Changes in ribosomal activity during incubation of Daucus carota root disks

Cytoplasmic monoribosomes from freshly cut and ‘aged’ carrot root disks were characterized relative to the Mg²⁺ optima for poly U (polyuridylic acid)-directed phenylalanine incorporation, the ease of dissociation by KCl in the presence of Mg²⁺, the ability to bind ³H-poly U, and acrylamide gel fractionation of the ribosomal proteins. The differences in in vitro amino acid incorporation by ribosomes and supernatant from fresh and ‘aged’ disks were confined to the ribosome fraction. The Mg²⁺ optima for poly U-directed ¹⁴C-phenylalanine incorporation was 16 mM for ribosomes from ‘aged’ disks compared to 20 mM for ribosomes from fresh disks. Monoribosomes from the fresh disks were easily dissociated into subunits (0·2 M KCl in 5 mM Mg²⁺) while the ribosomes from ‘aged’ disks were not completely dissociated even in 0·5 M KCl. Ribosomes from ‘aged’ disks were more effective in binding ³H-poly U than ribosomes from fresh disks. When the disks were subjected to an anaerobic environment prior to ribosome extraction (to strip monoribosomes of peptidyl-t RNA) the above effects of ‘aging’ were reversed. These results suggest that increased monoribosome activity associated with ‘aging’ may be related in part to an increase in the level of peptidyl-tRNA associated with the ribosomes. Acrylamide gel electrophoresis profiles of ribosomal proteins extracted from ribosomes of fresh and ‘aged’ tissue suggest that a change in the protein complement may also be important to the observed changes in ribosomal activity. The ribosomes from ‘aged’ disks contained at least two components not associated with ribosomes from fresh disks.     

Amino acid incorporation by nuclear membrane fraction of rat liver.

Nuclear membrane fraction of rat liver is able to incorporate ¹⁴C-leucine into its proteins $\text{in vitro}$. The incorporation of ¹⁴C-leucine into the nuclear membrane fraction was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable. RNase and DNase were not effective. The incorporation was also inhibited by several reagents known to interfere with energy metabolism. These characteristics of the incorporation of ¹⁴C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction. ¹⁴C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues. Acrylamide gel electrophoresis showed that three protein species were mainly labelled. The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 % of the control. The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of ¹⁴C-leucine by the nuclear membrane fraction was ruled out.     

Protein synthesis by isolated etioplasts and chloroplasts from pea and wheat and the effects of chloramphenicol and cycloheximide

Etioplasts capable of incorporating ¹⁴C-leucine into protein have been isolated from dark-grown pea and wheat plants. The requirements for leucine incorporation for etioplasts were similar to those for chloroplasts. An ATP-generating system, Mg²⁺, and GTP were required. The amino-acid-incorporation activity of etioplasts from wheat was comparable to that of chloroplasts on an RNA basis, whereas the activity of pea etioplasts was about 50% of the activity of pea chloroplasts. The incorporation of leucine into protein by etioplasts and chloroplasts from pea and wheat was inhibited by chloramphenicol, and to a slight extent by cycloheximide.     

Activation of protein synthesis by microsomes from aging beet disks

Microsomal fractions isolated from sterile, aged disks of red beetroot incorporate leucine into protein when supplemented with the supernatant fraction, ATP, GTP, and KCl; the incorporation is sensitive to RNase and is not due to bacteria. The microsomal activity is inhibited by puromycin and cycloheximide but is virtually insensitive to both d-threo and l-threo-chloramphenicol, as predicted from physiological studies.     

A comparative study in vivo and in vitro of the ability of ribosomes from Xenopus liver and ovary to incorporate L-[U-14C]leucine

1. A system for the incorporation in vitro of amino acids into protein is described for the South African clawed toad (Xenopus laevis laevis Daudin). 2. The incorporation of l-[U-(14)C]leucine by Xenopus-liver microsomes is very much greater per mg. of microsomal RNA than the incorporation by ovary microsomes. 3. The incorporation by Xenopus-liver and -ovary polysomes is approximately the same when expressed per mg. of polysomal RNA. 4. It was predicted from the above results that ovary microsomes should contain a ribosomal fraction inactive in protein synthesis. This was shown to be the case by a labelling experiment in vivo with l-[U-(14)C]leucine. 5. The labelling experiment in vivo also showed that the active polysomal fraction in ovary is associated with membranes and is liberated by treatment with deoxycholate; this is also true of liver microsomes in vivo. 6. The results are discussed in relation to previous work on the synthesis of proteins by amphibian ovarian tissue, and on the role of bound and free ribosomes in protein synthesis.     

