Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

Liver enzyme induction by 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) is accompanied by an increase in the specific activity of elongation factor 1.

Homogenates of liver were obtained from control rats and from rats that had received DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]. The postmicrosomal supernatant fractions were used for the purification of elongation factor 1 by hydroxyapatite chromatography and phosphocellulose chromarography. The amount of binding factor present was essentially the same for both groups of animals, but the specific activity, as measured by the binding assay, was about twice as high in the DDT-treated preparations. After sucrose-gradient sedimentation, the difference in specific activity was found to reside in the low-molecular-weight (50000) form of elongation factor 1. The implications of an increased reactivity of elongation factor 1 during the induction of membrane enzymes are discussed.     

Growth of liver accompanied by an increased binding of aminoacyl-transfer ribonucleic acids.

Homogenates of rat liver obtained 3 or 14 days after partial hepatectomy were used to prepare the postmicrosomal pH5-supernatant fraction and to prepare salt-wash fractions of the 40S ribosomal subunits and the 80S ribosomes. The factor-dependent binding of methionyl-tRNAfMet to ribosomes and the elongation-factor-1-dependent binding of phenylalanyl-tRNA to ribosomes were both increased after 3 days of growth, but not after 14 days of growth. An activity inhibitory to phenylalanyl-tRNA binding that was located in ribosomal wash fractions was decreased after 14 days of growth. Since the decreased inhibitory activity was obtained from the ribosomes and was tested against ribosomes and excess of pH5-supernatant fraction from control rat liver, its action was separate from the phenylalanyl-tRNA binding activities of the pH5-supernatant fractions from sham-operated and regenerating liver.     

Protein synthesis in aging soybean cotyledons. Loss in translational capacity

Ribosomes and supernatant fractions from soybean cotyledons of different ages were prepared to study the Poly U (polyuridylic acid)-directed phenylalanine incorporation. Ribosomes from younger cotyledons were more effective in phenylalanine incorporation compared to ribosomes from older cotyledons. Similarly, the supernatant fractions from younger cotyledons were more efficient, resulting in enhanced incorporation, than the older cotyledons. Substitution of wheat embryo supernatant fraction for soybean cotyledon supernatant fraction resulted in a several fold increase in amino acid incorporation activity, in ribosomes from all ages of soybean cotyledons. Such increase in activity was particularly significant in the older cotyledons. From these experiments it is concluded that in aging soybean cotyledons there is a loss in translational capacity.     

Mammalian mitochondrial ribosomes. Studies on the exchangeability of polypeptide chain elongation factors from bacterial and mitochondrial systems

Mammalian mitochondrial ribosomes from rat liver synthesised poly(phenylalanine) from [14C]-Phe-tRNA in the presence of a homologous 10(5) X gav supernatent fraction. The activity depended on the addition of synthetic template and was resistant to cycloheximide. The polyanion spermidine had a stimulatory effect on peptide synthesis in vitro. In contrast to Escherichia coli ribosomes, which also functioned with heterologous supernatant fractions, 55-S mitochondrial ribosomes were inactive when supplemented with heterologous supernatant fractions from E. coli or with purified bacterial elongation factors. EF-T slightly stimulated polyphenylalanine synthesis when added in combination with mitochondrial supernatant fractions. Two-dimensional electrophoretic analysis of the protein content of both supernatant fractions revealed considerable differences in the distribution of the species-specific proteins according to their isoelectric points. The mitochondrial supernatant proteins were in general more basic, and the few acidic proteins did not co-migrate with EF-Tu or EF-G from E. coli.     

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.     

