The presence of N-terminal methionine on nascent protein of rat liver and rabbit reticulocytes and its cleavage during polypeptide-chain elongation

1. The size of nascent globin peptides from which the N-terminal methionine residue is cleaved has been investigated by comparing the proportion of N-terminal methionine and valine in short and long chains. Nascent chains were labelled in rabbit reticulocyte lysates, fractionated according to length by chromatography on Sephadex G-50, and analysed by the Edman degradation of selected pooled fractions. It was found that different peptide fractions contained either methionine or valine, but not both, as the N-terminal residue. Methionine was present at the N-terminus of globin chains containing up to approx. 50 amino acids whereas valine was found to be the N-terminal amino acid of longer peptides. 2. In similar experiments with nascent proteins of rat liver, labelled either in vivo or in a cell-free system containing microsomal material and cell sap, evidence was obtained for the presence of methionine at the N-terminus of nascent chains up to approx. 65 amino acid residues long. Thus protein synthesis in liver appears to be initiated also by methionine, but in this case cleavage takes place somewhat later during peptide elongation than in globin synthesis.     

Compartmentation of free amino acids for protein synthesis in rat liver

The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [(3)H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2-3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.     

Studies on flavonoid metabolism. Metabolism of (+)-[14C]catechin in the rat and guinea pig

1. The fate of (+)-[U-(14)C]catechin and (+)-[ring A-(14)C]catechin has been studied in the guinea pig and rat. 2. (+)-[U-(14)C]Catechin was shown to give rise to labelled phenolic acids, labelled phenyl-gamma-valerolactones and (14)CO(2). 3. (+)-[ring A-(14)C]-Catechin did not give rise to labelled phenolic acids, but labelled phenyl-gamma-valerolactones were detected together with a higher proportion of (14)CO(2). 4. Administered [(14)C]delta-(3-hydroxyphenyl)-gamma-valerolactone gave rise to labelled m-hydroxyphenylpropionic acid in the rat whereas administered [(14)C]m-hydroxyphenylpropionic acid gave rise to a compound yielding labelled m-hydroxybenzoic acid on hydrolysis. 5. The distribution of radioactivity in the urine and faeces of (+)-[(14)C]catechin-fed animals is described; a high proportion of residual radioactivity was found in urine that had been exhaustively extracted with diethyl ether.     

Identification of the site of glycation of γ-II-crystallin by (14C)-fructose

Cataract formation in diabetes may be via non-enzymic glycosylation (glycation) of lens proteins due to increased concentrations of sugars present in the lenses of diabetic patients. The objective of this project was to identify the site(s) of glycation of bovine γ-II-crystallin by [¹⁴C]fructose. γ-II-crystallin was isolated from soluble lens nucleus proteins by gel chromatography, followed by ion-exchange chromatography and was then glycated by incubation with [¹⁴C]fructose. Radioactively labelled γ-II-crystallin was cleaved with trypsin. Affinity chromatography of the tryptic peptides gave a single main peak containing the majority of the radioactivity. This indicated that fructose had reacted at a single site on the protein. Amino acid analysis of this peptide showed it to contain only lysine and a trace amount of glycine. By relating the results of the amino acid analysis to the amino acid sequence of γ-II-crystallin, it was concluded that the labelled peptide corresponded to the N-terminal dipeptide. The site of glycation of bovine γ-II-crystallin by fructose was thereby identified as the α-NH₂ group of the N-terminal glycine.     

Compartmentation of free amino acids for protein biosynthesis. Influence of diurnal changes in hepatic amino acid concentrations of the composition of the precursor pool charging aminoacyl-transfer ribonucleic acid.

