Biosynthesis of ferritin in rat hepatoma cells and rat livers. II. Binding of iron by ferritin protein.

Ferritin and its protein subunits in rat hepatoma cell clone M-5123-C1 were biosynthetically labeled with [14C]leucine and 59Fe. Radioimmunoassays of ferritin/apoferritin and of protein subunits in the free polyribosome, membrane-bound polyribosome, smooth membrane, and cytosol fractions were done with ferritin-specific and subunit-specific rabbit IgG antibodies at various time intervals after pulsing. Much more 59Fe was bound by ferritin/apoferritin than by subunits in all of the cell fractions. Binding of iron to subunits may have been a random process. When hepatoma cells were simultaneously pulse-labeled with 59Fe and [14C]leucine, uptake of much of the 59Fe by ferritin occurred relatively early, in comparison to incorporation of [14C]leucine, in all of the cell fractions examined. Thus, 59Fe was readily incorporated into pre-existing ferritin. We conclude that most, if not nearly all, of the iron is incorporated after assembly of protein subunits.     

Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma.

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.     

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.     

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.     

Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver.

We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.     

Ornithine decarboxylase activity and DNA synthesis in Morris hepatomas 5123-C and 7800.

The activities of ornithine decarboxylase (ODC) and thymidine kinase (TK) and the rates of DNA synthesis were determined in hepatomas and livers of rats bearing Morris hepatoma 5123-C or 7800 and entrained to a schedule of 12 hours of light followed by 12 hours of darkness, with food (60% protein) available only during the first 2 hours of the dark period. ODC activity in hepatoma 5123-C displayed a diurnal oscillation, increasing 2-fold during the feeding period and then rapidly decaying to 20% of the peak level. The livers of rats bearing hepatoma 5123-C exhibited a similar oscillation of ODC activity, with peak values lower than in the hepatomas but higher than in the livers of control (non-tumor bearing) animals. TK activity and the rate of DNA synthesis in hepatoma 5123-C were low during most of the dark period but increased rapidly towards the end of the dark period. DNA synthesis reached a plateau at the dark-light interface and then rapidly declined, but TK activity remained high during the light period. Similar studies on hepatoma 7800 established that ODC activity in this hepatoma did not oscillate but remained at low levels throughout the day. Similarly, host livers of rats bearing hepatoma 7800 did not exhibit the diurnal oscillation of ODC activity characteristic of liver from control rats, but showed a slow increase in activity followed by a plateau and a slow decline to base-line levels. DNA synthesis in hepatoma 7800 was constant throughout the day, whereas TK activity may have increased during the dark period. In the livers of control rats and animals bearing hepatoma 5123-C or 7800, TK activity and rate of DNA synthesis were at low levels at all times studied and appeared not to oscillate.     

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.     

Distribution of ferritin mRNA and albumin mRNA between free and membrane-bound rat liver polysomes

Free and membrane-bound polyribosomes and their respective mRNAS were isolated from the livers of both normal rats and rats treated with a single large dose of iron a few hours before being killed. The membrane-bound and free polysomes were incubated in vitro with [³H]leucine and a pH 5 enzyme preparation, and peptide chains of albumin and ferritin were identified by immunoprecipitation. Albumin peptide chains were almost completely confined to the bound ribosomes, whereas ferritin peptide chains were found three times more frequently on free ribosomes than on bound ribosomes.Messenger RNA from each class of ribosome was translated in a wheat germ system, and the products were isolated by immunoprecipitation. Albumin mRNA was restricted almost exclusively to membrane-bound ribosomes. However, ferritin mRNA was equally abundant in the total mRNA of bound and free ribosomes. This finding contrasts with the low capacity of the bound ribosomes to synthesize ferritin in vitro, and suggests that the bound ribosomes of liver contain non-translated ferritin mRNA. Brief treatment of the rats with iron caused a sharp increase in the amount of nascent ferritin chains and l-ferritin mRNA in the free liver ribosomes, but failed to change the nascent chains or ferritin mRNA content of the bound polyribosome fraction.Only an apoferritin subunit of 19 000 daltons was formed by the free and membrane-bound ferritin mRNA. This has significance for the published observation of several sizes of protein subunits in ferritin isolated from tissue ferritins. These may represent modifications of the peptide chain after translation or artifacts of isolation.     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

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.     

Fasting does not abolish the diurnal oscillation of ornithine decarboxylase activity in Morris hepatoma 5123-C.

The activities of ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) were determined under conditions of feeding or fasting in the hepatomas and livers of rats bearing Morris hepatoma 5123-C. Prior to killing, the animals were entrained to a schedule of 12 hours of light followed by 12 hours of darkness with food (60% protein) available only during the first two hours of the dark period. With food available, ODC and TAT activities displayed diurnal oscillations in hepatomas and host livers, and in the livers of control (non-tumor bearing) animals, characterized by rapid increases in enzyme activity coincident with the onset of feeding followed by a decline to pre-feeding levels. When food was withheld the increase in ODC activity in host and control livers, and TAT activity in hepatoma, host and control livers was not evident. However, withholding food did not abolish the diurnal oscillation of ODC activity in hepatoma 5123-C.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