Hepatic Subcellular Fractions Lipids in Rats Fed Millet (Sorghum vulgarie) at Different Protein Levels

Effect of feeding defatted millet (Sorghum vulgarie) flour at 5, 10 and 14.5% protein levels respectively for six weeks has been studied on rat liver mitochondrial, microsomal and supernatant fractions total lipids, cholesterol, triglycerides, total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine. The results have been compared with rats fed casein at 10% level for the same period. The metabolism of liver subcellular fractions lipids of millet diet and casein diet fed rats has been studied by the incorporation of acetate-1-¹⁴C and . A significant increase in mitochondrial triglycerides of rats fed millet diet at 5 and 10% protein level, in microsomes of rats fed millet diet at 5, 10 and 15% protein levels and in supernatant fractions of rats fed millet diet at 5 and 15% protein levels was observed. A significant increase in total cholesterol in mitochondria and microsomes and a significant decrease in supernatant fraction of rats fed millet diet at 10% protein level was observed. A significant increase in mitochondrial total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine in rats fed millet diet at 10% protein level and a decrease in these in rats fed millet diet at 5 per cent protein level was observed. In microsomes total phospholipids were increased in rats millet diet at 10% protein level and phosphatidyl choline was increased in rats fed millet diet at 15% protein level. Total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine were significantly reduced in the supernatant fraction of rats fed millet at 10% protein level.

Incorporation of acetate-1-¹⁴C into nonsaponifiable fraction of mitochondria, microsomes and supernatant fractions of rats fed millet diet at 5 and 15 % protein levels was significantly greater, and in saponifiable fractions of the above subcellular fractions was greater in rats fed millet diet at 5 per cent protein level. The specific activity (counts/min/mg) of free cholesterol in mitochondria, microsomes and supernatant fractions of millet diet fed rats was significantly greater, whereas the specific activity of triglycerides was not significantly different from the controls. The acetate-1-¹⁴C specific activity of phosphatidyl choline and phosphatidyl ethanolamine was significantly greater in all the above subcellular fractions of millet diet fed rats (except of phosphatidyl choline in rats fed millet diet at 5 % protein level). The specific activities of phosphatidyl choline were significantly greater in mitochondria of rats fed millet diet at 5 % protein level and of phosphatidyl choline and phosphatidyl ethanolamine in microsomes and supernatant fractions of rats fed millet diet at 5 and 15% protein levels. The specific activities of phosphatidyl choline were significantly decreased in mitochondria and microsomes of rats fed millet diet at 10% protein level. The total acetate-1-¹⁴C activities (counts/min/g equivalent wet liver) of free and esterified cholesterol triglycerides, phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis from acetate-1-¹⁴C was either enhanced in millet diet fed rats or was comparable to the controls. The total activity of (counts/min/g equivalent wet liver) into phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis was decreased in microsomes of rats fed millet diet at 10% protein level, increased in rats fed millet diet at 5 and 15% protein levels.     

Effect of a modified separation procedure on the size and protein-synthetic activity of membrane-bound liver polyribosomes

1. Various subcellular fractions containing ribosomes were isolated from rat liver. 2. In the presence of [(14)C]leucine and Sephadex-treated cell sap the radioactivity incorporated into the synthesized protein resulting from the incubation of microsomal preparations or deoxycholate-treated polyribosomes was dependent on the amount of rRNA incubated. In contrast, when Sephadex-treated post-mitochondrial supernatant was incubated, the radioactivity incorporated into the synthesized protein was independent of the amount of rRNA incubated. 3. Microsomal preparations and membrane-bound ribosomes, prepared by the standard procedure, incorporated less [(14)C]leucine into protein, per mg of rRNA incubated, than free or deoxycholate-treated polyribosomes; accordingly, polyribosomes associated with the former fractions were found mainly as monomers. 4. If microsomal fractions or membrane-bound ribosomes were prepared by a simple modification of the standard procedure, i.e. by centrifugation on to a ;cushion' of 2m-sucrose, their protein-synthesizing activity was of the same order as that of the original post-mitochondrial supernatant, and membrane-free and deoxycholate-treated polyribosomes; in this case polyribosome profiles showed that very little degradation had occurred and compared well with those obtained for post-mitochondrial supernatant and isolated polyribosomes. 5. A method is described (Appendix) that provides a rapid and reliable assessment of the concentration of rRNA in subcellular fractions.     

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.     