To investigate further the mechanisms by which amino acids are segregated for protein biosynthesis, the distribution of a pulse of [3H]valine was monitored in hepatic amino acid pools at seven intervals in the diurnal cycle of meal-fed rats. Although each condition was characterized by a unique balance between intracellular and extracellular valine, in every case the specific radioactivity of valyl-tRNA at steady state was higher that that of intracellular valine but below the extracellular value. Further, the specific radioactivity of the valyl-tRNA could be accurately predicted if extracellular and intracellular valine were combined in proportions specified by the transmembrane concentration gradient. These observations not only substantiate our earlier conclusions that the amino acids used for protein synthesis do not originate exclusively from either the intracellular or extracellular pools, but also strengthen our theory that the membrane transport system is the physical basis for such compartmentation. On the basis of these data we present a method for measuring the specific radioactivity of the precursor pool for protein biosynthesis in cases where the actual isolation of the aminoacyl-tRNA is not technically feasible, and also suggest a theoretical basis for interpreting the unequal distribution of both total and [3H]valine between intracellular and extracellular fluids.     

Use of α-aminoadipic acid for the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. l-alpha-Amino[6-(14)C]adipic acid has been prepared from the dl-amino acid by oxidation of the l-isomer with l-amino acid oxidase to alpha-oxo[6-(14)C]adipic acid and by transamination of the latter with l-glutamic acid in an extract of a Cephalosporium sp. prepared by ultrasonic treatment of the mycelium. 2. The optical configuration of small amounts of (14)C-labelled alpha-aminoadipic acid from the mycelium of the Cephalosporium sp. has been determined by treatment with l-amino acid oxidase and measurement of the proportion of radioactivity subsequently retained on a column of a strong cation-exchange resin. 3. alpha-Aminoadipic acid which had been labelled in the mycelium from [1-(14)C]acetate appeared to contain more than 99% of the l-isomer. 4. l-alpha-Amino[(14)C]adipic acid (sodium salt) was taken up much more rapidly than the d-isomer, or alpha-oxo[6-(14)C]adipic acid, by suspensions of washed mycelium of the Cephalosporium sp. in water. The pool of intracellular alpha-aminoadipic acid was expandable. 5. Intracellular products found to be labelled with (14)C from l-alpha-amino[(14)C]adipic acid were delta-aminovaleric acid, saccharopine, lysine, protein, compounds which behaved like penicillin N, cephalosporin C and deacetylcephalosporin C respectively on paper chromatography and electrophoresis, and a peptide whose amino acid residues include alpha-aminoadipic acid, cysteine and valine. 6. l-alpha-Amino[(14)C]adipic acid acted as a precursor of the delta-(d-alpha-aminoadipoyl) side chains of extracellular penicillin N and cephalosporin C. 7. (14)C from d-alpha-amino[(14)C]adipic acid was incorporated into penicillin N and cephalosporin C, but the incorporation was accompanied by a relatively high dilution of specific radioactivity and some l-alpha-amino[(14)C]adipic acid was found in the intracellular pool. 8. These findings are discussed in relation to the origin of the d- configuration of the alpha-aminoadipoyl side chain of the antibiotics.     

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.     

Biosynthesis of peptides containing α-aminoadipic acid and cysteine in extracts of a Cephalosporium sp

1. Three intracellular peptides found in small amount in a Cephalosporium sp. were rapidly labelled when dl-[(14)C]valine was added to a shaken suspension of the organism. More (14)C was incorporated into peptide P3, delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine, than into peptide P2 (containing alpha-aminoadipic acid, cysteine, valine and glycine) or peptide P1 (containing beta-hydroxyvaline in place of the valine in peptide P2). 2. Peptides P3 and P2, but not peptide P1 were formed in a broken-cell system from the Cephalosporium sp. in the presence of delta-(l-alpha-aminoadipyl)-l-cysteine and dl-[(14)C]valine. No synthesis was observed in the presence of delta-(d-alpha-aminoadipyl)-l-cysteine or of dl-alpha-amino[(14)C]adipic acid and l-cysteinyl-l-valine or l-cysteinyl-d-valine. 3. The biosynthesis of these peptides was catalysed by the particulate fraction of the broken-cell system, whereas that of glutathione was catalysed by the supernatant fraction. 4. These results are discussed in relation to penicillin N and cephalosporin C biosynthesis.     

