Poly(A) polymerase activity in ethionine-intoxicated rats: the relative effectiveness of disaggregated and intact polyribosomes as primers for poly(A) polymerase

Ethionine intoxication causes a change in the metabolism of poly(A) sequences on the 3' OH terminus of mRNA in rat liver in vivo. In an attempt to determine the factors responsible for these changes, nuclear and cytoplasmic poly(A) polymerase activities and the state of the primer were examined in vitro. Requirements for optimal enzyme activities were determined. The nuclear and cytoplasmic enzymes had different K+, Mn2+, and poly(A) primer optima. The levels of nuclear and cytoplasmic poly(A) polymerase activity were shown to decrease following ethionine intoxication. Poly(A)+ RNA isolated from the livers of saline- and ethionine-treated rats served equally well as primers for the cytoplasmic poly(A) polymerase.Disaggregated polysomes were seven times more effective as primers than were intact polysomes. The results suggest that the mRNP particle which is released from polysomes as a result of ethionine intoxication functions better as a poly(A) polymerase primer than does the intact polysome.     

Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.     

Segregation of specific classes of messenger RNA into free and membrane-bound polysomes

Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA . mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane-bound polysomes contains a diverse set of messengers with a minimum of 500--2000 different species although approximately 5--8 messengers may constitute 25--30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA-bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane-bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These findings are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes.     

Comparison of the Effect of Intravenous Administration of d-Lysergic Acid Diethylamide on Free and Membrane-Bound Polysomes in the Rabbit Brain

Abstract: The intravenous administration of LSD to young adult rabbits resulted in the disaggregation of both free and membrane-bound classes of brain polysomes. Based on the analysis of LSD dosage and the time course of the LSD-induced brain polysome shift, it was found that free polysomes were more sensitive to the drug than the membrane-bound polysome fraction. LSD-induced hyperthermia may be involved in the disaggregation of free and membrane-bound polysomes, since a correlation was found between the extent of LSD-induced hyperthermia and the degree of brain polysome shift. Prevention of LSD-induced hyperthermia by maintaining the animal at 4°C blocked the disaggregation of both polysome classes. Induction of hyperthermia by elevation of ambient temperature also resulted in a shift in free and membrane-bound polysomes. In all cases the disaggregation of polysomes to monosomes was not caused by RNase activation. During polysome disaggregation, polyadenylated mRNA associated with both free and membrane-bound polysomes was not degraded but was relocalized from polysomes to monosomes.     

A simple and rapid procedure for high-yield isolation of essentially undegraded free and membrane-bound polysomes from rat liver

A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

RNA metabolism and membrane-bound polysomes in relation to globulin biosynthesis in cotyledons of developing field beans (Vicia faba L.).

In cotyledon cells of developing field beans the RNA content per cell does not change in the second half of developmental period 2, whereas globulin biosynthesis continues. The constant RNA content per cell results from an equilibrium between RNA synthesis and degradation. All types of RNA are synthesized until the end of globulin biosynthesis, but poly(A)-containing RNA was preferentially labelled during maximum globulin formation. During stage 2 of seed development of poly(A)-containing RNA fraction represents a discrete peak in the 12--18-S region on agarose gels and corresponds to the peak of poly(A)-containing RNA isolated from polysomes. alpha-Amanitin inhibits selectively the labelling of poly(A)-containing RNA and concomitantly globulin formation. Translation of total poly(A)-containing RNA, free and membrane-bound polysomes in a cell-free wheat germs demonstrates that the globulins are preferentially produced on membrane-bound polysomes and that poly(A)-containing RNA includes the mRNA for both vicilin and legumin.     

Translation in vivo and in vitro of proteins resembling apoproteins of rat plasma very low density lipoprotein.

Antibodies raised against rat plasma apoVLDL and a purified fraction of arginine-rich peptides (ARP) were labeled with Na125I and were shown to bind to polyribosomes isolated from rat liver. Antibody fractions enriched by selective affinity chromatography exhibited increased levels of binding to polysomes. Anti-apoVLDL immunoreactivity was further resolved into anti-ARP and anti apoB components, each reactive with a distinct polysome population. Binding was specific for rat polysomes, and was directed toward nascent polypeptide chains. About 2% of normal rat liver polysomes were recovered by indirect immunoprecipitation with anti-apoVLDL. Ribonucleic acid (RNA) extracted from this immunoprecipitate contained species with polyadenylate (poly[A] sequences characteristic of eukaryotic messenger RNA (mRNA). These species, purified by affinity chromatography on poly(U)-Sepharose, stimulated the in vitro synthesis of immunoprecipitable apoVLDL-like proteins by about 17-fold when compared to unfractionated rat liver mRNA. Most of the in vitro translation products precipitated by purified anti-ARP migrated identically on polyacrylamide gel electrophoresis with unlabeled purified ARP. Some implications of these findings with respect to plasma VLDL biosynthesis are discussed.     

