Effect of Sulfhydryl Reagents on the Ribosomes of Bacillus subtilis

The effect of various sulfhydryl reagents on the ribosomes of Bacillus subtilis was studied. The 70S ribosomes were completely dissociated into 30S and 50S subunits by appropriate concentrations of p-chloromercuribenzoic acid (PCMB) and 5,5'-dithio-bis-(2-nitro-benzoic acid). The N-ethylmaleimide and iodoacetamide failed to dissociate the ribosomes even at relatively high concentrations. The rate of dissociation of ribosomes by PCMB varied with the concentration of ribosomes. A progressive decrease in the rate of dissociation was observed as the concentration of ribosomes in the reaction mixture was increased. The PCMB-induced ribosomal subunits were unable to reassociate into 70S monomers unless they were dialyzed against buffer containing beta-mercaptoethanol. On the average, four molecules of PCMB per 70S ribosome and two molecules of PCMB per each 30S and 50S subunit were bound. The number of PCMB molecules bound per ribosome did not change with increasing concentrations of PCMB, even though higher concentrations of PCMB resulted in dissociation of ribosomes into subunits.     

Aminoacyltransferase I-catalysed binding of phenylalanyl-transfer ribonucleic acid to muscle ribosomes from normal and diabetic rats

The aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA (unfractionated Escherichia coli B tRNA acylated with radioactive phenylalanine and 19 non-radioactive amino acids) to skeletal-muscle ribosomes from diabetic rats was less than that to ribosomes from normal rats when the Mg(2+) concentration was low (7.5mm); whereas just the reverse was true when the concentration of the cation was higher (15mm). Thus the Mg(2+) dependency of aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA to ribosomes from normal and diabetic rats paralleled the effect of Mg(2+) concentration on synthesis of polyphenylalanine reported before. During incubation at 7.5mm-Mg(2+) phenylalanyl-tRNA was bound only to ribosomes bearing nascent peptidyl-tRNA. There are fewer such ribosomes in a preparation from the muscle of diabetic animals because diabetic animals synthesize less protein in vivo. Thus the difference in polyphenylalanine synthesis in vitro is adequately explained by the difference in enzyme-catalysed binding of phenylalanyl-tRNA to ribosomes, however, the basis of the difference in protein synthesis in vivo is still unknown.     

The attachment of polyuridylic acid to reticulocyte ribosomes

The attachment of polyuridylic acid to reticulocyte ribosomes was studied by using polyadenylic acid, which inhibits the attachment reaction only, while permitting translation of polyuridylic acid bound to ribosomes. After addition of polyadenylic acid the amount of polyphenylalanine synthesized under standard conditions was taken as a measure of the bound polyuridylic acid. In this way certain parameters of the attachment reaction and the subsequent translation of attached polyuridylic acid were defined: (1) polyuridylic acid-ribosome interaction at 37 degrees requires only Mg(2+) at an optimum concentration of 8mm; (2) K(+) (required for translation) is a non-competitive inhibitor of the attachment reaction; (3) optimum polyphenylalanine synthesis directed by attached polyuridylic acid occurs at 5mm-Mg(2+) concentration; (4) from kinetic studies single ribosomes appear to participate in the attachment reaction.     

Functional modification of rat liver ribosomes by the in vitro action of N-methyl-N'-nitro-N-nitrosoguanidine

The mechanism by which N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) inhibits protein synthesis has been studied in a rat liver cell free system. Using preformed aminoacyl-tRNA it was observed that incorporation of amino acid into polyribosomal protein was inhibited in the presence of low concentration of MNNG. This inhibition was not reversed by increasing the concentration of soluble factors. Transfer RNAs modified previously by treatment with MNNG and subsequently esterified with amino acids were transferred to polyribosomes with the same efficiency as those species which were not modified. Polyribosomes, on the other hand, lost activity to incorporate amino acids after pretreatment with MNNG. This inactivation was dependent on the concentration of MNNG with which polyribosomes were treated. When poly(U) was used with MNNG-treated polyribosomes, its translation, after correction for endogenous translation, was also found to be significantly low as compared to the case with untreated polyribosomes. Purified ribosomes stripped of endogenous mRNA when treated with increasing concentrations of MNNG progressively lost ability to support polyphenylalanine synthesis programmed by poly(U). The treated ribosomes, however, neither inhibited the activity of control ribosomes nor induced any loss of fidelity of translation by poly(U). It is concluded that MNNG inhibits protein synthesis through functional inactivation of ribosomes resulting from direct modification of ribosomal proteins possibly involving nitroguanidination of lysine residues.     

