was synthesized from beta-phenylpyruvic acid with [14C]-acetyl-CoA in the S10 fraction. [14C]-Benzylsuccinic acid was formed from beta-phenylpyruvic acid with [14C]-acetyl-CoA and ATP in this fraction, as was [14C]-arphamenine A from benzylsuccinic acid and L-[14C]-arginine. Thus, the pathway of arphamenine A biosynthesis was confirmed by the cell-free biosynthesis of this antibiotic.     

Effect of Chlorpromazine on Active Transport of Amino Acids in Tetrahymena*

SYNOPSIS. Low concentrations of chlorpromazine (～0.01 mM) inhibit growth and nucleic acid synthesis in the ciliate Tetrahymena pyriformis. Brief exposure of the cells to, e.g. 0.018 mM chlorpromazine, had very little effect on ¹⁴CO₂ production or on label incorporation into glycogen from [1-¹⁴C]glucetate, [6–14C]glucose, or [1-¹⁴C]leucine, but 17-h exposure of stationary phase cultures to this drug caused marked alterations in metabolism, including an almost complete loss of ability to decarboxylate L-[1-¹⁴C]leucine and L-[1-¹⁴C]tyrosine. It was shown that loss of ability to decarboxylate these amino acids results from loss of ability to transport them.     

EVIDENCE FOR THE BIOSYNTHESIS OF MANNOSYLPHOSPHORYLDOLICHOL AND N-ACETYLGLUCOSAMINYLPYROPHOSPHORYLDOLICHOL BY AN AXOLEMMA-ENRICHED MEMBRANE PREPARATION FROM BOVINE WHITE MATTER

An axolemma-enriched membrane fraction prepared by an improved procedure from bovine white matter catalyzes the enzymatic transfer of [¹⁴C]mannose and N-acetyl[¹⁴C]glucosamine from their nucleotide derivatives into a mannolipid and an N-acetylglucosaminyl lipid in the presence of exogenous dolichyl monophosphate. The labeled glycolipid products have the chemical and chromatographic characteristics of mannosylphosphoryldolichol and N-acetylglucosaminylpyrophosphoryldolichol. The initial rates of synthesis of the glycolipids by the axolemma-enriched membrane fraction have been compared with the initial rates of glycolipid formation catalyzed by a microsomal preparation and myelin in the presence or absence of dolichyl monophosphate. Essentially no glycolipid synthesis was observed when either GDP-[¹⁴C]mannose or UDP-N-acetyl[¹⁴C]glucosamine were incubated with myelin in the presence or absence of exogenous dolichyl monophosphate. A comparison of the initial rates of synthesis of the glycolipids using endogenous acceptor lipid revealed that the rate of formation of mannolipid was 7 times faster for the microsomal membranes than the axolemma-enriched membranes. In the presence of an amount of dolichyl monophosphate approaching saturation the initial rate of glycolipid synthesis was markedly enhanced for both membrane preparations. However, due to a more dramatic enhancement in the axolemma-enriched membranes the initial rate of mannolipid synthesis was only approx. 2.5 times greater in the microsomal membranes. A similar observation was made when the initial rates of N-acetylglucosaminyl lipid synthesis were compared for axolemma-enriched and microsomal preparations in the presence and absence of exogenous dolichyl monophosphate. These studies indicate that the axolemma-enriched membranes have a relatively lower content of dolichyl monophosphate than the microsomal membranes although the difference in the amount of mannosyltransferase is only two to three-fold lower. The presence of a sugar nucleotide pyrophosphatase activity capable of degrading GDP-mannose and UDP-N-acetylglucosamine has also been demonstrated in the axolemma-enriched membrane fraction.     

Protein and Ribonucleic Acid Synthesis by Plant Mitochondrial Systems

The incorporation of ¹⁴C-labelled phenylalanine into proteins of the mitochondrial systems obtained from 48-h germinating seeds of Vigna sinensis (L.) Savi can be stimulated by polyuridylic acid [poly (U)] and depressed by rifampicin, which is, however, ineffective if poly (U) is allowed to interact with the incorporating system before the antibiotic has access to it. A system consisting of a mitochondrial S-100 fraction and ribosomes from the same source with other cofactors can bring about polymerization of phenylalanine. The incorporation of ¹⁴C-labelled uracil into RNA by the plant mitochondria is greatly dependent on the exogenous addition of adenine, guanine, cytosine and also on 5-phosphoribosyl-l-pyrophosphate (5-PRPP). It is greatly suppressed by rifampicin and ethidium bromide.     