The formation of glycoproteins in tissues of higher plants. Specific labelling with d-[l-14C]glucosamine

1. Radioactivity from d-[l-(14)C]glucosamine is incorporated into ethanol-insoluble compounds of high molecular weight in a number of plant tissues, including roots of corn (Zea mays), callus cells of sycamore (Acer pseudoplatanus), axenic cultures of duckweed (Lemna minor) and germinating seedlings of corn, broad bean (Vicia faba) and barley (Hordeum vulgare). 2. Except in the case of Lemna, where some of the radioactivity was recovered in glucose, hydrolysis of these ethanol-insoluble materials with acid released [(14)C]glucosamine as the major radioactive product. 3. The labelled compounds isolated from Zea roots and the Acer cells are believed to be glycoproteins rather than polysaccharides on the basis of their solubility properties, their charge characteristics and their susceptibility to hydrolysis by 0.5m-potassium hydroxide and by the proteases trypsin and Pronase. Further, radioactive peptides were isolated and purified after Pronase treatment and shown to contain glucosamine as well as a number of amino acids. 4. The experiments therefore indicate that d-[(14)C]glucosamine can be used as a specific precursor of the amino sugar units of plant as well as animal glycoproteins.     

The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1. Rats were infused in vivo with [U-(14)C]glycine for periods of 2-6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [(14)C]glycine and [(14)C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-(14)C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.     

Microsomal components in relation to amino acid incorporation by preparations from the developing rat brain

1. After incorporation of [(14)C]valine in vitro, cerebral microsomes were separated into membrane-bound and free ribosomes by sucrose-density-gradient centrifugation. 2. In preparations from both 4-day-old and adult rats, free and bound ribosomes incorporated [(14)C]valine. Free ribosomes could be found as polysomes, which were highly active. 3. Microsomes labelled with [(14)C]valine in vitro were fractionated after deoxycholate treatment into a preliminary sediment, sedimented at 105000g (5min.), and ribonucleoprotein particles, sedimented at 150000g (70min.), to determine the role of membrane-bound ribosomes. In the adult the ribonucleoprotein particles retained most of the radioactivity, whereas in the young the preliminary sediment was as highly labelled as the ribonucleoprotein particles. 4. The labelled preliminary sediment from young preparations was both ribonuclease- and deoxycholate-resistant, and the nature of this material is discussed in terms of a possible structural component of microsomal membranes.     

A study of the nature of the immediate precursor of the extracellular α-amylase of Bacillus amyloliquefaciens. A reappraisal

1. A defined medium was devised for use in washed-cell experiments with post-exponential-phase cultures of Bacillus amyloliquefaciens. The medium allowed alpha-amylase to be secreted, bacterial concentration to increase and l-[U-(14)C]valine to be incorporated into protein at a linear rate, which was the same as in a post-exponential-phase culture, for up to 6h. 2. Determination of the specific radioactivity of l-[U-(14)C]valine in the medium, the intracellular amino acid pool, the cellular protein and the isolated alpha-amylase, after a 3h incubation of washed cells in the defined medium, showed that at least 76% of the alpha-amylase secreted was synthesized de novo. 3. By isolating the alpha-amylase formed during a 6h incubation in the presence of l-[U-(14)C]valine it was shown that the specific radioactivity of the N-terminal valine, within the limits of experimental error, was the same as that of the total valine residues from the complete alpha-amylase molecule. 4. A consideration of these results in relation to the whole literature on the subject strongly supports the idea that there is no reason to suppose that extracellular alpha-amylase is formed from a high-molecular-weight precursor in B. amyloliquefaciens and closely related organisms with identical characteristics of exoenzyme secretion.     

Source of valine for protein V biosynthesis in a producer of actinomycin C]

The specific activity of 14C-valine in valyl-tRNA formed during incubation of the actinomycin C-producing organism with 14C-valine was constant and lower than that of the whole cell pool. The constancy of the valyl-tRNA was indicative of the presence of a separate compartment for the valine pool used for protein biosynthesis. A lower specific activity of valine in valyl-tRNA as compared to that of the whole cell pool may be indicative of a low rate of valine metabolism in such separate compartment with exogenic 14-valine or a higher concentration of free valine in it as compared to the specific activity of this amino acid at average per cell.     