Soluble factor requirements for the Tetrahymena peptide elongation system and the ribosomal ATPase as a counterpart of yeast elongation factor 3 (EF-3)

Peptide elongation factor 3 (EF-3), which is widely present in yeasts and fungi (Eumycota), does not occur in another lower eukaryote, the unicellular protozoan Tetrahymena pyriformis, as was shown by the following findings: (a) there is no activity to satisfy the EF-3 requirement of yeast ribosomes in the post-ribosomal supernatant fraction from Tetrahymena, and (b) the Tetrahymena ribosomes displayed their full capacity for polyphenylalanine synthesis with purified EF-1 alpha and EF-2 alone from either Tetrahymena or yeast, and their activity on the Tetrahymena ribosomes was not further enhanced by the addition of yeast EF-3, in contrast to the case of the yeast ribosomes. However, as a substitute for the ribosome-activated nucleotidase activity of EF-3, Tetrahymena ribosomes were shown to harbor strong, firmly bound ATPase and GTPase activities, which probably involve the same active site. The ribosome-bound ATPase activity was inhibited by a polyclonal antibody raised against yeast EF-3 with the same inactivation profile as that of polyphenylalanine synthesis on Tetrahymena ribosomes, indicating that the ribosomal ATPase plays an essential role in the elongation process on Tetrahymena ribosomes as previously revealed in the yeast system. It was also shown that the ribosomal nucleotidase plays a pivotal role in the elongation cycle in other eukaryotes.     

A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.     

Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes.

Mice were immunized against ribosomal acidic proteins P1 and P2 from Artemia salina, and three kinds of monoclonal antibodies were isolated. One recognized P0 in addition to both P1 and P2 (anti-P). The other two recognized either P1 (anti-P1) or P2 (anti-P2) specifically and did not recognize P0. The anti-P antibody, but not anti-P1 or anti-P2, recognized a 22-amino acid peptide corresponding to the carboxyl-terminal sequence common to P1 and P2. This antibody, but not the others, inhibited poly(U)-directed polyphenylalanine synthesis. The anti-P1 bound to ribosomes but failed to inhibit polyphenylalanine synthesis: the anti-P2 did not bind to ribosomes at all. The anti-P and its Fab fragments inhibited the elongation step of protein synthesis, namely, the binding of elongation factors 1 alpha and 2 to ribosomes as well as their ribosome-coupled GTPase activities. Anti-P had little effect on the nonenzymatic phenylalanyl-tRNA binding to ribosomes and on peptidyltransferase activity. These results suggest the functional importance of the homologous carboxyl-terminal region of the three P proteins for the interaction of the ribosome with the two elongation factors. The epitope of anti-P1 must reside in a region of the protein which is not directly involved in its function.     

Reconstruction of the smaller subparticle of rabbit ribosomes from core-particle and split-protein fractions.

The smaller subparticle of rabbit reticulocyte ribosomes was shown to yield core-particle and split-protein fractions on treatment with 2.5 M-NH4Cl/61 mM-MgCl2. The core-particle fraction was inactive in poly(U)-directed polyphenylalanine synthesis, but activity was restored after recombination with the split-protein fraction. Optimum ionic conditions for the reconstruction of active subparticles were found to be 0.75 M-NH4Cl/19 mM-MgCl2 at 0 degrees C. Improved extents of reconstruction were obtained when the core-particles were isolated by methods that avoided pelleting. Core-particles isolated from subparticles pretreated with either proteinases or ribonucleases had diminished capacity to become re-activated.     

Dissociation of ribosomes and seed germination

Ribosomes from rice embryos (Oryza sativa) were dissociated into ribosomal subunits in vitro by systematic reduction of the Mg²⁺ concentration. Ribosomes from imbibed (28 C) embryos were more easily dissociated than those from nonimbibed embryos. This was not observed with ribosomes from either imbibed, nonviable embryos, or from embryos imbibed at 0 C. Ribosomes from embryos which had been imbided and subsequently dehydrated resembled ribosomes from nonimbibed embryos in their resistance to dissociation. The change in the resistance to dissociation was essentially complete after the first 20 minutes of imbibition at 28 C, and accompanied activation in vivo of protein synthesis as determined by amino acid incorporation in vitro. Ribosomes from either imbibed or nonimbibed embryos could be dissociated into subunits by 0.5 m KCl. These subunits were separated by density gradient centrifugation, and, if recombined, were active for polyphenylalanine synthesis in vitro. The individual subunits prepared from nonimbibed embryos could be replaced by the corresponding subunit fraction from imbibed embryos without loss of capacity to support polyphenylalanine synthesis. The change in the ease of dissociation of ribosomes appears to be a physiological process, and its possible relationship to the initiation of protein synthesis during seed germination is discussed.     

