Changes in Pool Sizes of Free Amino Acids and Amides in Leaves and Plastids of Zea mays during Leaf Development

The concentrations of free amino acids and amides within isolated maize (Zea mays L.) plastids were determined and compared with concentrations in the leaf tissue. The concentrations were different for each individual amino acid and varied between 1 and 10 millimolar. At five different developmental stages concentrations in the plastids were greater than those in the intact leaf tissue. During development, from the proplastid stage to the mature chloroplast, the amount of each amino acid per plastid remained relatively constant, but there were decreases in concentrations of plastid amino acids resulting from the developmental increase in plastid volume. In proplastids, the free amino acids were present in greater concentrations than those previously found to inhibit partially amino acid-synthesizing enzymes located in chloroplasts. In the chloroplasts, the molarities of the free amino acids were within the range known to inhibit amino acid-synthesizing enzymes.     

RIBOSOMAL ACTIVITY IN PRENATAL MOUSE BRAIN

Abstract— Regulation of protein synthesis is important for the proper growth and development of the brain. Our previous work on the regulation of protein synthetic activity in fetal mouse brain cell suspensions showed that the rate of protein synthesis decreased during the prenatal period. In the present study, ribosomal activity of cell-free homogenates and purified ribosomes obtained from fetal neural tissue was measured. The post-mitochondrial supernatant (PMS) fraction actively incorporated amino acids into polypeptides using either endogenous mRNA or polyuridylic acid as template. The protein synthetic activity was dependent upon the age of the fetus. Ribosomes purified from this fraction were also active in protein synthesis. Incorporation of phenylalanine was linear for 20 min, and dependent upon the concentration of ribosomes and the pH 5 enzyme fraction. The age dependent decrease in protein synthetic activity observed with the post-mitochondrial supernatant fractions was not found when these purified ribosomes were employed. Ribosomes obtained from fetal, newborn or adult neural tissue were compared and found equally active in their protein synthetic capacity.     

Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.     

In vitro translation with adenovirus polyribosomes.

Polyribosomes isolated from adenovirus type 2 (Ad2)-infected HeLa cells late in productive infection can be used for translation in cell-free systems. At least eight viral polypeptides are synthesized, including the precursors to virion polypeptides VI and VII. Separation of polyribosomes by zonal rate centrifugation followed by translation in a cell-free system reveals a correlation between the sizes of the polyribosomes and the polypeptides synthesized. The cell-free extracts incorporate amino acid linearly for only 10 min and show little or no capacity to reinitiate protein synthesis. The elongation efficiency measured as the number of amino acids incorporated per ribosome in 20 min is low, ranging from 10 to 100. The maximum chain elongation rate is estimated to be 10 to 20 amino acids per min. The limited elongation has been used to assess the relative concentration of mRNA's engaged in translation.     

Cell-free Synthesis of Globulin by Developing Oat (Avena sativa L.) Seeds

The primary storage protein synthesized during oat (Avena sativa L.) groat development is a globulin. Polysomes were isolated from oat groats 12 days after anthesis. These polysomes directed the incorporation of radioactive amino acids into protein in a cell-free protein synthesis system containing wheat germ supernatant. The Mg(2+) optimum was 4 mm, the pH optimum was 6-8, and the amount of amino acid incorporation depended on polysome concentration. Incorporation of amino acids was linear for about 10 min and approached a maximum after 20 min. Using the initiation inhibitor, T-2 toxin, it was determined that about 36% of the amino acid incorporation was due to the initiation of new polypeptide chains. The in vitro product co-electrophoresed with authentic oat groat globulin on polyacrylamide-sodium dodecyl sulfate (SDS) gels. The cyanogen bromide peptides of the in vitro product partially corresponded with those from authentic globulin when electrophoresed on polyacrylamide-SDS gels. These data suggest that the in vitro product is primarily oat globulin. The polysome population was separated into membrane-bound and free polysomes. Membrane-bound polysomes synthesized about twice the amount of protein as did free polysomes. Products synthesized in vitro on both types of polysomes were essentially the same.     

The synthesis of fatty acids in avocado mesocarp and cauliflower bud tissue.

1. Plastid and mitochondrial preparations were obtained by density-gradient centrifugation of homogenates made by gentle disintergration of avocado fruit mesocarp and cauliflower bud tissue. 2. The mitochondrial preparations had respiratory activity but did not incorporate [1-14C]acetate into fatty acids. 3. The plastid preparations incorporated [1--14C]acetate into the range of fatty acids found in the parent tissue. No fatty acid synthetase activity could be detected in the 12000g supernatant of these homogenates. 4. Homogenates produced by rupture of the tissue in an Ato-Mix blender and plastid preparations disintegrated by ultrasonic treatment both had fatty acid synthetase activity which did not sediment at 105000g and which formed mainly [14-C]stearate from [2-14C]malonyl-CoA. 5. It is concluded that the plastids are the principal site of fatty acid biosynthesis in the tissues studied.     