Biosynthesis of membrane polypeptides of rat liver peroxisomes

The biosynthesis of three major peroxisomal membrane polypeptides of rat liver was investigated. Total hepatic RNA extracted by the guanidinium/CsCl method from three control and three di(2-ethylhexyl)phthalate (a peroxisomal proliferator)-fed rats was translated in vitro in a rabbit reticulocyte lysate protein-synthesizing system. Translation products were immunoprecipitated by the antibodies against peroxisomal membrane polypeptides, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and analyzed by fluorography. The in vitro translation products of 70, 26, and 22 kDa peroxisomal membrane polypeptides were apparently of the same size as the respective mature polypeptides. The ratio of translatable mRNA levels for the 70, 26, and 22 kDa polypeptides in di(2-ethylhexyl)phthalate-fed rats to those in control rats were 5.4, 11.4, and 2.7, respectively. The synthesis of these three polypeptides with the free polysome fraction from di(2-ethylhexyl)phthalate-fed rats was more active than that with the membrane-bound polysome fraction, whereas the synthesis of albumin with the free polysome fraction was 27% of that with the membrane-bound polysome fraction. These results indicate that the peroxisomal major membrane polypeptides are synthesized on free polysomes and transported to peroxisomal membrane without any apparent proteolytic processing, and that the induction of these polypeptides by administration of a peroxisomal proliferator corresponds well to the induction of the peroxisomal beta-oxidation enzymes. The data also support the idea that peroxisomes are organized from pre-existing peroxisomes.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.     

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [¹⁴C]- or [³H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10⁴ and 2.1·10⁴ were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with $\text{N-[}{}^3\text{H]ethylmaleimide}$ or $\text{N-[}{}^{14}\text{C]ethylmaleimide}$, and the ³H to ¹⁴C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.     

Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes

The kinetics of labeled histone mRNA entry into polysomes was studied in nuclease-treated reticulocyte lysates. Added mRNA rapidly bound 1 or 2 ribosomes. However, the formation of full size polysomes required at least 16 min. The amount of mRNA bound to ribosomes reached a maximum (73%) within 2 min after mRNA addition and then declined slowly for the remainder of the experiment. Two initiation inhibitors, aurintricarboxylic acid and 7-methylguanosine 5'-triphosphate, were found to affect polysome size and the fraction of mRNA in polysomes in an opposite manner. These results suggest that initiation and reinitiation events may be intrinsically different. The relatively long time period required for the formation of large polysomes can be explained by large polysomes having higher initiation and/or reinitiation rates or slower elongation rates. These possibilities are not mutually exclusive. The results suggest that there exist several levels of control which can regulate polysome size and the fraction of mRNA in polysomes.     

Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3.

The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growing Tetrahymena thermophila, and about 4% in starved cells. Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes. Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution. Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection. These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes. A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation. Hybridization of 3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here. These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phase Tetrahymena is loaded onto polysomes while most of the mRNA is starved cells exists in a non-polysomal form.     

Poly(adenylic acid)-containing and -deficient messenger RNA of mouse liver

RNA was isolated and fractionated into poly(A)-containing and -deficient classes by oligo(dT) chromatography. Approximately 99% of the poly(A) material bound to the oligo(dT); that which did not bind contained substantially shorter poly(A) chains. All RNA fractions retained an ability to initiate cell-free translation, with the poly(A)-deficient fraction containing half the total translational activity, i.e., mRNA. Two-dimensional polyacrylamide gel analysis of the cell-free translation products revealed three classes of mRNA: 1, mRNA preferentially containing poly(A), including the abundant liver mRNA species; 2, poly(A)-deficient mRNA, including many mid- and low-abundant mRNAs exhibiting less than 10% contamination in the poly(A)-containing fraction fraction; and 3, bimorphic species of mRNA proportioned between both the poly(A)-containing and -deficient fractions. Poly(A)-containing and bimorphic mRNA classes were further characterized by cDNA hybridizations. The capacity of various RNA fractions to prime cDNA synthesis was determined. Compared to total RNA, the poly(A)-containing RNA retained 70% of the priming capacity, while 20% was found in the poly(A)-deficient fraction. Poly(A)-containing, poly(A)-deficient, and total RNA fractions were hybridized to cDNAs synthesized from (+)poly(A)RNA. Poly(A)-containing RNA hybridized with an average R0t 1/2 approximately 20 times faster than total RNA. Poly(A)-deficient RNA hybridized with an average R0t 1/2 approximately 3-4 times slower than total RNA. These R0t 1/2 shifts indicated that in excess of three-quarters of the total hybridizable RNA was recovered in the poly(A)-containing fraction and that less than one-quarter was recovered in the poly(A)-deficient RNA fraction. Abundancy classes were less distinct in heterologous hybridizations. In all cases the extent of hybridization was similar, indicating that while the amount of various mRNA species varied among the RNA fractions, most hybridizing species of RNA were present in each RNA fraction. cDNA to the abundant class of mRNAs was purified and hybridized to both (+)- and (-)poly(A)RNA. Messenger RNA corresponding to the more abundant species was enriched in the poly(A)-containing fraction at least 2-fold over the less abundant species of mRNA, with less than 10% of the abundant mRNAs appearing inthe poly(A)-deficient fraction.     

Membrane-bound and free polysomes in transformed and untransformed fibroblast cells

A rapid and quantitative fractionation procedure has been used to measure the amounts of free and membrane-bound polysomes in growing and stationary Py3T3 and 3T3 (mouse) cells. A comparison of growing 3T3 and Py3T3 cells does not reveal any significant differences with regard to the ratio of the two polysome fractions. The amount of free and membrane-bound polysomes decreases in both 3T3 and Py3T3 cells as they approach the stationary state, an effect which is much more pronounced for free polysomes. At greatly reduced growth rates or in stationary cells, however, the amount of membrane-bound polysomes doubles in Py3T3 cells while it decreases even further in 3T3 cells. By contrast, the amount of free polysomes remains at a reduced level in both 3T3 and Py3T3 cells when cell multiplication is inhibited.Based on the hypothesis that membrane proteins are selectively synthesized on membrane-bound polysomes, an attempt is made to relate the results to accumulated data in the literature and discuss its possible significance with respect to the loss of growth control in Py3T3 cells.     

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.