The metabolism of phosphatidylinositol in the thyroid gland of the pig

1. The metabolism of phosphatidylinositol in pig thyroid has been investigated as a basis for understanding the specific stimulation of the synthesis of this phospholipid in the gland by thyrotropin. 2. The gland contained an active Ca(2+)-dependent phosphatidylinositol-splitting enzyme with an optimum pH of 5.3-5.5. 3. The major water-soluble product (65%) formed by this catabolic enzyme was not phosphorylinositol but a related compound, which may be a cyclic phosphorylinositol. Both this and phosphorylinositol (35%) were released simultaneously from the phosphatidylinositol substrate. 4. The phosphatidylinositol-splitting enzyme was found almost exclusively in the supernatant fraction obtained by homogenization of the gland. It was not present in the acid-phosphatase-containing particulate fraction. 5. The incorporation of [2-(3)H(1)]inositol into phosphatidylinositol in the presence of either CDP-diglyceride or CTP+ATP was most active in the microsomal fraction. 6. When thyroidal microsomes were labelled with [(3)H]inositol and (32)P, and then incubated with unlabelled inositol, there was a dramatic loss of (3)H labelling from the phosphatidylinositol, which was not accompanied by an equivalent loss of (32)P from the phosphate moiety. This turnover of the inositol moiety required nucleotide coenzymes. It is postulated that the phosphatidylinositol is split into inositol and a phosphorus-containing lipid precursor of the phospholipid that remains on the microsomal membrane and is recycled. 7. Isolated thyroidal mitochondria synthesized phosphatidylinositol from [2-(3)H(1)]inositol only because of their contaminating microsomal component. 8. Some evidence has been obtained of a rapid transfer of phosphatidylinositol molecules from thyroidal microsomes to mitochondria when these were incubated together in the presence of a supernatant fraction. 9. Both phosphatidylinositol breakdown by the supernatant fraction of the gland and synthesis by the microsomes were totally inhibited by 1mm-chlorpromazine. This drug is known to suppress thyrotrophin-induced stimulation of activity in thyroid slices.     

Hemoglobin transition in erythrocytes of developing chick. Studies with cell-free protein-synthesizing systems.

Cell-free hemoglobin-synthesizing systems from erythrocytes of 4- and 17-day chick embryos have been developed. These systems have been used to investigate possible structural and functional differences in factors involved in protein synthesis obtained from these different developmental stages. Each cell-free system consists of three major cellular fractions i.e., the S-100 supernatant, the salt-washed ribosomes, and the 0.5 m KCl ribosomal wash. When the ribosomal wash fraction from one developmental stage is included in a cell-free system containing ribosomes and S-100 supernatant from the other developmental stage, a drastic reduction in the kinetics of [³H]leucine incorporation into globin products is observed, when compared to the homologous control cell-free systems. A similar depression of the kinetics of incorporation is observed when the mixed component is either the S-100 supernatant or the ribosomes. Control rates of incorporation can be reestablished when the corresponding homologous component is added back to the incubation mixture. The predominant types of hemoglobins produced in the salt-wash heterologous systems are those hemoglobins characteristic of the developmental stage of the salt wash. This seems to imply that the ribosomal salt-wash fraction may possess developmental stage specificity for the globins.     

In Vitro Protein Synthesis and Aging in Rhizoctonia solani

A study was made of the ability of cell-free protein synthesis systems from vegetative cells of different age of the fungus Rhizoctonia solani to produce polyphenylalanine. Polyuridylic acid-directed phenylalanine incorporation into peptides decreased linearly with cell age. The 105,000 x g supernatant fluid and ribosomal fractions were equally responsible for the total loss of synthetic activity of the older cells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase activity decreased with increasing cell age, which accounted for the defect of the supernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA was associated with the ribosomes of the older cells. In vitro systems from cells of different age transferred phenylalanine from phenylalanyl-tRNA to polyphenylalanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 increased and 5 decreased in specific activity with increased age; 3 others did not change during aging and 2 were below acceptable detectable levels.     

