The significance of promitochondrial structures in rat liver for mitochondrial biogenesis

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4-5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3-4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm(3). 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

Observations on iron uptake, iron metabolism, cytochrome c content, cytochrome a content and cytochrome c-oxidase activity in regenerating rat liver

1. Differential and density-gradient centrifugation were used to fractionate mitochondria and fluffy layer from normal and regenerating rat liver. The iron, cytochrome a and cytochrome c contents and cytochrome c-oxidase activity were studied as well as the uptake of (59)Fe into protein and cytochrome c. 2. A certain degree of heterogeneity was evident between the heavy-mitochondrial and light-mitochondrial fractions, and in their behaviour during liver regeneration. 3. The specific content of light-mitochondrial iron and cytochrome a was 1.3-1.4 times that of heavy mitochondria. Changes in cytochrome c-oxidase activity closely followed those of cytochrome a content during liver regeneration, but not for light mitochondria after 10 days. 4. Radioactive iron ((59)Fe) was most actively taken up by well-washed light mitochondria during early liver regeneration. After 22 days fluffy layer became preferentially labelled. This substantiates the view that fluffy layer partially represents broken-down mitochondria. 5. During early regeneration, light-mitochondrial fractions separated along a density gradient were about 3 times as radioactive, and showed distinct heterogeneity of (59)Fe-labelling, in contrast with near homogeneity for heavy mitochondria. 6. Immediately after partial hepatectomy fractions corresponding to density 1.155 were 5-10 times as radioactive as particles of greater density. The radioactivity decreased sharply after 6 days. 7. These particles of low density possessed higher NADH-cytochrome c-reductase (1.5-5-fold) and succinate-dehydrogenase (1.1-2-fold) activities than typical mitochondrial fractions. Their succinate-cytochrome c-reductase and cytochrome c-oxidase activities were slightly lower. 8. The results are discussed in relation to mitochondrial morphogenesis, and a possible route from submitochondrial particles is suggested.     

Properties of porcine white adipose tissue and liver mitochondrial subpopulations.

Properties of porcine white adipose tissue heavy and light mitochondrial subpopulations were investigated so as to identify any functional heterogeneity. Liver mitochondrial subpopulations were concurrently evaluated since their properties have been studied in some detail. Mitochondrial subpopulations were isolated by means of differential centrifugation and the relative purity estimated using marker enzymes. Due to the greater contamination of the light mitochondrial fractions, mtDNA content, determined by PCR analysis, was used as a basis to demonstrate any mitochondrial heterogeneity. Enzymatic activity, electron microscopy, lipid analysis and Western blotting were used to characterise the different populations. With the exception of liver cytochrome c oxidase, the enzymatic capacity of adipose and liver heavy mitochondria ranged between approximately two- and threefold higher than the corresponding light fraction. The cardiolipin content and mean mitochondrial diameters paralleled these differences, suggesting an increased mitochondrial mass rather than a functional difference. However, the cytochrome c oxidase activity of the liver heavy mitochondria was 4.75-fold higher relative to the light fraction. A strong correlation between cytochrome c oxidase activity and the subunit I content was evident. Adipose tissue mitochondrial subpopulations would seem to possess a comparable oxidative capacity per gram mitochondrial protein, while liver heavy mitochondria possess an increased oxidative capacity and mass.     

Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue

Apoptosis in myocardial tissue slices was induced by extended incubation under anoxic conditions. Mitochondria were isolated from the studied tissue. A new method of isolation of mitochondria in special conditions by differential centrifugation at 1700, 10,000, and 17,000g resulted in three fractions of mitochondria. According to the data of electron microscopy the heavy mitochondrial fraction (1700g) consisted of mitochondrial clusters only, the middle mitochondrial fraction (10,000g) consisted of mitochondria with typical for isolated mitochondria ultrastructure, and the light fraction consisted of small mitochondria (2 or 3 cristae) of various preservation. The heavy fraction contained unusual structural elements that we detected earlier in apoptotic myocardial tissue—small electron-dense mitochondria incorporated in bigger mitochondria. The structure of small mitochondria from the light fraction corresponded to that of the small mitochondria from these unusual elements—mitochondrion in mitochondrion. The most important functions of isolated mitochondria are strongly inhibited when apoptosis is induced in our model. The detailed study of the activities of the two fractions of the apoptotic mitochondria showed that the system of malate oxidation is completely altered, the activity of cytochrome c as electron carrier is partly inhibited, while succinate oxidase activity is completely preserved (complexes II, III, and IV of the respiration chain). Succinate oxidase activity was accompanied by high permeability of the internal membrane for protons: the addition of uncoupler did not stimulate respiration. ATP synthesis in mitochondria was inhibited. We demonstrated that in our model of apoptosis cytochrome c remains in the intermembrane space, and, consequently, is not involved in the cascade of activation of effector caspases. The possible mechanisms of induction of apoptosis during anoxia are discussed.     

Further characterization of rat liver mitochondrial fractions. Lipid composition and synthesis, and protein profiles.

1. Heavy and light mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their lipid composition and synthesis and protein profiles, as seen by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The light mitochondrial fraction was rich in total lipids, phospholipids and cholesterol. The cardiolipin content, however, was low. 3. Rates of [3H]glycerol incorporation into phospholipids of heavy mitochondria and microsomal fractions were almost identical. On the other hand, incorporation into the individual phospholipids in light mitochondria was about 4-6 times higher. Incorporation into cardiolipin of light mitochondria was about 10-fold higher than in the heavy mitochondria. 4. Analysis of protein profiles by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that the pattern obtained for the light mitochondria was similar to that for heavy mitochondria. However, the light fraction was relatively poor in high-molecular-weight proteins and rich in low-molecular-weight proteins. The microsomal protein profile was altogether different. 5. The significance of these findings is discussed in relation to mitochondrial biogenesis.     

Protein synthesis in mitochondria. 4. Preparation and properties of mitochondria from Krebs II mouse ascites-tumour cells

1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0·3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.     

Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.     

Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein, DNA and cytoplasmic protein in rat liver.

From rats fed ad libitum and kept under a 12 + 12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined avery four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphthol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max = 0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear non-histone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein,DNA and cytoplasmic protein in rat liver

Summary From rats fed ad libitum and kept under a 12+12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined every four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphtol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max=0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear nonhistone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

Mitochondria from human term placenta. I. Isolation and assay conditions for oxidative phosphorylation.

Human term placental mitochondria were resolved by differential centrifugation into three fractions, heavy mitochondria, light mitochondria and a third, less dense fraction. Approximately equal amounts of mitochondrial protein were found in the three fractions. These mitochondrial preparations differed in physical properties. ATPase and "ADPase" content and oxidative capacities. Assay conditions were developed which permitted the polarographic measurement of respiration and coupled phosphorylation carried out by all three mitocondrial preparations despite the variable nucleotide-phosphate phosphatase activities present. With heavy mitochondria, rates of respiration were consistently higher than those previously reported for unfractionated placental mitochondria. Respiratory control ratios were comparable to those of mitochondria from other steroid hormone-producing endocrine tissues and ADP/O ratios approaching the theoretical maxima were obtained. Both lighter placental mitochondrial fractions displayed somewhat lower respiration rates and respiratory control but their primary defect was a selective uncoupling of the third site of energy conservation. Modification of isolation procedures were evaluated in terms of quantitative yield and functional activity of the three fractions.     