Polypeptide Synthesis with Microsomes from Pressure-Treated Tetrahymena1

SYNOPSIS High hydrostatic pressure is known to interfere with mitosis, cytokinesis and synthesis of DNA, RNA and protein. In Tetrahymena, incorporation of phenylalanine and formation of polysomes are known to be pressure-sensitive. Microsomal preparations from Tetrahymena pyriformis GL can incorporate [¹⁴C]-phenylalanine into polypeptides. Incorporation was enhanced by addition of supernatant fraction and 14.5 mM Mg⁺⁺ An energy-generating system and exogenous messenger (poly U) were essential for polypeptide biosynthesis, Microsomes from pressurized cells (14,000 psi for 5 min) incorporated [¹⁴C]phenylalanine as efficiently as control microsomes. Microsomal function was not grossly damaged by pressure in a test system containing exogenous messenger, crude microsomal preparation, exogenous energy-generating system and supernatant fraction containing activating enzymes.     

Amino Acid incorporation in developing fucus embryos

The incorporation of ¹⁴C-leucine and ¹⁴C-amino acid mixture into protein in unfertilized eggs and developing embryos of the brown alga Fucus vesiculosus L. was studied. Bacterial contamination was initially a problem, but it was found that the addition of 40 μg/ml chloramphenicol to the incubation medium would inhibit bacterial protein synthesis without affecting early development of the Fucus embryos. The kinetics of uptake and incorporation of ¹⁴C-leucine into the trichloroacetic acid-soluble and -insoluble fractions indicated that the exogenous precursor did not equilibrate with the main soluble leucine pool before incorporation into protein. Uptake and incorporation of leucine by embryos 90 to 175 minutes old were proportional to exogenous leucine concentration over the range 5 × 10⁻⁶ m to 5 × 10⁻³ m. Unfertilized eggs will incorporate ¹⁴C-leucine into protein. The rate of this incorporation increases dramatically in newly fertilized eggs with a maximum rate at 3.5 hours, a period of cell wall formation and increasing metabolic rates. Thereafter, the rate of incorporation declines until approximately 15 to 17 hours when it increases again concurrently with the onset of rhizoid initiation and cell division.     

Biogenesis of microsomal membrane glycoproteins in rat liver. II. Purification of soluble glycoproteins and their incorporation into microsomal membranes

Sialoproteins isolated from the soluble fraction of rat liver could be incorporated into microsomal membranes. This incorporation was dependent on protein concentration, time, and temperature. Sodium dodecyl sulfate gel electrophoresis of membrane proteins after in vitro incorporation showed four major sugar-containing peaks and was similar to that found after in vivo labeling. Most of the incorporated protein was tightly bound to the microsomal membrane. Gel filtration and ion-exchange chromatography revealed the presence of several cytosolic glycoproteins that could be incorporated into microsomes. During prolonged centrifugation in a KBr solution with a density of 1.21 a highly labeled ([3H]glucosamine) protein (mole wt approximately to 70,000) that was actively incorporated into microsomes could be recovered in the upper region of the tube. These results demonstrate that several cytoplasmic glycoproteins of rat liver are transferred into microsomal membranes and that one of these is a lipoprotein.     

The Effect of Kinetin on Protein Level of Brassica Leaf Disks

The senescent leaf disks from Brassica rapa L. were floated on a medium containing ¹⁴C-L.-leucine in the presence or absence of kinetin. The increase in radioactivity in protein fraction of kinetin-treated disks was almost linear with time, while the increase in untreated disks incubated for 3 days started to slow down. The leaf disks were first incubated on the solution of ¹⁴C-L-leucine and then transferred to either kinetin solution or water in order to study whether the slower incorporation is due to the decreased rate of protein synthesis or that of protein retention. The decrease in radioactivity of disks floated on kinetin was slower than that of disks floated on water. The slower decrease in radioactivity caused by kinetin was not due to an increased turnover rate, since the same phenomena were observed in the presence of cold leucine or casein hydrolysate solution. These results suggest that kinetin retards the decomposition of protein in leaf disks and does not stimulate the synthesis of the bulk of protein.     