Protein synthesis by microsomal particles from regenerating rat liver

1. Washed microsome particles from regenerating liver were shown to incorporate [(14)C]leucine into protein more actively than similar preparations from normal liver. 2. The total incorporation in the preparations from regenerating liver increased linearly with the amount of protein incubated, whereas this was not so with preparations from normal liver. 3. The greater activity of regenerating-liver microsomes appeared to be associated with the bound polysomes. 4. The size distribution of polysomes obtained after removal of membrane with deoxycholate was the same in normal and regenerating liver. 5. In general the activity of polysome preparations from normal and regenerating liver was similar. 6. It is concluded that the greater activity of the particles in the microsome fraction from regenerating liver is to be attributed to the ribosomes bound to membrane and that their activity is controlled by factors present in the membrane.     

Guanine-deaminase activity in rat brain and liver

1. Guanine deaminase in rat brain and liver was distributed among all the subcellular fractions: nuclei, `heavy' mitochondria, `light' mitochondria, microsomes and the supernatant fluid. The greater part of the activity passed into the soluble fraction. Among the particulate components, the `light' mitochondria constituted the richest fraction. 2. The sum of the enzymic activities of the component fractions obtained on differential centrifugation was considerably greater than the activity of guanine deaminase in the whole homogenate. 3. The `heavy'-mitochondrial fraction had a powerful inhibitory effect on the guanine-deaminase activity of the supernatant fraction. 4. All the sedimented fractions, except the microsomes, gave rise to higher guanine-deaminase activity on treatment with Triton X-100. 5. The inhibitory capacity of the `heavy' mitochondria increased on treatment with Triton X-100; the detergent-treated nuclear fraction also brought about inhibition of the 5000g supernatant. 6. Guanine-deaminase inhibitor from the `heavy' mitochondria was solubilized by high-speed grinding of the particles, followed by treatment with Triton X-100. The inhibitor appeared to be protein in nature, since it was precipitated by trichloroacetic acid and by half-saturation with ammonium sulphate, and was non-diffusible. It was inactivated by heating at 50° for 5min. 7. It is possible that the guanine deaminase associated with particles differs from the soluble enzyme in its response to inhibitor.     

Studies on the effect of inflammation on the synthesis of glucosylated intermediates of glycoprotein biosynthesis in rat liver microsomes

Glycoprotein biosynthesis is substantially increased in liver during experimental inflammation. This study describes the effect of inflammation on the incorporation of labelled Glc from UDP-Glc into glucosylated lipid-linked intermediates of glycoprotein biosynthesis in liver microsomes. Maximum incorporation of Glc into lipid sugar and lipid oligosaccharide fractions occurred using microsome fractions from rats suffering from inflammation for 48-72 hr. Incorporation of Glc into lipid-sugar fractions was increased about three-fold over controls and incorporation into lipid-oligosaccharide fractions was increased about four-fold compared to controls. Maximum incorporation of Glc into lipid-sugar and lipid-oligosaccharide occurred at pH 6.0 and pH 5.2, respectively.     

Exchange of phospholipids between brain membranes in vitro

1. When unlabelled mitochondria from guinea-pig brain were incubated with a (32)P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from (32)P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. (32)P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [(32)P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a (32)P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.     

Incorporation of galactose from UDP-galactose into microsomal and golgi membranes of rat liver

Rough and smooth microsomes and Golgi membranes were incubated with UDP[¹⁴C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from β-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.     

Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues

A triacylglycerol and cholesteryl ester transfer activity has been isolated from rat liver. After homogenization, the liver cells were subfractionated into the 10 000 X g pellet, microsomal fraction and postmicrosomal supernatant. Most of the transfer activity appeared to be associated with the microsomal fraction. Rough and smooth microsomes contained nearly equal transfer activities. When isolated microsomes were subject to proteolytic attack, the transfer activity was not inactivated, unless it had been released from the microsomes prior to proteolytic treatment. This indicates that the activity is probably located within the microsomal vesicles. Similar transfer activities were found in the intestinal mucosa of rats, whereas little or no activity was detected in the brain, heart, kidney, or plasma.     

Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity.

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.