Amino acid incorporation by cell fractions from the oviduct of the laying hen and the synthesis of egg-white proteins

1. Homogenates of the magnum of the hen oviduct have been fractionated by differential centrifuging. 2. Centrifuging at 600g for 10min. gave a pellet containing most of the particulate material of the cell, but on washing this fraction some particles were removed from it. The washed 600g pellet contained most of the DNA of the homogenate. 3. Centrifuging the 600g supernatants at 10000g for 10min. gave particulate fractions (I particles) richer in RNA than other fractions which were active in incorporating amino acids into protein in isolation. When minced tissue was incubated with radioactive amino acids before homogenizing these particles were the most radioactive of the cell fractions. 4. The pellet obtained by centrifuging the 10000g supernatant at 105000g for 60min. was very small; its RNA/protein ratio was raised compared with that of the homogenate. It only incorporated amino acids in isolation to a small extent or not at all. 5. The 105000g supernatant contained a large proportion of the protein of the homogenate. 6. The I particles could be subfractionated by layering over denser sucrose to give a pellet with lower RNA content and incorporating activity, and also suspended material richer in both these properties. 7. Treatment of the I particles with deoxycholate gave rise to particles sedimenting at 105000g for 60min. containing most of the RNA of the original particles, but only about 34% of the protein, and with a high activity in incorporating amino acids. 8. The I particles, or particles derived from them by deoxycholate treatment, required GTP and phosphoenolpyruvate for the incorporation of free amino acids. The omission of ATP reduced the incorporation to varying extents. Chicken-liver cell sap stimulated the activity. 9. Radioactive amino acids linked to transfer RNA were transferred to protein by I particles. GTP and phosphoenolpyruvate were required for this transfer. The phosphoenolpyruvate requirement could not be replaced by increasing the GTP concentration. ATP partially inhibited the transfer. 10. After incorporation by the cell-free system, the hot-trichloroacetic acid extract, but not the lipid extract, was radioactive. Ribonuclease and puromycin inhibited at low concentrations. Lecithinase-C was much less inhibitory. Transfer of amino acid, from a radioactive lipid-amino acid complex prepared from hen oviduct, was not detected. 11. After short periods of incubation of minced tissue with [(14)C]lysine some of the radioactive protein of the isolated I particles behaved as ovalbumin. The distribution of radioactivity in the protein resembled that in ovalbumin in soluble extracts of the tissue obtained after longer periods of incubation.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.     

AMINO ACID INCORPORATION INTO PROTEIN IN NEURONAL CELL BODY AND NEUROPIL FRACTIONS IN VITRO

Abstract—

• 1 The incorporation of radioactivity from [³H]lysine into acid-insoluble material in vitro in mixed cell suspensions and isolated neuronal and neuropil fractions has been followed.
• 2 In the mixed cell suspension, incorporation was linear in fresh preparations for up to 60 min. In cold stored preparations, incorporation began to fall off after 30 min. Incorporation, at 4-11 pmol/mg protein/h, was intermediate between that in the tissue slice and in a cell-free preparation. Addition of a mixture of non-labelled amino acids at 1 mM produced a 30-40 per cent inhibition of incorporation. Molar rates of incorporation of glutamate and tryptophan into the mixed cell fractions were respectively 73 and 1-4 times that of lysine.
• 3 Only 8 per cent of the incorporated radioactivity could be recovered in soluble as opposed to particulate material. After hydrolysis of the protein, followed by paper chromatography and autoradiography, radioactivity was detected only in the position corresponding to lysine.
• 4 Incorporation in the separated cell fractions was not markedly reduced by the centri-fugation procedure. Incorporation into the neuronal fraction was 2-2-6 times that into the neuropil fraction, depending on the amino acid used. Incorporation into both was reduced by some 40 per cent by addition of an amino acid mixture.
• 5 Comparison of in vivo and in vitro data suggest that the differences in rate of incorporation are characteristic of neurons and neuropil in situ.

     

The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids.

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.     

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.     

The influence of electrical stimulation in vitro on protein synthesis and other metabolic parameters of rat extensor digitorum longus muscle

1. Apparatus is described in which rat extensor digitorum longus muscle can be incubated in buffer under conditions of light tension and be subject to contractures induced by electrical stimulation in vitro. Under these conditions the tissue retains its weight, its content of potassium and size of the extracellular space at values similar to those in vivo. 2. Though uptake of glucose was enhanced on addition of insulin, there was little increase in glucose consumption on stimulation. Breakdown of glycogen and enhancement of lactate output were found on stimulation. 3. Incorporation into protein of several labelled amino acids was diminished during stimulation. Accumulation of [(14)C]leucine was enhanced whereas that of glycine was decreased. 4. There were no very consistent changes in the content of free unlabelled amino acids during incubation with or without stimulation. Comparison of actual amino acid concentrations in tissue and incubation mixture with accumulation of (14)C-labelled amino acid indicated that full equilibration of the cell pool of amino amino acids with the medium is slow. 5. Substantial oxidation of several (14)C-labelled acids was observed. 6. The ATP content of the tissue declined a little during incubation and somewhat faster after a period of stimulation. 7. The results are discussed in relation to the way in which exercise can induce muscle hypertrophy.     