Transfer of proteins synthesized in the cytoplasm into mitochondria. Stimulation of mitochondrial protein synthesis by microsomal fraction]

The in vitro transport into mitochondria of proteins synthesized in the cytoplasm was studied. The system, in which the microsomes synthesize protein in the presence of mitochondria directly during the experiment proved to be the most efficient one. The microsomal fraction significantly stimulated the incorporation of 14C-valine into the isolated mitochondria proteins. The effects of EDTA treatment of the mitochondrial fraction, the dependence of protein synthesis stimulation on the ratio of mitochondria and microsomal proteins and the kinetic pattern of the reaction suggest that the stimulation of the labelled precursor incorporation into mitochondrial proteins is not probably due to the labelled microsomes adsorption on the mitochondria.     

The sequential synthesis of the polypeptide chain of serum albumin by the microsome fraction of rat liver

1. The isolated microsome fraction of regenerating rat liver was incubated with cell sap, a source of energy and [³⁵S]methionine, [¹⁴C]isoleucine or [¹⁴C]leucine for different periods of time, and microsomal albumin isolated. 2. The distribution of these isotopes in albumin was determined by separation of tryptic peptides from the protein. Radioactivity was measured in peptides either qualitatively by radioautography or quantitatively by labelling with both ³H and ¹⁴C. 3. A gradient of radioactivity existed at all times in albumin isolated after incubating microsomes. 4. The shorter the incubation time the fewer the peptides labelled in albumin, but the peptides with highest specific activity after short incubation times corresponded to those with highest specific activities after long incubation times. 5. Leucine released from the C-terminus of albumin had a higher specific activity than the mean specific activity of the remaining leucine residues in albumin. 6. The peptide with the highest specific activity in albumin is probably derived from the C-terminus of the protein. 7. [¹⁴C]Glutamic acid is incorporated into the N-terminus of albumin after incubating the microsome fraction with this isotopically labelled amino acid, cell sap and a source of energy. The specific activity of the N-terminal glutamic acid under these conditions is less than the mean specific activity of the remaining glutamic acid and glutamine residues in albumin. 8. The results are interpreted as reflecting a sequential synthesis of serum albumin in the isolated microsome fraction of rat liver. The direction of synthesis of albumin is from the N-terminus towards the C-terminus. 9. The bulk of incorporation of radioactive amino acid into albumin in the isolated microsome fraction is due to completion of partially completed, pre-existing peptide and polypeptide chains. A limited synthesis of new chains of albumin does, however, occur.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. III. Formation of Lamellar and Soluble Chloroplast Protein

Chloroplasts from leaves of plants which had been grown in the dark, and then illuminated for 12 hours were isolated, and allowed to incorporate ¹⁴C-leucine into protein, and the products of this incorporation were studied. Lamellar and soluble proteins are the principal products, and are formed in about equal amounts. Only some of the soluble proteins become heavily labeled. Those with highest specific activity have a molecular weight of the order of 140,000, while the higher molecular weight Fraction I protein has a much lower specific activity. The soluble protein as a whole does not serve as a precursor for the lamellar protein, and vice-versa, although a precursor-product relationship between a minor component of the soluble fraction and the lamellar fraction has not been ruled out. The relative protein synthesizing capabilities of chloroplasts and mitochondria are discussed with reference to the data presented.     

Base-exchange reactions for the synthesis of phospholipids in nervous tissue: the incorporation of serine and ethanolamine into the phospholipids of isolated brain microsomes

Abstract— The calcium-dependent incorporation of l -[3-³H]serine and [1,2-¹⁴C]ethanol-amine into the phospholipid of isolated subcellular fractions from chick brain was studied and the properties of incorporation were examined. The microsomal fraction was found to possess the highest rate of incorporation and was able to convert under optimal conditions about 120 nmol of labelled serine and 220 nmol of ethanolamine/g of fresh brain microsomes/h. The requirement for Ca²⁺ ion appeared to be absolute. Mg²⁺ ion caused a gradual reduction in the existing enzymic activity, only when pre-incubated with microsomes and labelled bases before adding Ca²⁺ ion. The incorporation of serine and ethanolamine was actively inhibited by Hg²⁺, Co²⁺, Cu²⁺ and Mn²⁺ ions, and was abolished by ethylenediamine tetra-acetate treatment. Ethanolamine, but not choline, inositol or carnitine, competitively inhibited serine incorporation, while d -serine had slight effect. Conversely, l -serine inhibited competitively the incorporation of ethanolamine. The greater part of the incorporated serine (85 per cent) was localized in microsomal phosphatidylserine, while a small percentage was found in phosphatidylethanolamine. Similarly, 90 per cent of the incorporated ethanolamine was confined to phosphatidylethanolamine and a small percentage was found in the plasmalogen derivative. The mechanism of serine and ethanolamine incorporation was investigated. The results are compared with those published for similar mammalian and non-mammalian systems.     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

