Protein synthesis by hepatic mitochondria isolated from carbon tetrachloride-exposed rats

Hepatic mitochondria isolated from rats 40 h after dosage with 1.1 ml/kg CCl4 are uncoupled and display structural damage. Mitochondrial function returns during hepatic recovery. Because the products of mitochondrial protein synthesis are essential to mitochondrial structure and function, the effects of CCl4 on the rate of mitochondrial protein synthesis, and on the products, was studied using mitochondria from CCl4-exposed rats during the early, maximum development and resolution stages of CCl4-induced mitochondrial damage. Rates of mitochondrial protein synthesis (incorporation of [35S]methionine) were elevated 300% over that of mitochondria from non-exposed rats 17 h after exposure; depressed by 50% at 40 h and above control at 113 h. When the radiolabeled products of incorporation were separated and examined by autoradiography, a novel, low-molecular-weight band, of approx. 9700, was apparent 40 h after CCl4 exposure. A band of similar molecular weight appeared when control mitochondria were incubated without an exogenous supply of ATP. Mitochondria from exposed rats which displayed rates of protein synthesis greater than control consistently had a relative increase in a band that corresponded in size to that of cytochrome oxidase subunit I. It was concluded that the loss of mitochondrial function induced by CCl4 could not be attributed to inhibition of mitochondrial protein synthesis, and that the mitochondria may not always synthesize protein in constant proportions.     

Inhibition of Protein Synthesis and Amino Acid Transport by Crystal Violet in Trypanosoma cruzi

ABSTRACT. [³⁵S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [³⁵S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [³H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.     

Brugia pahangi: inhibition of protein synthesis

The effects of inhibitors of protein synthesis and electron transport on the incorporation of [14C]leucine and [35S]methionine into protein by the filarial worm Brugia pahangi have been investigated. Cycloheximide inhibits the accumulation of both [14C]leucine and [35S]methionine by the worms and their incorporation into protein. In addition, inhibitors of electron transport and some anti-parasitic compounds also significantly inhibit filarial protein synthesis. Antimycin A and cyanide inhibit [14C]leucine incorporation into protein 63 and 72%, respectively, without affecting either motility or lactate production. Interestingly, the anti-malarial compounds chloroquine and quinacrine also significantly inhibit both accumulation and incorporation of [14C]leucine by B. pahangi. In addition, fluorographs of sodium dodecyl sulfate-polyacrylamide gels of homogenates from filariids incubated in [35S]methionine and cycloheximide with and without chloramphenicol indicate that there is a discrete population of proteins, possibly mitochondrial in origin, that are synthesized in the presence of cycloheximide and are not inhibited by chloramphenicol.     

Synthesis of Na+/K+ ATPase by the preimplantation rabbit blastocyst

The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (beta- and gamma-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished approximately 79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (less than 0.06 mM) and stage-related differences in blastocoele volume. Analysis of beta- and gamma-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P less than 0.001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst.     

Site of synthesis of the proteins of mammalian mitochondrial ribosomes. Evidence from cultured bovine cells

In order to determine the sites of synthesis of the proteins of the mammalian mitochondrial ribosome (mitoribosome), bovine (MDBK) cells were labeled with [35S]methionine in the presence of inhibitors of mitochondrial and cytoplasmic protein synthesis. Labeling in the absence of cytoplasmic protein synthesis produced a "blank" fluorogram, indicating that there is no mitochondrial product. Additionally, incorporation of [35S]methionine into the enumerated mitoribosomal proteins continued in the absence of mitochondrial protein synthesis. Finally, it was demonstrated that mitoribosomal proteins can be both translated and assembled into complete mitoribosomes in the absence of mitochondrial protein synthesis. These results indicate that in mammals, as opposed to lower eukaryotes, all of the mitoribosomal proteins are products of cytoplasmic protein synthesis.     

Expression of the mammalian mitochondrial genome. Role for membrane potential in the production of mature translation products

Protein synthesis was investigated in isolated mitochondria under conditions which either inhibited electron transport or uncoupled oxidative phosphorylation. In a medium containing an exogenous source of ATP and oligomycin, an inhibitor of the ATP synthase complex, incorporation of [35S]methionine into proteins is stimulated in the presence of inhibitors of the electron transport chain; substituting uncouplers of oxidative phosphorylation for the latter leads, in contrast, to a decrease in the rate of incorporation of the labeled amino acid into mitochondrial translation products. Studies on the metabolic stability of mitochondrial translation products revealed that "mature" polypeptides made in isolated mitochondria are stable as indicated by the absence of degradation during a 50 min "chase" period. Under conditions which reduce or dissipate the membrane potential, 50-60% of the newly made polypeptides (pulse) are degraded within 50 min. The kinetics of the degradation process for individual mitochondrial gene products reveal that the largest proportion of polypeptides degraded to an acid-soluble form during the chase period are abnormal proteins, likely the result of premature chain termination. Emerging as a common denominator in these studies is a role for a transmembrane potential across the inner membrane in the production of mature "stable" mitochondrial gene products.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma

