Transcortin in rat kidney tissue: distribution in microsomal fractions

During chromatography of renal tissue cytosolic proteins on DEAE-cellulose the protein specifically binding [3H]corticosterone is eluted within the potassium phosphate concentration range of 0.08-0.10 M. Analysis of kidney slices revealed the synthesis of [3H]transcortin whose electrophoretic mobility was close to that of the blood plasma protein. Using radioimmunochemical methods, it has been found that transcortin-specific [125I]IgG antibodies interact with growing polypeptide chains of membrane-bound polyribosomes. Free polyribosomes do not bind antibodies against transcortin.     

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro.

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.     

Endogenous ADP-ribosylation of elongation factor 2 in polyribosome fraction of rabbit reticulocytes

Several polypeptides of about 120, 96, 85, 60 and 38 kDa are shown to be radiolabeled during incubation of the mono- and polyribosome fraction of rabbit reticulocytes with [32P]NAD. Among them is a polypeptide coinciding with elongation factor 2 (EF-2) in its electrophoretic mobility in SDS-polyacrylamide gel. The addition of pure EF-2 to the polyribosome fraction results in an increase of the radioactive label in this polypeptide band. From this it is concluded that both endogenous and added EF-2 is ADP-ribosylated by an enzyme associated with polyribosomes. A possibility of regulation of protein synthesis through endogenous ADP-ribosylation in vivo is considered.     

Ferritin mRNA is found on bound as well as on free polyribosomes in rat heart

Free and endoplasmic reticulum-bound polyribosomes from rat heart were examined for their ferritin mRNA content. A procedure for separation and purification of the two ribosome populations that produced good yields of homogeneous mono- and polyribosomes with no contaminating ultrastructures and gave distinctive sedimentation profiles in 15-50% sucrose gradients was developed. 14C-labeled free and bound polyribosomes added to heart preparations indicated that only 3% of free and 5.5% of bound polyribosomes cross-contaminated the bound and free fractions, respectively. RNA from both polyribosome populations hybridized with [32P]cDNA for rat ferritin. The extent of hybridization with mRNA from endoplasmic reticulum (ER)-derived polyribosomes was much greater than what could be accounted for by cross-contamination with free polyribosomes. This indicates that heart ferritin is synthesized not only on free polyribosomes for internal use in iron storage but also on ER-bound polyribosomes, where it may be destined for secretion into the plasma.     

ROLE OF RIBONUCLEASE ACTION IN PHENYLALANINE-INDUCED DISAGGREGATION OF RAT CEREBRAL POLYRIBOSOMES

—l -phenylalanine (1 mg/g body wt) or physiological saline (0.9% NaCl) was given intraperitoneally to infant (7-day old), immature (14-day old), and adult (42-day old) rats. The state of ribosomal aggregation was determined in the cerebral postmitochondrial supernatant and purified polyribosome fractions prepared in the presence of rat liver ribonuclease inhibitor. Polyribosomes isolated from cerebral cortices of infant and immature rats 30 or 60 min after administration of phenylalanine were partially disaggregated, whereas the state of aggregation of polyribosomes from mature cerebrum was unchanged. In contrast, little or no evidence of phenylalanine-induced polyribosome disruption was noted in the postmitochondrial supernatant fractions, from which the cerebral polyribosomes were prepared, in any of the animals. Omission of the ribonuclease inhibitor resulted in polyribosome disaggregation in the postmitochondrial supernatant fractions prepared from saline-treated as well as phenylalanine-treated infant rats, but the disruption was more profound in the latter group. Ribonuclease activities in cerebral postmitochondrial supernatant preparations from infant and immature rats were higher than the corresponding values in preparations from adult animals. In addition, the administration of phenylalanine resulted in increases in ribonuclease activities in cerebral postmitochondrial supernatant preparations from the younger animals, but had no effect on these activities in adult animals. These results suggest that alterations in structure and function of polyribosomes from the infant rat cerebrum following a loading dose of phenylalanine were related to exposure of the polyribosomes during isolation to elevated activities of cerebral ribonucleases resulting from this treatment. This hypothesis was supported by the finding that phenylalanine treatment had no effect on the incorporation in vivo of intracisternally-administered radioactive lysine into total, soluble or ribosomal protein of infant cerebrum. However, when cerebral ribosomal RNA was differentially labelled in phenylalanine-treated and saline-treated infant rats by the intracisternal administration of [³H] or [¹⁴C]uridine, and polyribosome fractions were then prepared from the pooled cerebral cortices of both groups, radioactive ribosomes derived from saline-treated rats were more highly aggregated than those derived from phenylalanine-treated animals. It is concluded that gross alterations in cerebral polyribosome structure and function do not occur in vivo in young rats given a large amount of phenylalanine intraperitoneally. However, this treatment, in addition to increasing ribonuclease activity in cerebral cell-free preparations, also sensitizes cerebral polyribosomes to subsequent breakdown upon exposure to ribonucleases during isolation.     

