Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

Comparative study on fucosylation of chicken liver and virus-induced hepatoma Mc-29

Comparative studies on fucoprotein metabolism of chicken liver and hepatoma Mc-29 have been carried out and the following parameters were determined: the incorporation rate of [14C]fucose into hepatoma and liver total tissue homogenate, acid-soluble and acid-insoluble fractions, acid-soluble nucleotide fraction and into plasma-membrane acid-precipitable fraction; the activity of microsomal and plasma-membrane fucosyltransferase; the electrophoretic pattern of hepatoma and liver plasma-membrane proteins and the incorporation of [14C]fucose into the glycoprotein fractions in both plasma-membrane preparations. It was found that the labelling of hepatoma tissue homogenate and plasma membranes was higher than that of the same liver preparations 3 hr after the [14C]fucose injection. This finding was supported by a considerably elevated hepatoma fucosyltransferase activity. The labelling rate of numerous fucoproteins from hepatoma plasma membranes was greatly increased and some of the individual glycoprotein bands were labelled to a higher extent compared with liver. The data presented show specific alterations of fucose and fucoprotein metabolism which could be considered as a characteristic feature of chicken viral-induced hepatoma Mc-29.     

The role of the plasma membrane in fatty acid uptake by rat liver parenchymal cells

1. Suspensions of isolated rat liver parenchymal cells incorporate [(14)C]palmitic acid into glycerides at about 40% of the rate obtained with liver slices. 2. At short time-intervals most of the incorporation is into phosphatidylcholine and this is recovered mainly in the plasma-membrane fraction. 3. At later times (5min to 2h) the [(14)C]palmitic acid is mainly found in triglyceride, but this is not recovered in the plasma-membrane fraction. 4. Addition of lysophosphatidylcholine increases incorporation of palmitic acid into both phosphatidylcholine and triglyceride, with maximum effect at about 0.1mm. 5. In vivo, 1min after injection of [(14)C]palmitic acid, radioactive phosphatidylcholine is concentrated in the plasma-membrane fraction, but the proportion present in this fraction declines rapidly. 6. The phosphatidylcholine of the plasma-membrane fraction has, at 1min after injection, a specific radioactivity 30-fold greater than that of the whole tissue. 7. This phosphatidylcholine reaches its maximum specific radioactivity before the tissue phosphatidic acid or diglyceride. 8. The phosphatidylcholine of the plasma-membrane fraction has a very rapid turnover. 9. It is proposed that the rapid formation of phospholipids in the plasma membrane is by acylation of their lyso-derivatives and the role of this process in fatty acid uptake is discussed.     

The biosynthesis of cytochrome c. Sequence of incorporation in vivo of [14C]lysine into cytochrome c and total proteins of rat-liver subcellular fractions

1. In order to determine the initial intracellular site of synthesis of cytochrome c in the liver cell, groups of rats were injected with [(14)C]lysine and killed 7.5, 15, 30 and 60min. later. The livers were homogenized in 0.3m-sucrose and subcellular fractions obtained. The mitochondrial fraction was further subfractionated. Pure cytochrome c was isolated from extracts of each fraction, obtained first with water at pH4.0 and then with 0.15m-sodium chloride. 2. A comparison of the kinetics of incorporation of [(14)C]lysine into total protein for each particulate fraction showed the usual two different kinds of kinetics. Incorporation into all the mitochondrial subfractions and the nuclear fraction rose gradually to a plateau value at about 20min., in contrast with that into the two microsomal fractions which rose rapidly to a peak value about seven times that for the mitochondrial fractions. The kinetics for the incorporation into mitochondrial cytochrome c showed a plateau value at 30min. about three times that for the total mitochondrial protein. There was no difference in the specific radioactivity of the mitochondrial cytochrome c extracted with water or 0.15m-sodium chloride or between the different mitochondrial subfractions. In contrast, the cytochrome c isolated from water extracts of the microsomal fractions had a lower specific radioactivity than that obtained from the 0.15m-sodium chloride extract. The specific radioactivity of the latter showed a rapid rise to a peak value about four times that for the mitochondrial cytochrome c, and the shape of the curve was similar to that for the total protein of the microsomal fraction. The results suggest that cytochrome c is synthesized in toto by the morphological components of the microsomal fraction. It seems first to be bound tightly to a microsomal particle, passing then to a looser microsomal binding and being finally transferred to the mitochondria. The newly synthesized cytochrome c in the mitochondrion could not be differentiated from the old by its degree of extractability at pH 4.0.     

The effect of electrical stimulation on the incorporation of l-[U-14C]valine into the protein of chopped tissue from guinea-pig cerebral cortex

1. Chopped tissue from guinea-pig cerebral cortex carried out an energy-dependent incorporation of [(14)C]valine into protein. 2. At all times studied the nuclear fraction of the homogenized tissue accounted for about 25% of the total labelled protein. 3. Electrical stimulation at first increased, but subsequently decreased, the rate of incorporation of [(14)C]valine into protein of the chopped tissue. 4. The initial increase in the incorporation of [(14)C]valine into protein occurred in the nuclear fraction. At later times electrical stimulation decreased the incorporation into all the subcellular fractions, but the relative contribution of the nuclear fraction to the total labelled protein increased. 5. These changes are discussed in relation to the changes in the rates of respiration, glycolysis, high-energy phosphate content and intracellular Na(+) and K(+) concentrations, which were measured under the same experimental conditions as those used to study protein synthesis.     

