Effect of phenylalanine on protein synthesis in the developing rat brain

1. Inhibition of the rate of incorporation of [(35)S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [(35)S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [(35)S]methionine and [(14)C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [(14)C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [(14)C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.     

The origin of the sulfur atom of thiamin

The incorporation of the sulfur atom of 35S-labeled amino acids into thiamin in Escherichia coli and Saccharomyces cerevisiae was studied. The specific radioactivity of the S atoms was incorporated at similar levels into thiamin and cysteine residues in cell proteins. However, the specific radioactivity of the S atoms from [35S]methionine was not incorporated into thiamin but into methionine residues in cell proteins. Thus, the origin of the S atom of thiamin was established as being the S atom of cysteine. No activity from [U-14C]cysteine was recovered in thiamin, proving that the carbon skeleton of this amino acid was not utilized in synthesizing the thiazole moiety of thiamin.     

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [³⁵S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [¹⁴C]glycine into the free pool was observed. The corresponding incorporation of ³⁵S and ¹⁴C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

Relative significance of exogenous and de novo synthesized fatty acids in the formation of rumen microbial lipids in vitro

Mixed rumen microorganisms (MRM) or suspensions of rumen Holotrich protozoa obtained from a sheep were incubated anaerobically with [1-(14)C]linoleic acid, [U-(14)C]glucose, or [1-(14)C]acetate. With MRM, the total amount of fatty acids present did not change after incubation. An increase in fatty acids esterified into sterolesters (SE) and polar lipids at the expense of free fatty acids was observed. This effect was intensified by the addition of fermentable carbohydrate to the incubations. Radioactivity from [1-(14)C]linoleic acid was incorporated into SE and polar lipids with both MRM and Holotrich protozoa. With MRM the order of incorporation of radioactivity was as follows: SE > phosphatidylethanolamine > phosphatidylcholine. With Holotrich protozoa, the order of incorporation was phosphatidylcholine > phosphatidylethanolamine > SE. With MRM the radioactivity remaining in the free fatty acids and that incorporated into SE was mainly associated with saturated fatty acids, but a considerable part of the radioactivity in the polar lipids was associated with dienoic fatty acids. This effect of hydrogenation prior to incorporation was also noted with Holotrich protozoa but to a much lesser extent. Small amounts of radioactivity from [U-(14)C]glucose and [1-(14)C]acetate were incorporated into rumen microbial lipids. With protozoa incubated with [U-(14)C]glucose, the major part of incorporated radioactivity was present in the glycerol moiety of the lipids. From the amounts of lipid classes present, their radioactivity, and fatty acid composition, estimates were made of the amounts of higher fatty acids directly incorporated into microbial lipids and the amounts synthesized de novo from glucose or acetate. It is concluded that the amounts directly incorporated may be greater than the amounts synthesized de novo.     

Incorporation of methylsulfonylmethane sulfur into guinea pig serum proteins

Methionine, an essential amino acid, and cysteine are the major sulfur-containing amino acids in the body and both are thought to be synthesized predominantly in plants and micro-organisms. Methylsulfonylmethane (MSM) is a natural constituent of the environment in which it is found in plants, in milk and urine of both bovines and humans, is a normal oxidation product of dimethyl sulfoxide (DMSO) also in the natural environment and may be part of the natural global sulfur cycle. To determine whether sulfur from methylsulfonylmethane (MSM) is incorporated into sulfur amino acids, I fed 35S-MSM to guinea pigs. 35S was incorporated into peptidyl methionine and cysteine of guinea pig serum proteins. The specific activity of 35S-methionine was 30% greater than for 35S-cysteine, suggesting a precursor-product relationship. Total specific activity of serum proteins was increased by only 30% with a 100% increase of administered 35S-MSM, suggesting a limiting step in synthesis. Approximately 1% of the radioactivity was recovered in serum proteins, none in the feces and most was excreted in the urine. Microorganisms of intestinal lumen may be responsible for the incorporation of the 35S of MSM into sulfur amino acids. MSM may provide a source of sulfur for essential animal methionine by mechanisms not yet elucidated in either animals or micro-organisms.     

Biosynthesis of thiazole from thiamine in Escherichia coli]

The incorporation into the thiazole moiety of thiamine of several labeled compounds has been studied on short time incubations of washed-cells suspensions. No incorporation of radioactivity from [G-14C] methionine was found in a mutant auxotrophic for methionine. No radioactivity was incorporated from [U-14C] aspartate or from [U-14C] serine. The incorporation of 35S from sulphate was lowered by cysteine or glutathione but was unaffected by methionine or homocystine. Although the synthesis of thiazole is dependent on methionine, neither the sulphur atom nor the carbon chain of thiazole originate from methonine in E. coli. No carbon originates from cysteine which is the likely direct donor of sulphur.     

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.     

The formation of sulphur-containing amino acids in germinating seeds of rape (Brassica napus L.)

Sodium [(35)S]sulphide was fed to batches of germinating rapeseed, in some instances with the addition of unlabelled cysteine. Both the total radioactivity and specific radioactivity of the free sulphur-containing amino acids were examined. Cysteine and homocysteine were rapidly labelled; label subsequently appeared in cystathionine and methionine. The results obtained indicated that both the sulphydration and trans-sulphuration pathways were operating. This conclusion was reinforced by the results of experiments in which batches of rapeseed were incubated with l-[(14)C]homoserine. These showed the formation of labelled homocysteine, cystathione and methionine. It was thought the trans-sulphuration pathway was making the greater contribution to the biosynthesis of methionine in germinating rapeseed.     