Interaction of arginine with the ribosomal peptidyl transferase centre.

Arginine inhibits the formation of acetylleucyl-puromycin from C(U)-A-C-C-A-LeuAc and puromycin ('fragment reaction'), catalized by Escherichia coli and yeast ribosomes. From 18 different L-amino acids assayed, arginine was the most effective in producing inhibition (50% inhibition at 20 mM, with 1 mM puromycin). L-Argininamide and D-arginine gave about the same inhibition as L-arginine. The inhibition by L-arginine is competitive with respect to puromycin. The plot of the slopes obtained in a Lineweaver and Burk representation versus [Arg]2, and the plot of 1/v versus [Arg]2 at a fixed concentration of puromycin, are linear, which seems to indicate that two arginine molecules must interact at the puromycin binding site to produce inhibition. In addition to the 'fragment reaction', arginine inhibits the non-enzymatic binding of AcPhe-tRNA, C(U)-A-C-C-A-Leu and C(U)-A-C-C-A-LeuAc to ribosomes. However, it does not inhibit poly(U)-directed polyphenylalanine synthesis or the reaction of puromycin with AcPhe-tRNA previously bound to the peptidyl site. The results agree with arginine binding to the acceptor site, and with a sequential mechanism for the 'fragment reaction', puromycin binding first.     

Resistance of the peptidyltransferase centre of rabbit ribosomes to attack by nucleases and proteinases.

Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either ribonucleases (I or T1) or proteinases (trypsin or chymotrypsin), and their capacity to function in poly(U)-directed polyphenylalanine synthesis and in the puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-protein fractions were also examined. The protein moiety of proteinase-treated L-subparticles was analysed by one-dimensional sodium dodecyl sulphate/polyacrylamide- and two-dimensional polyacrylamide-gel electrophoresis. The introduction of 16--100 scissions in the RNA moiety had no effect on the activity of the L-subparticles in polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-salt-shock core-particles and split-protein fractions. In contrast, L-subparticles treated with low amounts of trypsin (0.56 ng of trypsin/microgram of L-subparticle) were inactive in polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the puromycin reaction was increased by 70% as a result of trypsin treatment (280 ng of trypsin/microgram of L-subparticle). At least two of the acidic proteins implicated in the translocation function were not affected by trypsin treatment. Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The protein(s) needed for S-subparticle binding were shown to be present in high-salt-shock cores. At least six proteins associated with the core-particles were attack during trypsin treatment of L-subparticles. An examination of L-subparticles isolated from trypsin-treated polyribosomes showed that the amount of trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by trypsin to fragments of approx. 40 000 daltons. This protein was also cleaved by chymotrypsin.     

Effects of sulfhydryl inhibitors on depolarizations-contraction coupling in frog skeletal muscle fibers

We have studied the effects of the sulfhydryl reagents on contractile responses, using either electrically stimulated single muscle fibers or short muscle fibers that were voltage-clamped with a two-microelectrode voltage-clamp technique that allows the fiber tension in response to membrane depolarization to be recorded. The sulfhydryl inhibitors para- chloromercuribenzoic acid (PCMB) and parahydroximercuriphenyl sulfonic acid (PHMPS), at concentrations from 0.5 to 2 mM, cause loss of the contractile ability; however, before this effect is completed, they change the fiber contractile behavior in a complex way. After relatively short exposure to the compounds, < 20 min, before the fibers lose their contractile capacity, secondary tension responses may appear after electrically elicited twitches or tetani. After losing their ability to contract in response to electrical stimulation, the fibers maintain their capacity to develop caffeine contractures, even after prolonged periods (120 min) of exposure to PHMPS. In fibers under voltage-clamp conditions, contractility is also lost; however, before this happens, long-lasting (i.e., minutes) episodes of spontaneous contractile activity may occur with the membrane polarized at -100 mV. After more prolonged exposure (> 30 min), the responses to membrane depolarization are reduced and eventually disappear. The agent DTT at a concentration of 2 mM appears to protect the fibers from the effects of PCMB and PHMPS. Furthermore, after loss of the contractile responses by the action of PCMB or PHMPS, addition of 2 mM DTT causes recovery of tension development capacity.     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.     

