Kinetic studies on the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurospora crassa.

The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques. In pulse and pulse-chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins. The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside-binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics. Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria. Mitochondrial matrix proteins labelled in pulse and pulse-chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor-product type relationship. The results suggest that newly synthesized mitochondrial proteins exist in an extra-mitochondrial pool from which they are imported into the mitochondria.     

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.     

Individual ribosomal protein pool size and turnover rate in Escherichia coli

The pool size of free individual ribosomal proteins present in the cell sap of Escherichia coli has been determined by pulse-labelling a culture before a chase with cold marker.Ribosomes plus ribosomal precursor particles were prepared together and the proteins from this fraction purified. The specific radioactivity of each 30 S and 50 S protein was measured at the time of pulse and at the various times of chase: unequal labelling was already observed at the time of pulse; the kinetics of chase of most 30 S proteins reached a plateau very rapidly; the kinetics of 50 S proteins were more variable. Precise calculation of individual pool size was carried out using the mathematical model described in the Appendix. Almost all ribosomal 30 S proteins have a pool size close to zero. Only four 30 S proteins (S10, S16, S17 and S18) have a sizeable pool (2 to 6% of the corresponding ribosomal protein). Most 50 S proteins have a small pool size (1 to 2%). The free ribosomal proteins of the pool are transferred to mature ribosomes; the half-life of these proteins in the pool has been calculated (0 to 1·4 min). Finally, as judged from the kinetic data, no degradation of ribosome-bound protein was apparent. The significance of the results is discussed with respect to the function of ribosome and the process of ribosome biogenesis.     

Nuclear protein synthesis in a temperature-sensitive Chinese hamster ovary cell line at 40 degrees C

We examined the incorporation of radioactive amino acids into nuclear proteins occurring at nonpermissive conditions in tsH1 Chinese hamster ovary cells with a temperature-sensitive defect in cytosol nonmitochondrial protein synthesis. In leucine-free medium at 40 degrees C, total cellular protein synthesis declined by 1-1.5%/min. As reported by others, preincubating these cells at 42 degrees C for 5-10 min sharply increased the rate of decline. The synthesis of acidic nuclear proteins at nonpermissive conditions (40 degrees C + 300 micrograms/ml cycloheximide) was demonstrated by the nuclear incorporation of 3H-tryptophan. Radioactivity, seen by autoradiography to be associated with these isolated Triton-X-100-washed nuclei, was released after incubating labelled nuclei with proteolytic enzymes. During incubation of tsH1 cells at nonpermissive conditions, pulse/chase experiments were consistent with the loss of some nuclear radioactivity into the cytoplasm. The distribution of cytosol and nuclear proteins, labelled at permissive or nonpermissive conditions and separated by isoelectric focusing, differed quantitatively and probably qualitatively, confirming the residual synthesis of acidic nuclear proteins at 40 degrees C in the presence of cycloheximide. Most newly synthesized acidic proteins retained by nuclei from cells labeled at nonpermissive conditions were present in a transciptionally active chromatin fraction. Although under these conditions the apparent rate of cellular RNA synthesis was unchanged, inhibiting residual cycloheximide-resistant nuclear protein synthesis with puromycin proportionately reduced RNA synthesis. Preincubating cells with 20 micrograms/ml of actinomycin D did not inhibit residual labelling of nuclear proteins; effects on residual nuclear labelling of impaired mitochondrial respiration were ambiguous. Nuclear proteins labelled under nonpermissive conditions probably included some of the 'prompt' heat shock proteins recently described. Provided certain assumptions are correct, our results are consistent with very limited protein synthesis associated with and even intrinsic to cell nuclei. They also suggest that this residual cycloheximide-resistant protein synthesis could be concerned with optimum synthesis or processing of certain nuclear RNA species.     

Inhibition of protein synthesis by glucagon in different rat muscles and protein fractions in vivo and in the perfused rat hemicorpus.

The effect of glucagon on the rate of muscle protein synthesis was examined in vivo and in the isolated perfused rat hemicorpus. An inhibition of protein synthesis in skeletal muscles from overnight-fasted rats at various plasma concentrations of glucagon was demonstrated in vivo. The plantaris muscle (Type II, fibre-rich) was more sensitive than the soleus (Type I, fibre-rich). Myofibrillar and sarcoplasmic proteins were equally sensitive in vivo. However, protein synthesis in mixed protein and in sarcoplasmic and myofibrillar fractions of the heart was unresponsive to glucagon in vivo. In isolated perfused muscle preparations from fed animals, the addition of glucagon also decreased the synthesis of mixed muscle proteins in gastrocnemius (Type I and II fibres) and plantaris, but not in the soleus. The sarcoplasmic and myofibrillar fractions of the plantaris were also equally affected in vitro. Similar results were observed in vitro with 1-day-starved rats, but the changes were less marked.     