The rate of protein synthesis in needles of Norway spruce (Picea abies): Light stimulation, regulation through photophosphorylation, stress enhancement

The incorporation of ¹⁴C-leucine into the total-protein fraction of needles of Norway spruce (Picea abies [L.] Karst.) during short time incubation was used as a measure of protein synthesis in the light and in the dark. Light saturation curves, obtained for needles of different ages (new flush and 1 and 2 years old) or at different seasons (summer-winter) followed the Michaelis-Menten algorithm, exhibiting marked differences with regard to light saturation (V_max) and the half-saturation constant (K_{5. 2}). The light saturation curves of ATP level (mg g^?1 fresh weight) and of leucine incorporation into protein (nmol mg^?1 h^?1) matched each other, suggesting that photophosphorylation may be decisive for the rate of protein synthesis in the light. This is confirmed by the action spectrum of leucine incorporation. which resembled an action spectrum of leaf photosynthesis, and also by partial inhibition of protein synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of non-cyclic photophosphorylation. Light stimulated protein synthesis showed pronounced seasonal fluctuations with a summer maximum. Furmigation of 5 years old spruce trees for 3 months with SO₂ in combination with O₃ and/or NO₂ caused a distinct enhancement of the protein synthesis rate in the light and, at a reduced absolute level, also in the dark. A similar result was obtained for 40 to 70 years old spruce stands when healthy and sick trees were compared: the latter being afflicted by the novel type of forest decline, which is characterized by yellowish bronze discolouration of sun-exposed older needles and partial loss of older needle generations (3 to 4 years old). The 1 year old needles of the unhealthy trees showed a markedly increased ¹⁴C-leucine incorporation rate which, in the dark, was even more pronounced than in the light. Stress-physiological mechanisms, which could possibly explain this stimulation, are discussed.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Effect of K+ concentration on the rate of synthesis of fast-labeling proteins in the adrenal cortex

The effect of K+ concentration on protein biosynthesis and 32P-incorporation into the acid-insoluble fraction of dog and guinea pig adrenal cortex slices was studied. An increase in K+ concentration in the incubation medium from 3 to 8-11 mM induced after 15-20 min of incubation a significant stimulation of 14C-leucine incorporation into the acid-insoluble fraction of post-mitochondrial supernatant. More extensive labelling of this fraction with 32P was observed. Addition of valinomycin caused a shift in the maximum of 14C-leucine incorporation towards lower K+ concentrations. The Na+,K+-ATPase inhibitors--ouabain and strophantin K--reduced the K+-stimulated protein synthesis. These data suggest that K+ transport into the cell is necessary for the stimulating effect to be manifested. Chelation of Ca2+ strongly decreased the incorporation of 14C-leucine into proteins in the presence of 5 mM K+. However, protein labelling increased with a gradual rise in K+ concentration up to 25 mM.     

Phytochrome-controlled Leaf Unrolling and Protein Synthesis

In the primary leaf sections of etiolated wheat (Triticum aestivum L.) seedlings, red light-induced unrolling is accompanied by an increase in incorporation of ¹⁴C-leucine into protein. By differential centrifugation, the unrolling response was found to be closely related to incorporation of the amino acid into the supernatant fraction (105,000g). Cycloheximide and chloramphenicol inhibit both leaf unrolling and synthesis of the supernatant protein, although chloramphenicol exerts its effect more strongly on the fraction which presumably contains the plastids. In a barley (Hordeum vulgare L.) albino mutant completely devoid of ribulose diphosphate carboxylase activity, only incorporation of ¹⁴C-leucine into the supernatant fraction is substantially promoted by red light. This mutant exhibits the photoresponse of leaf unrolling.