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1.
Incubation of isolated rat liver mitochondria with radioactive amino acids resulted in the charging of tRNAs for arginine, asparagine, leucine, lysine, methionine, proline and valine. The aminoacyl-tRNAs were shown to be distinct from their cytosolic counterparts by chromatography on RPC-5. By electrophoresis on urea polyacrylamide slab gels it was found that all these mitochondrial aminoacyl-tRNAs were about 70-76 nucleotides long. The unique mitochondrial asparaginyl- and prolyl-tRNAs, not previously identified in mammalian cells, were shown to hybridize to mtDNA. Mitochondrial leucyl-tRNA separated into 3 peaks on RPC-5 and the first species was shown to be different than a combination of the other two by molecular size and partial RNase T1 digestion patterns. Each was coded by a separate gene on mtDNA as shown by partial additivity of hybridization. Separate genes for mitochondrial tRNAMetm and tRNAMetf, separated by RPC-5 chromatography, were also demonstrated. These results bring to 21 the number of individual tRNAs coded by mammalian mtDNA. 相似文献
2.
The products of mitochondrial protein synthesis in established cell lines of various mammalian species were labelled with [35S]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10,000 and 50,000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells. 相似文献
3.
Two leucyl-tRNA synthetases (EC 6.1.1.4) have been purified to near homogeneity, the one from mitochondria and the other from cytoplasm of Tetrahymena pyriformis. Both enzymes were found to be structurally unrelated, single polypeptides with molecular weights of approximately 100,000 as determined by gel permeation, sucrose gradient centrifugation, and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These enzymes behaved differently in elution profiles through hydroxyapatite- and diethylaminoethyl cellulose-column chromatography and isoelectric focusing. The two enzymes also showed some differences in responses to various salts for charging and in pH optima and temperature sensitivity, but no significant difference was found in their affinities (Km) for ATP and leucine. These enzymes recognized different leucyl-tRNA isoaccepting species as revealed by reversed-phase column chromatography. The mitochondrial enzyme can charge six isoaccepting leucyl-tRNA species, while the cytoplasmic enzyme can recognize only four species. 相似文献
4.
The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation. 相似文献
5.
Ribosomal subunits are isolated from potato tuber mitochondria devoid of contaminating organelles. The sedimentation constants of the two mitochondrial ribosomal subunits are 33S and 50S respectively. The apparent sizes of the high molecular weight RNAs are 19S and 25S.The proteins of these ribosomes have been analyzed by two-dimensional electrophoresis in SDS polyacrylamide gels to determine their number and molecular weights. The small subunit contains 35 protein species ranging from 8 to 60 kDa. The 50S large subunit contains 33 protein species ranging from 12 to 46 kDa. These preliminary results are the first analysis made on mitochondrial ribosomes from a higher plant. 相似文献
6.
The cytoplasmic and chloroplast ribosomes from the marine diatom Cylindrotheca fusiformis were isolated and characterized. The cytoplasmic ribosomes sedimented in sucrose at 84S and dissociated into subunits of 64S and 42S in the absence of Mg2+. It contained ribosomal RNAs with molecular weights of 1.31×106 and 0.70×106. The chloroplast ribosomes sedimented at 70S only in the presence of high Mg2+ concentrations (25–100 mM). No stable subunits were routinely observed and at very high levels of Mg2+ (>100 mM) the 70S species was converted to a form sedimenting at 55S. At 4°C ribosomal RNAs with molecular weights of 1.1×106 and 0.40×106 were detected on polyacrylamide gel electrophoresis. When the RNAs were resolved at room temperature the large molecular weight component disappeared while RNA with molecular weights of 0.65×106 and 0.53×106 were observed. Apparently the large chloroplast RNAs dissociated into two pieces of unequal molecular weight. These properties of the diatom's chloroplast ribosomes are very similar to those of the counter parts in unicellular green algae, which suggests that both types of algae have a common phylogenetic ancestor. 相似文献
7.