Polyribosome size analysis. Measurement of number-average polyribosome sizes

The analysis of translational efficiencies of specific mRNAs requires a determination of the polyribosome size. The appropriate value to use in such calculations is the number-average size. A method is described for accurately measuring the number-average size of total and of specific protein synthesizing polyribosomes using isokinetic sucrose density gradients and 125I-labeled antibodies. By this method, we demonstrated that albumin synthesizing polyribosomes from a serum albumin secreting mouse hepatoma cell line exist over a broad range from trimers to 20-mers (mean 6-10). The specificity of antibody interaction with polyribosomes was demonstrated using cells not synthesizing mouse serum albumin, and by demonstrating that 125I-anti ovalbumin does not bind to mouse hepatoma polyribosomes. Treatment of the mouse hepatoma cells with 1 MUM cycloheximide shifted practically all of the monomers into polyribosomes resulting in an increase in the number-average size of the albumin synthesizing polyribosomes. Cycloheximide treatment, however, did not eliminate the size heterogeneity in the albumin synthesizing polyribosomes.     

Transport and intracellular localization of cortisol-asialotranscortin complexes in rat liver

The influence of desialylation of human transcortin on transport of the transcortin-cortisol complex into the liver cells and its intracellular distribution was investigated in perfused rat liver. Under experimental conditions used the half-time of cortisol in perfusion medium was decreased more than 200 times in presence of asialotranscortin compared to that of native transcortin. Experiments with [3H]cortisol and [131I]asialotranscortin demonstrated a simultaneous uptake of cortisol and asialotranscortin by the hepatocytes. Distribution of [3H]cortisol and [131I]asialotranscortin in subcellular fractions showed the following pathway of cortisol-asialotranscortin complex: cell membrane, lysosomes, cytoplasm. The complex dissociates in lysosomes.     

Polyribosomes, isolated from rat liver in a low ionic strength medium, capable of autonomic translation in a cell-free system without the addition of cellular fluid

Polyribosomes isolated from the liver in the presence of 10 mM KCl and purified by centrifugation through 2 M sucrose were shown to incorporate [3H]leucine both into aminoacyl-tRNA and polypeptides in a cell-free system without cell sap. The incorporation of [3H]leucine showed a linear increase within 80-100 min and was then levelled off. The system was sensitive to cycloheximide, puromycin and ethionine and needed ATP, GTP and unlabeled amino acids. The quantitation of tRNA in polyribosomes (the fraction which did not sediment with the subparticles after polyribosome dissociation) revealed more than two tRNA molecules per 80S monosome. It is likely that this tRNA excess as well as the earlier established presence of aminoacyl-tRNA synthetases and elongation factors promote the autonomic translation of polyribosomes.     

Transport and intracellular distribution of components of the cortisol-asialotranscortin complex in the liver

The effect of desialation of human transcortin on the transcortin-cortisol complex transport into hepatocytes and on subsequent intracellular distribution of the complex components was studied in model experiments with perfused rat liver. It was demonstrated that in the presence of desialated transcortin the time of [3H]cortisol incubation in the perfusion medium decreased more than 200 times as compared with the native protein. [3H]cortisol and [131I]asialotranscortin trapping by hepatocytes occurs simultaneously. The content of the [3H]cortisol--[131I]asialotranscortin complex in rat liver plasma membranes reaches a maximum 3 min after beginning of perfusion. Then the intact complex is transported into lysosomes, where it dissociates with a subsequent release of asialotranscortin and cortisol metabolites into the cytoplasm.     

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2-3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[(14)C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.     

Diversity and nature of ribosomal pools in hepatoma 7800 and host liver

1. The ribosomal components in the postmitochondrial supernatant of a rat hepatoma (hepatoma 7800) and the corresponding host liver were examined for diversity and functional competence. 2. The ;free' and ;membrane-bound' polyribosomes of both tissues were equally active in vivo and had equilibrated with newly synthesized ribosomes 4hr. after administration of [6-(14)C]orotic acid. 3. The inactive monomer-dimer pool in hepatoma 7800 was unattached to membranes and a larger fraction of the polyribosomes was free in hepatoma than in liver. 4. By using sensitivity to puromycin as a criterion, evidence was obtained that most of the polyribosomes in hepatoma 7800 were active in vivo. 5. Actinomycin, azaguanine and carbon tetrachloride caused marked conversion of polyribosomes into inactive monomers and dimers in the host liver and moderate conversion in the hepatoma. 6. Significant accumulation of ferritin and shifts in the mean polyribosome size to the lighter species occurred in the host liver of rats bearing large hepatomas.     

Three-Dimensional Organization of Polyribosomes–A Modern Approach

Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed tworow forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.     

Phosphorylation of elongation factor 1 in polyribosome fraction of rabbit reticulocytes

A single protein, Mr approximately 50000, is shown to be phosphorylated during incubation of a mono- and polyribosome fraction of rabbit reticulocytes with [gamma-32P]ATP at a low ionic strength. This protein has been identified as the elongation factor 1 alpha (EF-1 alpha). The phosphorylated EF-1 alpha, in contrast to the unmodified factor, is not detected in complexes with mono- and polyribosomes. It is suggested that the phosphorylation of EF-1 alpha can result in its decompartmentation from polyribosomes and thus affect the rate of protein synthesis.     