Ultrasensitive fluorescence-based detection of nascent proteins in gels

The most common method of analysis of proteins synthesized in a cell-free translation system (e.g., nascent proteins) involves the use of radioactive amino acids such as [(35)S]methionine or [(14)C]leucine. We report a sensitive, nonisotopic, fluorescence-based method for the detection of nascent proteins directly in polyacrylamide gels. A fluorescent reporter group is incorporated at the N-terminus of nascent proteins using an Escherichia coli initiator tRNA(fmet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, we find that the protein translational machinery accepts BODIPY-FL, a relatively small fluorophore with a high fluorescent quantum yield, as an N-terminal modification. Under the optimal conditions, fluorescent bands from nanogram levels of in vitro-produced proteins could be detected directly in gels using a conventional UV-transilluminator. Higher sensitivity ( approximately 100-fold) could be obtained using a laser-based fluorescent gel scanner. The major advantages of this approach include elimination of radioactivity and the rapid detection of the protein bands immediately after electrophoresis without any downstream processing. The ability to rapidly synthesize nascent proteins containing an N-terminal tag facilitates many biotechnological applications including functional analysis of gene products, drug discovery, and mutation screening.     

THE NATURE OF THE AMINO ACID POOL USED FOR PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The incorporation into brain slice protein of externally provided [1-¹⁴C]valine was measured at varying levels of valine in the medium, under conditions of constant protein synthesis and equilibration of intracellular valine specific activity. The results indicate that the valine pool used for protein synthesis is not identical to the pool of total free valine. Neither does the incorporation solely occur from an extracellular pool which is in equilibrium with the incubation medium. The data are compatible with a two-site activation model in which aminoacylation of tRNA occurs at both an internal site utilizing amino acid from the intracellular pool and an external (possibly membranous) site converting extracellular valine directly to valyl-tRNA. A good fit to the experimental observations is also provided by a compartmented intracellular valine pool model.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland

1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.     

Metabolism of U14C leucine and U14C valine by adult female Glossina morsitans during pregnancy

After U¹⁴C leucine or U¹⁴C valine injections into haemolymph of adult female Glossina morsitans during late pregnancy, radioactivity was detected in the post-parturient female and its larval offspring in the injected material, lipids, and a range of non-essential amino acids. The level of radioactivity recorded from the third instar larva was higher than that remaining in the injected adult, and the activity was higher in amino acids than in the lipid fraction. Radiometric analysis of oöcyte and intra-uterine progeny 24 hr after haemocoelic administration to females of labelled leucine or valine revealed a pattern of radioactivity coincident with growth characteristics of these young stages. Rate of leucine uptake by the in utero third instar larva was slightly higher than that of valine, and this instar continues feeding even only a few hours before parturition. For both labelled materials, expired carbon dioxide and excreta from remales in early pregnancy showed significantly higher radioactivity than those in late pregnancy. Uric acid is the main nigrogenous waste of leucine and valine metabolism, though small amounts of these amino acids are also lost during excretion, with valine elimination being higher than leucine.     

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells has been studied. When these cells are synchronized for initiation by fluoride treatment and then double labeled with [³⁵S]methionine and either tritiated proline, phenylalanine, or valine for short pulses, the percentage of N-terminal methionine incorporated in the nascent peptides compared to total incorporation is significantly higher than that of the tritiated amino acids tested. The data indicate that methionine is the initiator amino acid for the synthesis of poliovirus-specific proteins.     

The effect of electrical stimulation on the incorporation of l-[U-14C]valine into the protein of chopped tissue from guinea-pig cerebral cortex

1. Chopped tissue from guinea-pig cerebral cortex carried out an energy-dependent incorporation of [(14)C]valine into protein. 2. At all times studied the nuclear fraction of the homogenized tissue accounted for about 25% of the total labelled protein. 3. Electrical stimulation at first increased, but subsequently decreased, the rate of incorporation of [(14)C]valine into protein of the chopped tissue. 4. The initial increase in the incorporation of [(14)C]valine into protein occurred in the nuclear fraction. At later times electrical stimulation decreased the incorporation into all the subcellular fractions, but the relative contribution of the nuclear fraction to the total labelled protein increased. 5. These changes are discussed in relation to the changes in the rates of respiration, glycolysis, high-energy phosphate content and intracellular Na(+) and K(+) concentrations, which were measured under the same experimental conditions as those used to study protein synthesis.