H De Vries J C De Jonge J M Schneller R P Martin G Dirheimer A J Stall 《Biochimica et biophysica acta》1978,520(2):419-427
Total mitochondrial tRNA from Neurospora crassa was characterized by base composition analysis, one- and two-dimensional gel electrophoreses and reversed-phase chromatography on RPC5. The guanosine + cytidine content was about 43%, as compared to 60% for cytoplasmic tRNA. The modified nucleoside content was low and about the same as that of total yeast mitochondrial tRNA, though the G + C content is very different. We found psi, T, hU, t6A, m1G, M2G, m22G. Neither the eukaryotic "Y" base, nor the prokaryotic s4U were present. On two-dimensional polyacrylamide gel electropherograms about 25 species were separated. One species for phenylalanine, two for leucine and two for methionine could be located. Neurospora crassa mitochondrial tRNA does not hybridize with yeast mitochondrial DNA. 相似文献
8.
The major and modified base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method. The base composition of these mitochondrial 4 S RNA preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs. The results of these studies are: 1. When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are characterized by high (A + U)/(G + C) ratios and low overall degrees of base methylation and modification. 2. The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4-S RNAs than from their cytoplasmic counterparts. Thus, several modified constituents found in both cytoplasmic and mitochondrial 4 S RNA are absent from the bacterial 4-S RNAs. 3. Mitochondrial 4S RNA from both hepatomas was found to be under-methylated and undermodified when compared with normal liver mitochondrial 4S RNA. This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepatoma 5123D (i.e., 8% undermethylation). These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors. 相似文献
9.
Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo. 相似文献
10.
Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic Acid 总被引:6,自引:2,他引:4
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Pring DR 《Plant physiology》1974,53(5):677-683
Mitochondria were prepared from etiolated maize shoots (Zea mays L. var. McNair 508) by homogenization followed by differential centrifugation and equilibrium banding in discontinuous sucrose or Renografin-sucrose gradients. Mitochondria prepared by sucrose banding showed better physiological integrity than those prepared by renografin-sucrose banding, although both procedures yielded mitochondria that showed respiratory control and coupling of oxidation to phosphorylation of ADP. Mitochondria prepared by Renografin-sucrose banding were free of dectectable cytoplasmic ribosomal RNA, while sucrose banding resulted in a low level of contamination. Ribosomes isolated from mitochondria sedimented at about 78S, with subunits sedimenting at 60 and 44S. Using Escherichia coli ribosomal RNA as internal standards, the molecular weights of mitochondrial ribosomal RNAs were found to be 0.74 to 0.75 and 1.26 × 106 daltons by polyacrylamide gel electrophoresis, before or after denaturation in formaldehyde. Cytoplasmic ribosomal RNA molecular weights were 0.70 and 1.26 × 166 before denaturation, and 0.68 and 1.5 × 106 after denaturation, suggesting an unusual reaction of the heavy ribosomal RNA to formaldehyde. 相似文献
11.
The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver. 相似文献
12.
The temperature-sensitive Chinese hamster ovary cell mutant tsH1, has been shown previously to contain a temperature-sensitive leucyl-tRNA synthetase. At the non-permissive temperature of 40 degrees C cytosolic protein synthesis is rapidly inhibited. The protein synthesis which continues at 40 degrees C appears to be mitochondrial, since: (a) whole-cell protein synthesis at the permissive temperature of 34 degrees C is not inhibied by tevenel, the sulfamoyl analogue of chloramphenicol and a specific inhibitor of mitochondrial protein synthesis; however, whole-cell protein synthesis at 40 degrees C is inhibited by tevenel, (b) Protein synthesis by isolated mitochondria from tsH1 cells is not significantly inhibited at 40 degrees C. (c) At 40 degrees C [14C]leucine is incorporated predominantly into the mitochondrial fraction of tsH1 cells. (d) The incorporation of [14C]leucine at 40 degrees C into mitochondrial proteins of tsH1 cells is inh-bited by tevenel but not by cycloheximide. These results suggest that the mitochondria of tsH1 cells contain a leucyl-tRNA synthetase which is different from the cytosolic enzyme. The inhibition of cytosolic, but not of mitochondrial protein synthesis in tsH1 cells at 40 degrees C allows the selective labelling of mitochondrial translation products in the absence of inhibitors. The mitochondrial translation products labelled in tsH1 cells at 40 degrees C and at 34 degrees C in the presence of cycloheximide have been compared by sodium dodecylsulphate-polyacrylamide gel electrophoresis. Both conditions of labelling give similar profiles. The mitochondrial translation products are resolved into two components, one with an apparent molecular weight range from 40,000 to 20,000 and a second with an apparent molecular weight range from 20,000 to 10,000. 相似文献
13.
The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57,500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform-methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h. 相似文献
14.