Gender-related differences in morphology and thermogenic capacity of brown adipose tissue mitochondrial subpopulations

To investigate the possible existence of a gender dimorphism in the morphology and functionality of brown adipose tissue (BAT) mitochondrial subpopulations, we obtained three mitochondrial fractions - heavy, medium and light - by differential centrifugation. Electron microscopic analysis was carried out and mitochondrial protein content, cytochrome c oxidase and ATP synthase activities, mitochondrial DNA content and UCP1 protein levels were measured in each mitochondrial fraction. Female rats showed a greater mitochondrial size than males, with a different distribution pattern of the subpopulations. These differences were accompanied by higher oxidative and thermogenic capacities and a higher protein content in female rat BAT. This tissue also showed a greater tendency to respiratory chain uncoupling, as well as a close coordination between the oxidative, phosphorylative and thermogenic processes. These differences were found in the heavy subpopulation but not in the light one. Our results demonstrate that female rat BAT shows a highly differentiated mitochondrial pool, with the heavy mitochondrial subpopulation as the main responsible for the greater thermogenic activity of this tissue. In addition, it seems that there is a differential regulation of the mitochondrial growth cycle between genders in BAT, which leads to enhanced thermogenic capacity in female rat mitochondria.     

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons : II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA

There are two types of mitochondria present in imbibed peanut cotyledons: a light type (density 1.182 grams per cubic centimeter) and a heavy type (density 1.205 grams per cubic centimeter). The membrane fractions from these two types can be distinguished using sucrose density gradient analysis, and differences in membrane density between the light and heavy types are reflected in differences in their protein N and phospholipid P composition. With increasing time after imbibition, there is a substantial increase in the amount and activity of the light type of mitochondria due to their de novo synthesis. The membrane density of the light mitochondrial fraction declines over 5 days after the start of imbibition as the phospholipid P to protein N ratio increases. The heavy mitochondrial fraction declines during the first 3 days after the start of imbibition, and then it remains at a low, but constant, level thereafter. Even during the decline, however, there is synthesis of proteins comparable to that into light mitochondria. The mitochondrial biogenesis that has been observed in peanut cotyledons is of the light type, the function and physiological importance of the minor heavy type is not known.     

Two membrane sites for DNA synthesis in Pneumococcus.

Summary A DNA membrane fraction extracted from pneumococci can be separated into two subfractions with respect to macromolecular composition and DNA synthesis by centrifugation in a 30–60% w/v neutral sucrose gradient. Each fraction can be rebanded in a sucrose gradient or centrifuged to equilibrium in a CsCl density gradient without altering the ability of the fractions to synthesize DNA. The fast sedimenting (heavy) fraction contains 45% of the DNA, and the bulk of the phospholipid, protein, and RNA. The light fraction contains 50% of the DNA, and lower, but significant amounts of phospholipid, RNA, and protein. Both fractions contain a DNA replication complex consisting of a number of enzymes involved in synthesizing DNA or DNA precursors, as well as RNA polymerase activity. However, the specific activity of DNA polymerase in the light fraction is much greater than that in the heavy fraction. In addition, the following results suggest that the former is concerned primarily with replication of the genome while the latter has characteristics of a repair function for the genome. (1) newly synthesized DNA can be detected within 30 s in the light fraction but not until 4 min in the heavy fraction. (2) an RNA-DNA single-stranded hybrid can be demonstrated during initial stages of DNA synthesis in the light, but not heavy fraction. (3) extensive semiconservative DNA replication occurs in the light fraction, whereas little such replication is detected in the heavy fraction. (4) DNA polymerase activity in the light fraction has several of the characteristics of a polymerase identified by others as being concerned with normal DNA replication, such as inhibition by N-ethylmaleimide, and relatively high rates of chain elongation (4.9×10⁴ nucleotides/min). In contrast, DNA polymerase activity in the heavy fraction has characteristic properties associated with DNA polymerase I, a possible repair enzyme. These include higher activity for a d(A-T)n template than that detected in the light fraction, no effect of N-ethylmaleimide, and relatively low rates of chain elongation (9×10³ nucleotides/min).     

Biochemical studies on sub-fractions of rat liver mitochondria

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Transcription of chromatin from mouse fibroblasts.

Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended "light" and a compact "heavy" component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II). Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.     

Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.