Isolation of preparative amounts of polyribosomes from normal rabbit and guinea pig spleen]

Preparative amounts of polyribosomes were isolated from normal rabbit and guinea pig spleen; up to 40 optical units of the polyribosome preparation could be obtained by centrifugation in a Spinco L-2B centrifuge with SW-27 rotor. The amount of polyribosomes isolated from spleens of immune animals was 2-3 higher than that isolated from normal animal spleens. Concentration of polyribosomal preparations by lyophylization and the storage of dried preparations do not alter the sedimentation properties of the polyribosomes. The distribution pattern of normal rabbit spleen polyribosomes in a linear sucrose gradient and the sedimentation constants of the polyribosome peaks are in good agreement with data reported by some other authors for plasmocytome polyribosomes. Using electrophoresis in agarose-polyacrylamide gel the radioactive proteins synthesized in the cell culture of normal rabbit spleen it was shown that in normal spleen the average amount of globulins makes up to 35% of total protein synthesis, as reported by some authors.     

POLYRIBOSOMES IN PROLIFERATING AND NONPROLIFERATING ROOT MERISTEM CELLS

Pea root meristem cells which are actively dividing contain higher proportions of polyribosomes than do cells whose progression through the mitotic cycle has been arrested by starvation. The initiation of cell division which follows the addition of sucrose to starved roots is accompanied by an increase in polyribosome content. This reformation of polyribosomes is insensitive to actinomycin D; it is suggested that preexisting mRNA molecules, synthesized prior to starvation, participate in polyribosome reformation, but that those mRNA species necessary for movement of cells through the mitotic cycle are gradually lost during the starvation period.     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

Identification and properties of renal mineralocorticoid receptors in relation to glucocorticoid binders in rat liver and kidney.

The binding of the natural mineralocorticoid aldosterone and the glucocorticoid corticosterone to macromolecules in rat liver and kidney cytoplasmic fractions was compared by various chromatographic procedures. Equilibration of kidney cytosol with 10nM-aldosterone, either alone or in the presence of a competing steroid, was ideal for ionexchange chromatography of DEAE-cellulose DE-52, and revealed the presence of four sorts of binding components. One of these, eluted in the 0.001M-phosphate pre-wash, and another, less abundant, forming a peak at 0.006M-phosphate, did not bind corticosterone at equimolar concentrations, and appear to constitute the mineralocorticoid-specific 'MR' receptor in rat kidney. They could not be detected in the liver. Radioactivity eluted in the 0.02 and 0.06M-phosphate regions on DEAE-cellulose DE-52 appears to be due to [3H]aldosterone binding to glucocorticoid-specific 'GR' receptors and to transcortin respectively, since labelling was greater with corticosterone even at 10 nM than with the mineralocorticoid at 100nM and since [14C]corticosterone bound to blood serum transcortin was always co-chromatographed in the 0.06M-phosphate region. These two components appear to be identical with those in the liver and could be labelled maximally only by 100nM-corticosterone. The separation between specific mineralo- and glucocorticoid-binding species was less clear when chromatography was attempted on DEAE-Sephadex A-50 columns, possibly because of disaggregation into subunits in the presence of the high KC1 concentrations required for elution. Competitive binding followed by filtration through Sephadex G-200 gel indicated that cellular MR binders, unlike GR receptors, exist mostly as high-molecular-weight aggregates, although both appear to exhibit a comparable monomeric molecular weight of approx. 67000.     

Investigation of the corticosteroid receptor system in rat hippocampus by ion exchange fast protein liquid chromatography

In order to study the receptor system for adrenocortical steroids, hippocampal cytosolic preparations--containing both type I and type II receptors--were subjected to anion exchange fast protein liquid chromatography (FPLC). With running buffer containing Tris, EDTA, and glycerol three peaks (1-3) were eluted from the column at 220, 400 and 560 mM NaCl respectively regardless of whether [3H]corticosterone or [3H]RU 28362 had been used as radiotracer. None of the peaks was caused by serum transcortin as revealed by control studies. However, the sequestering influence of transcortin on receptor binding of corticosterone could be demonstrated by the FPLC technique with mixtures containing serum and hippocampus cytosol. Competition experiments with cytosolic samples revealed that type I receptor was present only in peaks 2 and 3 while type II was found in all three peaks in variable amounts, depending on the presence of molybdate. When molybdate was added to the running buffer only two peaks (2 and 3) were eluted, both containing type I and type II receptors. Peak 1 was attributed to the activated type II receptor while peak 2 represented nonactivated receptors. The origin of peak 3 remains uncertain. The data indicate that molybdate must be present in the cytosolic preparation and in the running buffer to keep type II receptor in its nonactivated form. Type I receptor was probably not transformed into the activated form in the absence of molybdate but lost binding capacity and/or affinity for corticosterone.     

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.