F Gasnier R Rousson F Lerme E Vaganay P Louisot O Gateau-Roesch 《European journal of biochemistry》1992,206(3):853-858
Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria. 相似文献
15.
The changes in agarose gel electrophoretic velocities of several RNAs of varying molecular weight and base composition with concentration of the denaturant, methylmercuric hydroxide (MMH), have been studied. At intermediate MMH concentrations, the mobility of any one species is intermediate between the “native” (no MMH) and fully denatured (5 mm MMH) values. A + U-richer RNAs are partially denatured at lower concentrations of MMH than are G + C-richer RNAs. Electrophoresis at intermediate MMH concentrations is useful for resolving some RNA species that are not well resolved either in the absence of MMH or under fully denaturing (5 mm MMH) conditions. It is shown that it is possible to carry out MMH electrophoresis in polyacrylamide gels; this is useful for low molecular weight RNAs. An equation to correlate changes in mobility between the native and denatured states is proposed. 相似文献
16.
1. By a procedure involving adsorption to barium sulfate, chromatography on DEAE-Sephadex and QAE-Sephadex and preparative polyacrylamide gel electrophoresis, decarboxyfactor X was purified from plasma of phenprocoumon-treated cows. No contaminants could be detected in the final preparation by polyacrylamide gel electrophoresis and zone-electrophoresis. 2. The molecular weight of decarboxyfactor X, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis is approximately 55 000, which is equal to that of factor X. The protein consists of two polypeptide chains with molecular weights of 44 000 and 17 000. 3. Decarboxyfactor X has antigenic determinants in common with normal factor X. 4. The amino acid composition and aminoterminal amino acids of normal factor X and decarboxyfactor X are identical. 5. Less than one residue of gamma-carboxyglutamate could be detected per mole of decarboxyfactor X. 6. In the absence of Ca2+, normal factor X has a slightly higher electrophoretic mobility than decarboxyfactor X. In the presence of Ca2+ the mobility of factor X decreases considerably while the mobility of decarboxyfactor X remains unaltered. 相似文献
17.
Shirley E. Hawkins 《Journal of theoretical biology》1976,62(2):299-313
Progress on various aspects of nucleic acids and protein synthesis in amoebae has been reviewed. The RNA molecules involved in the character changes seen after micro-injection of non-homologous cytoplasmic fractions have been isolated after polyacrylamide gel electrophoresis, and their approximate molecular weights calculated. Injection of these RNA molecules was shown to alter the response of recipient cells to growth in streptomycin and neomycin.The relative molecular weights of cytoplasmic ribosomal RNAs have been estimated using both aqueous and formamide gel electrophoresis. Some attempts to characterize the nuclear RNAs seen on aqueous polyacrylamide gels, and to evaluate this data with that published by other workers have been made. Results from assays of DNA- and RNA-directed DNA polymerase activity are considered in relation to those from other eukaryotes.Problems arising after attempts to use rabbit globin messenger RNA to direct globin synthesis in amoebae, and the possibilities of using minature gel systems and small cell numbers to identify proteins and RNAs after various experimental treatments are discussed. 相似文献
18.
The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose /2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation. 相似文献
19.
Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900. 相似文献
20.
MITOCHONDRIAL RNA FROM CULTURED ANIMAL CELLS : II. A Comparison of the High Molecular Weight RNA from Mouse and Hamster Cells
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Bland S. Montenecourt Margaret E. Langsam Donald T. Dubin 《The Journal of cell biology》1970,46(2):245-251
Discrete RNA fractions sedimenting slightly slower than 18s ribosomal RNA have been found in mitochondrial preparations from both hamster (BHK-21) and mouse (L-929) cells. This RNA could be separated into two components, present in approximately equimolar amounts, by prolonged zonal centrifugation or acrylamide gel electrophoresis. The hamster components had sedimentation constants averaging 16.8 and 13.4, and molecular weights (estimated by gel electrophoresis) averaging 0.74 and 0.42 x 106 daltons. Mixed labeling experiments showed that the mouse components sedimented and electrophoresed 3–6% more slowly than the corresponding hamster components. The RNA from both cell lines resembled mitochondrial ribosomal RNA from yeast and Neurospora in being GC poor, and in addition the larger and smaller components resembled each other in base composition. These results, taken with those of other recent studies, are compatible with the idea that our high molecular weight mitochondrial RNA is ribosomal; such RNA would then constitute a uniquely small size-class of ribosomal RNA. 相似文献