Iron prevents ferritin turnover in hepatic cells

It has long been assumed that iron regulates the turnover of ferritin, but evidence for or against this idea has been lacking. This issue was addressed using rat hepatoma cells with characteristics of hepatocytes subjected to a continuous influx of iron. Iron-pretreated cells were pulsed with [(35)S]Met for 60 min or with (59)Fe overnight and harvested up to 30 h thereafter, during which they were/were not cultured with ferric ammonium citrate (FAC; 180 microm). Radioactivity in ferritin/ferritin subunits of cell heat supernatants was determined by autoradiography of rockets obtained by immunoelectrophoresis or after precipitation with ferritin antibody and SDS-PAGE. Both methods gave similar results. During the +FAC chase, the concentration of ferritin in the cells increased linearly with time. Without FAC, the half-life of (35)S-ferritin was 19-20 h; with FAC there was no turnover. Without FAC, the iron in ferritin had an apparent half-life of 20 h; in the presence of FAC there was no loss of (59)Fe. Without FAC, concentrations of ferritin iron and protein also decreased in parallel. We conclude that a continuous influx of excess iron can completely inhibit the degradation of ferritin protein and that the iron and protein portions of ferritin molecules may be coordinately degraded.     

The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [(14)C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [(14)C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize alpha-lactalbumin. The polyribosomes were incubated in the presence of [(3)H]leucine and alpha-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of alpha-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

Intracellular transport of a newly synthesized asialoglycoprotein receptor in rat liver

Intracellular transport of a newly synthesized asialoglycoprotein receptor was studied biochemically using a monospecific antibody for the receptor. Pulse-labeling by intravenous injection of [3H]leucine and pulse-chasing after 10 min by cycloheximide injection resulted in the maximal labeling of the receptor in the rough microsomes at 15 min, in the smooth microsomes and the heavy Golgi subfraction (GF3) at 25 min and in the intermediate plus light Golgi subfraction (GF1+2) at 30 min. By 60 min, the labeling in GF1+2 had decreased and leveled off. In the plasma membrane fraction, the labeled receptor first appeared at 20 min, increased rapidly and also reached a constant level at 40-60 min. Intracellular movement of the newly synthesized receptor in the GF1+2 and plasma membrane fractions was also investigated by purifying the receptor protein from the GF1+2 and plasma membrane fractions by affinity chromatography. It was revealed that the specific radioactivities of the receptor in the two fractions become equilibrated after 60-120 min. The receptor of the various membrane fractions was also pulse-labeled in vivo for 20 min simultaneously with [3H]glucosamine and [14C]leucine, and pulse-chased for the following 40 min. After pulse-labeling for 20 min, the ratio of the radioactivity of [3H]glucosamine or [3H]sialic acid to [14C]leucine of the receptor from the rough and smooth microsomes, and GF3, GF2, and GF1 increased in that order. That of the receptor from the plasma membrane fraction was infinitely higher, because, while a significant amount of 3H-radioactivity was incorporated into the receptor in the Golgi apparatus, only a negligible amount of 14C-radioactivity was incorporated into the same receptor in the plasma membrane due to the delay in the arrival of [14C]leucine labeled receptor to the plasma membrane. After chasing for 40 min, however, the same radioactivity ratios of the GF1 and plasma membrane fractions approached each other. All these results strongly suggest that the distribution of the newly synthesized receptor becomes rapidly equilibrated between the trans-Golgi components and plasma membranes probably by repeated recycling of the receptor protein between the two membranes.     

The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.

Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.     

Pathways in the binding and uptake of ferritin by hepatocytes

The binding and uptake of rat liver ferritin by primary cultures of rat liver hepatocytes was studied in order to assess the relative importance of saturable, high-affinity pathways and nonspecific processes in the incorporation of the protein by the cells. To minimize artifacts, ferritin not subjected to heat treatment and labeled in vivo with 59Fe was used. Binding to cell membranes was estimated from incubations performed at 4 degrees C. After 2 h, when a steady state in cell-associated ferritin had been achieved, approx. 4-10(4) binding sites per cell were observed, with an affinity constant for ferritin of 1 x 10(9) M-1. At 37 degrees C, the maximal uptake from these sites was 1.3 x 10(5) ferritin molecules/cell per h. For ferritin molecules bearing an average of 2400 iron atoms, this uptake amounts to 5 x 10(6) iron atoms/cell per min. Half-maximal uptake was achieved at a ferritin concentration, or KM1, of 3 x 10(-9) M. Although uptake rates at least a thousand times greater could be achieved by binding to the much larger number of low-affinity sites, the apparent KM2 for such 'nonspecific' uptake was 4 x 10(-7) M. At ferritin concentrations up to 2 nM, at least 90% of ferritin bound and taken up by hepatocytes involves saturable, high-affinity sites, presumably true ferritin receptors.     

ATP- and ATP+ ubiquitin-stimulated proteolysis in rat liver and Yoshida ascites hepatoma.

To gain insight into the activity of cytosolic proteases in tumors, the ATP-dependent proteolysis of cell sap and the ATP- and ubiquitin-dependent proteolysis of Fraction II (a cytosolic subfraction freed of endogenous ubiquitin) were measured in the anaplastic Yoshida ascites hepatoma AH 130. Hepatoma cell sap showed only low, although significant, ATP-stimulated proteolysis, as best seen by comparisons with rat liver made on the basis of wet weight. Much of the basal proteolytic activity of cell sap and of its subfraction enriched in high Mr complexes (Fraction X) peaked near 18S in sucrose gradients. In contrast with cell sap, Fraction II from hepatoma degraded [14C]methylcasein more efficiently than Fraction II from normal liver, but the activities for liver and tumor did not differ on a wet weight basis. Altered polypeptide patterns shown by SDS-PAGE in the Yoshida hepatoma suggested that some abundant hepatoma-specific cytosolic protein might interfere with degradation of the [14C]methylcasein by hepatoma.     

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.