The kinetics of incorporation in vivo of [14C]acetate and [14C]carbon dioxide into the fatty acids of glycerolipids in developing leaves

1. The patterns of incorporation of (14)C into glycerolipid fatty acids of developing maize leaf lamina from supplied [1-(14)C]acetate and from (14)CO(2) during steady-state photosynthesis were similar. Oleate of phosphatidylcholine and palmitate of phosphatidylglycerol attained linear rates of labelling more rapidly than did other fatty acids, particularly the linoleate and linolenate of monogalactosyl diacylglycerol. 2. After the transfer of lamina from labelled to unlabelled acetate, there was a decrease in labelled oleate and linoleate of phosphatidylcholine and a concomitant increase in the amount of radioactivity in the linoleate and linolenate of monogalactosyl diacylglycerol. 3. The rapidly labelled phospholipids, phosphatidylcholine and phosphatidylglycerol, were shown by differential and sucrose-density-gradient centrifugation to be associated with different organelles, the former being mainly in a low-density membrane fraction, probably microsomal, and the latter mainly in chloroplasts. 4. During a 48h period after supplying spinach leaves with [(14)C]acetate, radioactivity was lost from the oleate of phosphatidylcholine present in fractions sedimented at 12000g and 105000g, and accumulated in the linolenate of monogalactosyl diacylglycerol of the chloroplast. 5. It is proposed that the phosphatidylcholine of some non-plastid membranes is intimately involved in the process of oleate desaturation and that this lipid serves as a donor of unsaturated C(18) fatty acids to other lipids, principally monogalactosyl diacylglycerol, of the chloroplasts.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

The distribution of surface antigens during fractionation of mouse liver plasma membranes

1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified gamma-globulin antibody fraction was iodinated with (125)I. The reaction of the (125)I-labelled gamma-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The gammaglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. ;Light' and ;heavy' mouse liver plasma-membrane subfractions bound similar amounts of gamma-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO(3)-Na(2)CO(3) buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro.

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5'-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

14C] Alanine incorporation into proteins and their contents in the tissue of intact and adrenalectomized rabbits after hydrocortisone and cortitropin administration

A single administration of hydrocortisone to intact rabbits increases the incorporation of [14C] alanine into proteins of the brain and liver tissue homogenates and soluble fractions as well as in blood plasma proteins and reduces the label incorporation into the brain tissue proteins and reduces its incorporation into the blood plasma proteins. Adrenalcetomy is followed by an increase in the incorporation of [14C] alanine into proteins of the brain and muscle tissue homogenates and soluble fraction and into proteins of blood plasma and liver tissue homogenates as well as by reducing the label incorporation into the spleen soluble fraction proteins. ACTH administered to adrenalectomized rabbits reduces incorporation of [14C] alanine into the brain and muscle tissue proteins, total proteins of liver tissue homogenate and increases it into the proteins of the spleen tissue soluble fraction. Multiple administration of the soluble fraction hormones both to intact and adrenalectomized rabbits inhibits the label incorporation into the studied tissue proteins. Parallel with the change in [14C] alanine incorporation into proteins under the hormones effect certain shifts in their contents were also established.     

Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies.

1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain.     

Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [(32)P]orthophosphate, [(14)C]choline and [(14)C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with (32)P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with (32)P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis.     

The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm.

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.     

A cyto-chemical study on the pancreas of the guinea pig. III. In vivo incorporation of leucine-1-C14 into the proteins of cell fractions

DL-leucine-1-C(14) was administered by intracardiac injection to guinea pigs and its in vivo incorporation into the proteins of various pancreatic cell fractions followed over a period of 2 hours. The pancreas was homogenized in 0.88 M sucrose and fractionated by differential centrifugation to give nuclear, zymogen, mitochondrial, microsomal, postmicrosomal, and final supernatant fractions. The proteins of these fractions, obtained by precipitation with trichloroacetic acid followed by washing, were counted. The proteins of the microsomal fraction showed the highest early specific activity and were followed by those of the zymogen and mitochondrial fractions. The microsomal fraction was broken up into two subfractions: one consisting of detached RNP particles, the other representing mainly the microsomal content and membranes. The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancreas of fasted animals. A tentative cytological picture of protein synthesis and transport based on these findings is presented.     

The uptake and intracellular distribution of [35S]trithiomolybdate in bovine liver in vivo

[35S] trithiomolybdate was administered intravenously to a group of four steer at two dose rates, 1 and 26 mg Mo per animal. Radioactivity appeared rapidly in the liver and was distributed in all the subcellular fractions examined. Examination by Sephadex G-100 gel-filtration of the cytosol fraction showed that distinct 35S-binding protein peaks were present. The protein-bound radioactivity was displaceable and was identified as [35S]thiomolybdates. No radioactivity eluted with metallothionein, but 35S was associated with the high molecular weight copper fraction, eluted in the void volume of the column, which increased transiently after the administration of the higher dose. It was suggested that the presence of protein-bound thiomolybdates in the liver gave rise to new ligands, which altered the equilibrium of copper between the different metal-binding proteins. This might be similar to the alteration in the copper-binding of albumin produced by the presence of thiomolybdates.     

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

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

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