Proteoglycan synthesis by cultured chondrocytes from the Swarm rat chondrosarcoma was examined after treatment with 0.1 mg/ml of cycloheximide which inhibited [3H]serine incorporation into total protein by greater than 90%. Incorporation of [35S]sulfate into proteoglycans decreased with nearly first order kinetics (t 1/2 = 96 +/- 6 min) with an accompanying increase in the size of the proteoglycan molecules, primary due to an increase in chondroitin sulfate chain sizes. After 5 h of cycloheximide treatment, when [35S]sulfate incorporation was inhibited by about 90%, addition of 1 mM beta-D-xyloside restored 76% of the incorporation into chondroitin sulfate observed in cultures treated only with xyloside. This suggests that the biochemical pathways for the affected by cycloheximide treatment. Cultures were prelabeled for 15 min with either [3H]serine or [35S]-methionine, and then cycloheximide was added to block further protein synthesis. Both precursors appeared in completed proteoglycan molecules with nearly first order kinetics with t 1/2 values of 92 +/- 8 and 101 +/- 11 min for [3H]serine and [35S]methionine, respectively, values in close agreement with the t 1/2 from the [35S]sulfate data. These results suggest that after cycloheximide treatment, the rate of [35S]sulfate incorporation into proteoglycan, after a correction for increases in chondroitin sulfate chain size, was directly proportional to the size of the intracellular pool of core protein. From the steady state rate of proteoglycan synthesis (estimated to be about 80 ng/min/10(6) cells in separate experiments) and a corrected t 1/2 value of 60 min, the amount of precursor core protein can be calculated to be about 500 ng/10(6) cells in these experiments.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Rapid and selective effects of thyrotropin on [35S]methionine incorporation into cytoplasmic and nuclear proteins in calf thyroid tissue slices

Previous studies have indicated that thyrotropin may induce general increases in RNA and protein synthesis in calf thyroid tissue slices. In this report, we show that thyrotropin selectively stimulates [35S]methionine incorporation into small numbers of specific cytoplasmic and nuclear proteins. We provide data on the time course of stimulation and on relative molecular masses and isoelectric points of hormone-response proteins. Calf thyroid tissue slices were incubated for 3 h, 6 h or 9 h in the presence or absence of thyrotropin (50 mU/ml); [35S]methionine (50-75 microCi/ml) was added for the final 3 h of incubation. Cytoplasmic and nuclear fractions were then prepared, and analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Thyrotropin increased [35S]methionine incorporation into two cytoplasmic and four nuclear proteins within 3 h; hormonal effects on the labeling of five of these six proteins were transient, and no longer evident after 6-9 h. In contrast, a second group of two cytoplasmic and four nuclear proteins exhibited increased labeling after a delay of 6-9 h. Our results suggest that thyrotropin selectively stimulates the synthesis of specific cytoplasmic and nuclear proteins in calf thyroid tissue slices, and that stimulation involves at least two mechanisms (one rapid, the other delayed).     

Synthesis of ribonucleic acid by isolated rat liver mitochondria

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.     

Effects of Carboxylemethylation and Polyamine Synthesis Inhibitors on Methylation of Trypanosoma brucei Cellular Proteins and Lipids

ABSTRACT. The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S-adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL-α-difluoromethylomithine (Ornidyl, DFMO) the AdoMet decarboxylase inhibitor 5′-{[(Z)-4-amino-2-butenyl]-methylamino}-5′-deoxyadenosine or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [¹⁵S] or [U-¹⁴C]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fraction. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 μM) and sinefugin (2 nM) on [³⁵S] and [U-¹⁴C]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5-fold through incorporation of [U¹⁴C]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U-¹⁴C]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U-¹⁴C]methionine as AdoMet. Exogenous AdoMet had no effect on [³⁵S]methionine incorporation. The agents studied are curative for African trypnosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.     