Proteome dynamics in complex organisms: using stable isotopes to monitor individual protein turnover rates

The complete definition of changes in a proteome requires information about dynamics and specifically the rate at which the individual proteins are turned over intracellularly. Whilst this can be achieved in single-cell culture using stable isotope precursors, it is more challenging to develop methods for intact animals. In this study, we show how dietary administration of stable isotope-labelled amino acids can obtain information on the relative rates of synthesis and degradation of individual proteins in a proteome. The pattern of stable isotope-labelling in tryptic peptides can be deconstructed to yield a highly reliable measure of the isotope abundance of the precursor pool, a parameter that is often difficult to acquire. We demonstrate this approach using chickens fed a semisynthetic diet containing [(2)H(8)]valine at a calculated relative isotope abundance (RIA) of 0.5. When the labelling pattern of gel-resolved muscle proteins was analyzed, the intracellular precursor isotope abundance was 0.35, consistent with dilution of the amino acid precursor pool with unlabelled amino acids derived from degradation of pre-existing proteins. However, the RIA was stable over an extended labelling window, and permitted calculation of the rates of synthesis and degradation of individual proteins isolated by gel electrophoresis. For the first time, it is feasible to contemplate the analysis of turnover of individual proteins in intact animals.     

The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane

Thiol-affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS-PAGE and 2D-PAGE in conjunction with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devised to label substrate protectable protein with the thiol specific reagent [14C] N-ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol-affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by [14C]NEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross-reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol-affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.     

Synthesis of soluble protein during the cell cycle of the fission yeast Schizosaccharomyces pombe

Many enzymes show a pattern of increase in activity through the cell cycle which is different from the continuous exponential pattern of total protein synthesis. A group of proteins at an intermediate level between single enzymes and total protein, the soluble proteins, was examined to resolve this anomaly. The synthesis of the pH 8.1 soluble proteins of Schizosaccharomyces pombe through the cell cycle was followed by pulse labelling with ³H-leucine in synchronous cultures. The soluble proteins were analysed by electrophoresis on acrylamide gels. Soluble proteins represent 30% of the total proteins of S. pombe and the rates of synthesis showed a continuous increase through the cell cycle. Individual groups of proteins, represented by a single band after electrophoresis, showed a similar continuous increase in synthesis through the cell cycle. Any proteins which may be synthesised discontinuously, such as some enzymes, represent such a small proportion of any one protein group in the electrophoretic separation that their effect was not detectable. These results are different from those described for mammalian cells.     

Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid

The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein‐by‐protein basis. The ability to measure protein synthesis and degradation rates on a proteome‐wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well‐established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L‐leucine was replaced with a stable isotope labelled analogue ([²H₇]L‐leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.     

HeLa cell plasma membranes : IV. Iodination of membrane proteins in synchronized cells

Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The ¹²⁵I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated.     

Sucrose inhibits vascular smooth muscle cell proliferation/migration--protein synthesis is maintained

Abstract.  We have previously shown that the onset of smooth muscle cell proliferation in tissue cultures is triggered independently of serum. The aim of the present study was to investigate if this process was affected by osmotic stress. Vascular explants from 8-month-old male rats were cultured under serum-free conditions using collagen I as migration substrate. Sucrose was added to the culture medium in concentrations varying from 1 to 3% (30–90 mOsM). Cell migration from aortic explants onto the culture dishes was totally inhibited at a sucrose concentration of 90 mOsM. A significant dose-dependent decline in proliferation was shown for cells in explants pulse labelled with ³H-thymidine. In contrast, pulse labelling with ³⁵S-methionine revealed that protein synthesis was maintained in the presence of sucrose. The results indicate that osmotic pressure affects smooth muscle cell protein synthesis, proliferation and migration.     

Short-term effects of ACTH on protein synthesis in adrenal cortex cells of young rats

Two units of ACTH were administered intraperitoneally to young 20 gm-rats which received an intravenous injection of L-leucine-3H thirteen min later. ACTH-injected rats, and control rats which received the isotope alone, were killed at 2-, 10-, 30- and 60-min intervals. Electron microscope autoradiographs in control animals showed strong amino-acid uptake at pulse time (2-min) in the cytoplasm of adrenal zona fasciculata cells. Label was shared between the endoplasmic reticulum (ER) and mitochondria, and a lower but still considerable uptake was seen in nucleoli. At first chase time interval (10-min) cytoplasmic labelling declined, while nuclear and nucleolar labelling increased, both changing little thereafter, and there was a 10-30 min Golgi peak. ACTH administration provoked an overall increase in amino-acid incorporation into cytoplasm, nucleus and nucleolus at pulse time, with no changes in the distribution of the reactions among organelles. Intensification of labelling was most evident over nucleoli, the grain density of which was four-times as high as in controls. The short-term increase in ER and mitochondrial protein synthesis observed after ACTH injections was considered to be consistent with the hypothesis that most newly-formed proteins in these cells may be involved in the regulation of steroidogenesis. The marked increase in nucleolar labelling suggested the presence of proteins involved in RNA synthesis.     