The use of synthetic tRNAs as probes for examining nascent peptides on Escherichia coli ribosomes.

The polyuridylic acid-dependent syntheses of polycysteine and polyserine were carried out on Escherichia coli ribosomes using two new synthetic tRNA species. The peptides were initiated with N-acetyl or N-acyl coumarin derivatives of either Ser-tRNA or Phe-tRNA. The properties of the resulting nascent peptides were compared to those of nascent polyphenylalanine chains synthesized under similar conditions. This was accomplished by following changes in the fluorescence properties of the probes covalently linked to the amino-terminus of each of the nascent polypeptides as they were formed on the ribosomes. Nascent polycysteine and polyserine peptides appeared quite different from those of polyphenylalanine, as indicated by the anisotropy of fluorescence from the amino terminal probe. In contrast to serine and cysteine peptides, the synthesis of all the polyphenylalanine peptides was insensitive to inhibition by erythromycin, even though these peptides were initiated with N-acyl serine. The results support the hypothesis that nascent polyphenylalanine peptides have atypical physical and chemical properties and demonstrate the utility of using modified tRNAs to study ribosome function and the synthesis of proteins.     

The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit

The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.     

The activity of the acidic phosphoproteins from the 80 S rat liver ribosome

The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes.     

Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes.

Factor requirements for yeast and rat liver ribosomes were determined in several different reactions using either yeast or liver factors. In polymerization assays yeast ribosomes required a factor in addition to elongation factor 1 (EF-1) and elongation factor 2 (EP-2). The third factor (EF-3) requirement was observed with EFs from either yeast or liver for both poly(U)-directed polyphenylalanine synthesis and elongation of endogenous peptidyl-tRNA. No significant effect of EF-3 was observed with liver risomes in either assay. In contrast to results with polypeptide synthesis EF-3 was not required for EF-1 dependent binding of [3H]Phe-tRNA or the translocation-dependent formation of N-acetylphenylalanylpuromycin. Up to 2-fold stimulation of the binding reaction was observed with saturating levels of either yeast or liver EF-1. No effect of EF-3 was observed on ribosome-EF-2-GDP-fusidic acid complex formation. The data suggest that the yeast EF-3 may be a loosely bound ribosomal protein which is not required for a specific step in the elongation cycle but is involved in the coordination of the partial reactions required for polymerization.     

The nature of the differential inhibition of polyphenylalanine synthesis in extracts from Artemia salina and rabbit reticulocytes by the Phytolacca americana protein

The difference in sensitivity of polyphenylalanine synthesis in extracts from Artemia salina and rabbit reticulocytes to inhibition by the Phytolacca americana protein (PAP) has been found to be linked to the source of the supernatant enzyme fraction and not the ribosomes. In the presence of reticulocyte supernatant enzyme fraction polyphenylalanine synthesis is less sensitive to inhibition by PAP than that observed in the presence of A. salina supernatant enzyme fraction. The results suggest that reticulocyte elongation factor 2 is responsible for this effect.     

Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.     

Protein synthesis in the liver of rats injected with cholesteryl 14-methylhexadecanoate

1. Rats were injected intraperitoneally with cholesteryl 14-methylhexadecanoate and killed after various intervals of time up to 3 days; ribosomes and cell sap were isolated from their liver tissue. These fractions were tested for their ability to participate in protein synthesis. 2. Protein synthesis in complete systems containing ribosomes, cell sap and all necessary cofactors was significantly enhanced at 12 and 72h after the injection and significantly inhibited at 24h. At early times after injection isolated ribosomes had a slightly enhanced ability to bind nRNA. Peptide-elongation processes (i.e. binding of aminoacyl-tRNA to ribosomes, peptidyl transfer and polyphenylalanine synthesis) showed significant stimulation or inhibition depending on the time after injection of the ester. 3. A correlation was found between the ability of cell sap to stimulate polyphenylalanine synthesis and the relative cholesteryl 14-methylhexadecanoate content in the postmicrosomal supernatant at different time-intervals after administration of the ester. No significant changes were found in its content in the whole liver tissue. 4. Since the injected ester has previously been shown to accumulate in some enzymic fractions, the changes in its relative content may represent a regulatory mechanism modulating the rate of protein synthesis.