Isolation and detailed characterization of human cell lines resistant to -threo-chloramphenicol

The isolation and characterization of chloramphenicol resistant derivatives of the human cell line HeLa B is described. Growth of resistant lines was unaffected in the presence of 100 μg/ml -threo-chloramphenicol, whereas growth of the parental cells was inhibited at 12.5 μg/ml. The incorporation of [³⁵S]methionine into mitochondrial protein of intact resistant cells continued normally in the presence of 100 μg/ml chloramphenicol (cytoplasmic protein synthesis was blocked by addition of 50 μg/ml emetine). Under these conditions the electrophoretic profile of labelled, presumptive mitochondrially-made proteins was similar to that of the parental cell line labelled in the absence of chloramphenicol. The cell lines selected in the presence of chloramphenicol also showed increased resistance to some other inhibitors of mitochondrial protein synthesis, e.g. carbomycin and mikamycin. [¹⁴C]Chloramphenicol was found to have normal access to the interior of resistant cells and it is therefore unlikely that resistance results from altered cell permeability. No modification of the drug by acetylation or glucuronide conjugation mechanisms was observed. The possibilities remain that resistance is mediated by altered permeability of the mitochondrial membrane, or from modification to a component of the mitochondrial protein synthetic system.     

Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

Mitochondrial DNA expression in Drosophila melanogaster: neosynthesized polypeptides in isolated mitochondria

The expression of mitochondrial genome of D. melanogaster in isolated mitochondria was followed by incorporation of 35S methionine in neosynthesized polypeptides. A high level of protein synthesis was obtained after optimization of all the incubation parameters. Two kinds of energy-generating systems were used: an endogenous system where an oxidizable substrate were added for ATP synthesis; an exogenous system with an energy-rich compound for ATP regeneration, the latter proved to be the most effective. The effect of the oxidative phosphorylation uncoupler (Clccp), and an ATPase inhibitor (oligomycine) allow us to postulate the role of the electrochemical potential in the expression of the mitochondrial genome. Electrophoresis and autoradiography of neosynthesized mitochondrial proteins exhibits 18 to 24 protein bands, ranging from 6.5 to 65 Kd; incubation of KC 0% drosophila cells with 35S methionine and cycloheximide gave similar results. Both our results and those published elsewhere suggest that the expression of mitochondrial genome in higher organisms could be more complex than simple translation of the 13 genes presents on these genomes.     

Preparation of a cell-free extract from rat brain which can initiate protein synthesis in vitro

A cell-free protein synthesis system, derived from brains of 3 mo-old male Fischer-344 rats, has been characterized. The optimum conditions for amino acid incorporation in the system were 5 mM magnesium ion and 200 mM potassium ion. Incorporation depended on the addition of ATP, GTP, and an enegy-generating system, and was sensitive to addition of the drugs aurintricarboxylic acid and sodium fluoride, inhibitors of initiation of protein synthesis. Both 40S and 80S initiation complexes were labeled in vitro, using [³⁵S]methionine. Such labeling was sensitive to the protein synthesis inhibitors, aurintricarboxylic acid and sodium fluoride. The system, which can initiate protein synthesis, should be of use for examining mechanisms which underlie alterations in rat brain protein synthesis induced by various treatments.     

An efficient and prolonged in vitro translational system from isolated cucumber etioplasts

Etioplasts were isolated from dark grown cucumber cotyledons pretreated with kinetin and gibberellic acid. When incubated in a cofactor enriched medium these etioplasts incorporated [35S] methionine into a hot trichloroacetic acid-insoluble fraction; this incorporation was linear for 8 h of incubation and was inhibited by chloramphenicol but not by cycloheximide. Over the same time period, the etioplasts showed continued linear synthesis of the chlorophyll precursors protochlorophyllide, Mg-protoporphyrin and protoporphyrin IX. Analysis of products of in vitro protein synthesis by etioplasts and cotyledons showed the thylakoid membrane polypeptide profiles to be identical. Continued incorporation of [35S] methionine into the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) for 8 h has been confirmed further by immunoprecipitation with anti-spinach RuBisCO. This competent in vitro translation system should be useful for future studies of chloroplast protein synthesis and gene expression.     

Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of chronic ethanol consumption on the synthesis of polypeptides encoded by the hepatic mitochondrial genome

Liver mitochondria from rats fed ethanol chronically demonstrate an impaired ability to incorporate [35S]methionine into polypeptide products in vitro. This ethanol-induced effect on mitochondrial translation in vitro could not be attributed to significant differences in the methionine precursor pool sizes of ethanol and control mitochondria or to the acute effects of residual ethanol. The observed reduction of radiolabeled methionine incorporation into mitochondrial gene products of ethanol mitochondria in vitro reflects a decrease in the synthesis of all the mitochondrial gene products. However, the percentage of total radiolabel incorporated into each gene product is unaffected by ethanol, suggesting an ethanol-induced coordinate depression of mitochondrial protein synthesis. Moreover, SDS-PAGE and densitometry of submitochondrial particles from ethanol-fed and control rats demonstrated that the steady-state concentration of each of the mitochondrial gene products is decreased in ethanol-fed rats. This reduction of the steady-state concentration of the mitochondrial gene products may be related to the observed depressions of oxidative phosphorylation activities associated with hepatic mitochondria from ethanol-fed rats.