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Microsomal preparations from rat adipose tissue catalyse the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to an endogenous acceptor forming a [¹⁴C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [¹⁴C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K_m and V_max. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [¹⁴C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

     

Polyprenol phosphates and mannosyl transferases in Phaseolus aureus

The transfer of mannose from GDP[¹⁴C]mannose to lipid and to insoluble polymer by a particulate preparation of Phaseolus aureus has been investigated. The evidence favours the lipid being a prenol phosphate mannose. Of a range of prenol phosphates tried, betulaprenol phosphate was the most effective exogenous acceptor of mannose. Most of the insoluble [¹⁴C]polymer formed was glycoprotein in nature although small quantities of ¹⁴C were associated with glucomannan and galactoglucomannan fractions. Time studies failed to reveal a typical precursor-product relationship between the lipid and polymer fractions but on incubation of [¹⁴C]mannolipid with the particulate fraction a small transfer (0·5–0·7%) of [¹⁴C] to polymer was detected. p-Hydroxymercuribenzoate inhibited (by 90%) the transfer of [¹⁴C] from GDP[¹⁴C]-mannoseto polymer and simultaneously increased (3-fold) the [¹⁴C] recovered in the lipid fraction. The effect was nullified by mercaptoethanol. Attempts to solubilize the transfer system were only partially successful. The formation of a chromatographically identical mannolipid was demonstrated in particulate fractions of Codium fragile and tomato roots.     

Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study

Abstract— A technique for the isolation of pure neuronal perikarya and intact glial cells from cerebral cortex has been developed for routine use. The yield of neuronal perikarya and glial cells was greater from highly immature (5–10 days) rat cerebral cortex than from the cortex of older rats (18–43 days). The perikarya/glia yield ratio decreased with age indicating that, as the glial population matured, the procedure succeeded in isolating a gradually smaller proportion of the existing neurons. The perikarya/glia ratio was highest for the 5-day-old cortex in which no mature glial cells could be identified. After a 10-min pulse in vivo of intrathecally injected [¹⁴C]phenylalanine, the specific radioactivity of the neuronal proteins was higher than that of the glial proteins in the 5-, 10- and 18-day-old rat but was lower in the 43-day-old rat. The values for absolute specific radioactivity of the ¹⁴C-labelled proteins in both cell types were greater, the younger the brain. The ¹⁴C-labelling of neuronal and glial proteins in the 18-day-old rat was assessed in vivo as a function of time by determining the incorporation of [¹⁴C]phenylalanine into such proteins at 5, 10, 20 and 45 min after administration of the amino acid. The rate of incorporation of [¹⁴C]phenylalanine into the glial cells was faster than into the neurons since higher specific radioactivities of the glial proteins could be achieved at earlier times. Also, a biphasic pattern of ¹⁴C-labelling of the glial proteins was noted, suggesting, perhaps, a sequential involvement of the oligodendrocytes and astrocytes. Homogenates of prelabelled neuronal perikarya were fractionated into the nuclear, mitochondrial microsomal and soluble cell sap fractions. In the 18-day-old cerebral cortex, the proteins of the microsomal fraction exhibited the highest specific radioactivity at the end of 10 min, whereas by 20 min proteins of the mitochondrial fraction were most highly labelled. The specific radioactivity of the nuclear proteins increased over the entire 45-min experimental period. On the contrary, the proteins of the soluble cell sap, in which the specific radioactivity was at all times by far the lowest, were maximally labelled by 5 min. Examination of the labelling of the neuronal subcellular fractions as a function of age revealed that at 10 min after administration of [¹⁴C]phenylalanine, the specific radioactivities of all ¹⁴C-labelled proteins were highest in the youngest (5-day-old) neurons. The proteins of the microsomal fraction were most rapidly labelled at all ages. During this interval the proteins of the soluble cell sap were only moderately labelled in the 5-day-old neurons and were totally unlabelled in the 43-day-old neurons, indicating age-dependent differences in the rate of utilization of the amino acid precursor by the neurons.