Thein vitro activity of the microsomal fraction isolated from the spleen and the lymph nodes after immunization of the donor

The microsomal fraction from the spleen (after perfusion) of immunized rabbits incubated for 20 min at 37° C under usual conditions in the presence of energy sources incorporates¹⁴C-labelled amino acids both into the solubilized (by adding deoxycholate), and into the nonsolubilized part (15%). The cell supernatant incorporates under these conditions the¹⁴C-labelled amino acids into total proteins in the absence of microsomes but in a lower degree. The cell supernatant contains gamma globulin detectable by immunoelectrophoresis. Gamma globulin obtained by specific precipitation of the solubilized microsomal fraction with antigamma-globulin serum had an measurable radioactivity. The precipitate of gamma globulin obtained from the supernatant of the incubation medium in the same manner (after removing the microsomes) had a specific activity twice as high. On separating the microsomal fraction extract and the incubation medium supernatant on DEAE cellulose most fractions show on extinction maximum at 260 nm in the first case and at 280 nm in the second case. The microsomal fraction isolated from the spleen and lymph nodes of immunized pigs-48 and 72 h after revaccination, when incubatedin vitro, incorporated¹⁴C-labelled amino acids into total protein. After ultrasonic disintegration in 0.14m NaCl and filtration through a Sephadex G 25 column it is specifically precipitated with the antigammaglobulin serum. Gamma globulin isolated after incubation of the microsomal fraction had a measurable radioactivity. AntiHSA antibodies determined by adsorption on immunosorbent did not possess significant radioactivity. Only the concentrated supernatant of the incubation medium showed minute radioactivity of 75–94 counts/min /ml. The problem of investigating the formation of nascent specific antibodies on a subcellular levelin vitro during the early period of secondary response to the antigen is discussed, in particular the problem of their detection. An erratum to this article is available at .     

Factors controlling amino acid incorporation by ribosomes from Krebs II mouse ascites-tumour cells

1. A ribosome-cell sap system capable of supporting the incorporation of (14)C-labelled amino acids into protein has been prepared from Krebs II mouse ascites-tumour cells. The requirements of this system for optimum activity and response to added messenger RNA have been investigated. One such system has been obtained for which amino acid incorporation is almost wholly dependent on the addition of suitable messenger RNA. 2. Ribosomes of widely different but predictable activities in the cell-free system have been prepared from Krebs cells pretreated in a variety of ways. The factors in the pretreatment of the cells responsible for these differences have been investigated. 3. The structural and functional properties of these different ribosome preparations and their response to exogenous messenger RNA have been examined and are discussed in the light of modern concepts of the control of protein synthesis.     

Chloroplast Fatty Acid transformations in nitrogen-deficient and senescent tissues

The fatty acids of plastids from several types of mineral-deficient and senescent tissues were analyzed. Incorporation of acetate into long-chain fatty acids of leaf tissue and of plastids from nitrogen-deficient and normal plants was determined. In general, the senescent and nitrogen-deficient chloroplasts contained a higher ratio of saturates to unsaturates than did plastids from younger tissues and from tissues grown on a complete nutrient.

Nitrogen-deficient leaf tissue and plastids were capable of rapidly incorporating acetate into some of the fatty acids, especially palmitic and oleic acids. However, the comparative rate of acetate incorporation into linolenic acid in nitrogen-deficient chlorophyllous tissue was less than in tissue grown on a complete nutrient. With the addition of UDP-glucose to a reaction mixture containing added cofactors for noncyclic photosynthetic phosphorylation the relative incorporation of acetate into linolenate as compared to palmitate was increased in both the nitrogen-deficient and normal leaf tissue. This would indicate that nitrogen-deficient tissues have the enzymic systems for forming long-chain fatty acids but that the reduced photosynthesis limits the amount of precursors for the formation of lipids, especially galactolipids. However, nothing is known about the rate of fatty acid degradation under these conditions.

     

RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5 m -KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m -KCI was performed, purified ribosomal subunits were obtained. Cross-contamination of subunits was less than 5%. Re-associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell-free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos

The aim of this work was to determine in what form carbon destined for starch synthesis crosses the membranes of plastids in developing pea (Pisum sativum L.) embryos. Plastids were isolated mechanically and incubated in the presence of ATP with the following ¹⁴C-labelled substrates: glucose, fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate. Glucose 6-phosphate was the only substrate that supported physiologically relevant rates of starch synthesis. Incorporation of label from glucose 6-phosphate into starch was dependent upon the integrity of the plastids and the presence of ATP. The rate of incorporation approached saturation at a glucose 6-phosphate concentration of less than 1 mM. It is argued that glucose 6-phosphate is likely to enter the plastid as the source of carbon for starch synthesis in vivo.Abbreviations ADPG PPase ADP-glucose pyrophosphorylase - DHAP dihydroxyacetone phosphate