Dengue virus-induced modifications of host cell membranes.

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.     

Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline.

Methods for measurement of rates of collagen synthesis in vivo have thus far been technically difficult and often subject to quite large errors. In this paper a simplified method is described for obtaining synthesis rates of collagen and non-collagen proteins, for tissues of rabbits. This involves an intravenous injection of [3H]proline, administered with a large dose of unlabelled proline, and measurement of the specific radioactivity of proline and hydroxyproline in body tissues up to 3 h later. The specific radioactivity of [3H]proline in plasma and the tissue free pools rises rapidly to a plateau value which is maintained for at least 2 h, when the specific radioactivity of the type I collagen precursors, isolated from the skin, was similar to that of the plasma and tissue-free pool. Furthermore, over this period, the increase in the specific radioactivity of proline in collagen and non-collagen protein was linear with respect to time. These results suggest that the large dose of proline floods the precursor pools for protein synthesis, and that this effect can be maintained for quite long periods of time. Such kinetics greatly simplified the method for obtaining collagen synthesis rates in vivo, which were calculated for lung, heart, skin and skeletal muscle, and shown to be quite rapid, ranging between about 3 and 10%/day. The lung was a particularly metabolically active tissue, with synthesis rates of about 10%/day for collagen and 35%/day for total non-collagen proteins, indicating rapid turnover of both intracellular and extracellular proteins of this tissue.     

Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krüppel

We have identified early embryo proteins related to the segmentation gene Krüppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krüppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krüppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krüppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krüppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krüppel function may involve post-translational modification of proteins.     

Comparison of the turnover patterns of total and individual muscle proteins in normal mice and those with hereditary muscular dystrophy

1. The incorporation of amino acids into hindleg muscle proteins of normal and dystrophic mice was measured (1/2)h to 16 days after administration of the radioactive pulse. 2. Dystrophic animals showed a faster initial rate of incorporation into total and soluble proteins in the first few hours after injection, but the extent of incorporation relative to the size of the amino acid pool was similar in both. There was little difference between the overall degradation rates although this started later in the dystrophic proteins. An initial fast phase of degradation reached a plateau after 3 days whereupon the residual label in the protein remained constant up to 16 days after injection. 3. Analyses of individual radioactive proteins fractionated by polyacrylamide-gel electrophoresis showed that the distribution of label was similar in all the soluble proteins from normal and dystrophic muscle. Time-course experiments revealed that in dystrophic mice the two major soluble proteins of the muscle, creatine kinase and adenylate kinase, initially incorporated 2-3 times more label relative to the initial size of the precursor pool. This label was then lost equally rapidly and the final plateau value was much less than that in normal mice. This initial peak of activity was not observed in normal mice. 4. A group of dehydrogenases showed similar initial turnover patterns in both dystrophic and normal mice but the final plateau value was much higher in the former. 5. The results provide support for the hypothesis that there is no obvious defect in the protein synthetic machinery of dystrophic muscle. However, certain proteins do show anomalous turnover patterns relative to those in normal animals. A single structural gene mutation giving rise to one particularly unstable and readily degradable muscle protein is excluded as the cause of the dystrophy.     

Changes in protein synthesis due to an inflammatory challenge

Rates of protein synthesis in various chick tissues were examined 16 hr after an inflammatory challenge. Protein synthetic rates were calculated from the rate at which [14C]leucine was incorporated into protein and the specific activity of [14C]leucine in the precursor pool. An injection of either Escherichia coli or sheep red blood cells (SRBC) decreased the rate of protein synthesis in the gastrocnemius muscle, and increased the rate in liver, bursa, spleen, and thymus. E. coli, but not SRBC, decreased protein synthesis in the pectoralis muscle. E. coli significantly decreased the aggregation of pectoralis muscle polysomes and increased the aggregation of polysomes in the thymus, bursa, and spleen. E. coli increased the aggregation of free, but not bound, polysomes in liver, suggesting an increase in synthesis of export proteins. SRBC significantly increased polysomal aggregation in bursa and spleen only. A crude preparation of leukocyte endogenous mediator, isolated from peritoneal macrophages, decreased muscle-polysomal aggregation. These studies indicate that tissue-specific changes in protein synthesis occur after a noninfectious inflammatory challenge. These changes may be part of a homeostatic mechanism which supports the immune response.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.