Role of Methionine in Cephalosporin Synthesis

Methionine has an almost unique stimulatory effect on biosynthesis of cephalosporins (by Cephalosporium acremonium). No other sulfur-containing compound tested, except dl-methionine-dl-sulfoxide, replaced methionine. dl-Methionine stimulated the synthesis of cephalosporins when added after the growth phase. The utilization of inorganic sulfate was repressed by methionine. Experiments with l-methionine-S(35) showed that essentially all the sulfur in the cephalosporins was derived from methionine. Sulfur-labeled compounds found in the soluble pool from cells grown with methionine-S(35) were methionine, homocysteine, taurine, cystathionine, cysteic acid, glutathionine, and cysteine. dl-Serine-3-C(14) was incorporated into the antibiotics, and its utilization was stimulated by methionine. l-Cysteine had a sparing effect on the incorporation of methionine-S(35) and serine-C(14) into the antibiotics. The data are consistent with the hypothesis that a cystathionine-mediated pathway is operative in the transfer of sulfur between methionine and cysteine.     

Relationship between intracellular amino acids and protein synthesis in the extensor digitorum longus muscle of rats

1. The incorporation into protein, and the accumulation into the free amino acid pools, of radioactive l-leucine and glycine was studied in rat extensor digitorum longus muscle. 2. The tissue was incubated first with (14)C-labelled and then with (3)H-labelled amino acid. 3. The experimental results were consistent with a model based on the premise that the amino acids in protein were incorporated directly from the extracellular pool.     

Method for Measuring Microbial Growth in Rumen Content

Radioactive sodium sulfide was used to label the sulfide pool of rumen contents in vitro. Microbial protein synthesis was calculated from the size and rate of dilution of label in the sulfide pool, and from the radioactivity incorporated into protein together with a conversion factor specifying the nitrogen-sulfur ratio determined for microbial protein. The microbial cell yield, calculated on the basis of the adenosine triphosphate (ATP) available from fermentation, was 13 to 14 g (dry weight) per mole of ATP, which is in good agreement with the values obtained for pure cultures of bacteria. Calculation of microbial protein yield per kilogram of ration agreed fairly well with previous estimates for similar rations.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Developmental Changes in the Free Amino Acid Pool and Total Protein Amino Acids of Pea Cotyledons (Pisum sativum L.)

Changes in the levels of twenty-two free amino acids and in the amino acid composition of the total protein were measured throughout the development of cotyledons of a dwarf garden pea, Pisum sativum cv Greenfeast, grown in a constant environment. A sensitive double-isotope dansylation technique was used. Fresh weight, dry weight, and protein content were also followed. Twenty of the amino acids showed synchronous changes in levels, giving a developmental pattern containing four peaks; major peaks occurred very early and very late in development. The amino acid composition of the total protein, which was always very different from that of the free amino acid pool, showed early changes to one consistent with the final storage protein composition of the seed. These changes included a 50% drop in methionine content and a 70% rise in cysteine. While the maximum free methionine level occurred early in development, that of cysteine was late.     

Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits.

We describe a procedure that allows cysteine and methionine content to be determined on microgram amounts of partially purified protein. The only requirements are that the protein can be obtained as a pure band after electrophoresis on a polyacrylamide gel and that some data on amino acid content be available. This method involves double labeling by growing bacterial cells with [3H]leucine and [35S]SO4 and determining the ratio of these radioisotopes incorporated into the ribonucleic acid polymerase subunits. The relative specific activities of [3H]leucine and [35S]cysteine and methionine are determined from the ratio of these isotopes incorporated into beta-galactosidase, the leucine, cysteine, and methionine contents of which are known. We have used this procedure to determine the sulfur content of the subunits of Escherichia coli ribonucleic acid polymerase. These new data are necessary to quantitate the rates of synthesis of these subunits by in vivo labeling with [35S]SO4.     

Methionine overproduction by Saccharomycopsis lipolytica.

Six ethionine-resistant (Etr) regulatory mutants of Saccharomycopsis lipolytica Sl/1 overproducing methionine have been isolated. Five of them are also resistant to seleno-methionine. The activity of homocysteine synthase (O-acetyl-L-hormoserine-acetate lyase, adding hydrogen sulfide) is derepressed in these mutants and is not susceptible to the methionine-mediated repression. The pool of free methionine in Etr mutants is enhanced 1.5 to 18 times, and incorporation of 35S into methionine is 1.5 to 50 times higher than that in the wild strain. Neither accumulation of endogenous free methionine in Etr mutants nor the uptake of exogenous methionine is accompanied by an increase in the S-adenosylmethionine pool. This implies compartmentation of methionine metabolism in S. lipolytica.     

The origin of the sulfur atom in thiamine.

The mode of biosynthesis of the thiazole moiety of thiamine, 4-methyl-5beta-hydroxyethyl thiazole (MHET) was studied using Salmonella typhimurium as test organism. It was shown by isotope incorporation experiments, that the sulfur atom, but not carbon-3, of cysteine is incorporated into MHET, indicating a separation of the sulfur atom of cysteine from the carbon chain during incorporation. Isotope competition experiments revealed that the incorporation of [35S]cysteine is not significantly diluted by the presence of methionine, homocysteine, and glutathione. No incorporation of label from [14C]glutamate and [14C]formate was observed, leaving the origin of the five-carbon unit still in doubt.     

Accumulation of free amino acids in growing Xenopus laevis oocytes

The sizes of amino acid pools in growing Xenopus laevis oocytes have been measured. The total free amino acid content per oocyte increases nearly 25-fold during oocyte growth. Together, glutamic acid and aspartic acid account for approximately 59-75% of the total amino acid pool in Xenopus oocytes. On the other hand, methionine and cysteine are the least abundant of the amino acids detected, each accounting for less than 0.7% of the total pool in developing oocytes. It is argued that the acid-extractable amino acid pool represents the precursor pool used in protein synthesis.