Regulation of fatty acid synthesis during the cessation of phospholipid biosynthesis in Escherichia coli.

In 1975, Cronan et al. (J. Biol. Chem. 250:5835-5840) reported that free fatty acids accumulated during glycerol starvation of an Escherichia coli glycerol auxotroph. On the basis of labeling experiments showing significant incorporation of [14C]acetate into the fatty acid fraction of glycerol-starved cells, these authors concluded that fatty acid synthesis proceeded normally in the absence of phospholipid synthesis. Since these findings might have been due to an increase in the intracellular specific activity of the [1-14C]acetyl coenzyme A pool of the glycerol-starved cells, we reexamined the effect of glycerol starvation on fatty acid synthesis. We found that (i) the incorporation of 3H2O and/or [2,3-14C]succinate into the fatty acid fraction of glycerol auxotrophs is severely reduced during starvation, (ii) the incorporation of [1-14C]acetate into the lipid fraction of an acetate-requiring glycerol auxotroph is inhibited by 95% during glycerol starvation, and (iii) the accumulation of fatty acids, as measured by microtitration, in glycerol-starved cells is less than 10% that of glycerol-supplemented cells. These results indicate that fatty acid synthesis is inhibited in the absence of phospholipid synthesis of E. coli.     

Deoxyribonucleic Acid Synthesis in FV-3-infected Mammalian Cells

Deoxyribonucleic acid (DNA) synthesis and virus growth in frog virus 3 (FV-3)-infected mammalian cells in suspension were examined. The kinetics of thymidine incorporation into DNA was followed by fractionating infected cells. The cell fractionation procedure separated replicating viral DNA from matured virus. Incorporation of isotope into the nuclear fraction was depressed 2 to 3 hr postinfection; this inhibition did not require protein synthesis. About 3 to 4 hr postinfection, there was an increase in thymidine incorporation into both nuclear and cytoplasmic fractions. The nuclear-associating DNA had a guanine plus cytosine (GC) content of 52%; unlike host DNA it was synthesized in the presence of mitomycin C, it could be removed from nuclei by centrifugation through sucrose, and it was susceptible to nuclease digestion. This nuclear-associating DNA appeared to be a precursor of cytoplasmic DNA of infected cells. The formation of the latter DNA class could be selectively inhibited by conditions (infection at 37 C or inhibition of protein synthesis) that permit continued incorporation of thymidine into nuclear-associating DNA. The cytoplasmic DNA class also had a GC content of 52%, was resistant to nuclease degradation, and its sedimentation profile in sucrose gradients corresponded to that of infective virus. Contrary to previous reports, we found that (i) viral DNA synthesis can continue in the absence of concomitant protein synthesis, and (ii) viral DNA synthesis is not abolished at 37 C. The temperature lesion in FV-3 replication appeared to be in the packaging of DNA into the form that appears in the cytoplasmic fraction of disrupted cells.     

FACTORS INFLUENCING THE FREQUENCY OF MESOSOMES OBSERVED IN FIXED AND UNFIXED CELLS OF STREPTOCOCCUS FAECALIS

Mesosomes of Streptococcus faecalis (American Type Culture Collection 9790) were seen about 92% less frequently in freeze fractures of unfixed cells than in freeze fractures and sections of fixed cells. This difference in frequency was not related to any period of unbalanced macromolecular synthesis induced by chemical fixation. All measured synthetic processes (DNA, RNA, and protein synthesis, and glycerol incorporation) were halted with either osmium tetroxide (OS) or glutaraldehyde fixation. That fewer mesosomes were seen in freeze fractures of unfixed cells was probably due to the difficulty of observing cross-fractured mesosomes in this organism in the unfixed state. Unfortunately, mesosomes probably preferentially cross fracture in the unfixed state and therefore are usually only observed, infrequently, in those cases where the freeze fracture follows the surface layer of a mesosomal membrane. However, the addition of glycerol to unfixed cells, especially in the chilled state, greatly increased the frequency of observation of cytoplasmic mesosomes in freeze fractures. It is thought that glycerol, like chemical fixation, increases the number of surface-fractured mesosomes, which in turn increases the frequency of mesosome observation. It was also observed that cellular autolysis occurring during OS fixation seemingly reduced the number of mesosomes observed in thin sections and freeze fractures of OS-fixed cells.     

Effect of Glycerol Deprivation on the Phospholipid Metabolism of a Glycerol Auxotroph of Staphylococcus aureus

A study of the effects of glycerol deprivation on the content and metabolism of the phospholipids of a glycerol auxotroph of Staphylococcus aureus showed that (i) there was an increase in the proportions of lysylphosphatidylglycerol (LPB) and a concomitant decrease in the proportion of phosphatidylglycerol. The total phospholipid content per sample and the proportion of cardiolipin did not change, but the phosphatidic acid increased transiently and then fell to pretreatment levels. (ii) The loss of (32)P from the lipids during the chase in a pulse-chase experiment was essentially the same in phosphatidylglycerol, cardiolipin, and phosphatidic acid during glycerol deprivation or growth in the presence of glycerol. LPG lost half the radioactivity in slightly more than two doubling times when grown with glycerol. In the absence of glycerol, (32)P accumulated in LPG for about 20 min and then stopped, after which time there was no apparent turnover. (iii) During glycerol deprivation, the initial (32)P incorporation decreased sixfold compared to that of the control with glycerol. The initial incorporation into LPG decreased only 2.5-fold, whereas that of PG decreased 45-fold. (iv) During glycerol deprivation, the free fatty acid content increased from 1.2 to 12.5% of the total extractable fatty acids and then slowly decreased. The increase was largely iso- and anti-iso-branched 21-carbon-atom fatty acids. In glycerol-supplemented cultures, the major fatty acids were branched 14- to 18-carbon fatty acids. The decrease in longer chain free fatty acids after 60 min represented their esterification into lipids. (v) During glycerol deprivation ribonucleic acid synthesis and cell growth continued for 40 min and protein synthesis continued for 90 min. Then synthesis and growth stopped. (vi) After the addition of glycerol to glycerol-deprived cells, (32)P and (14)C-glycerol were incorporated into the phospholipids without lag; ribonucleic acid, protein synthesis, and cell growth began after a 5- to 10-min lag at the pretreatment rate. The initial rate of lipid synthesis after the addition of glycerol was three times greater than the growth rate. This rapid rate continued for about 25 min until the lipid content and proportions of LPG and phosphatidylglycerol were restored.     

Synthesis of DNA in permeabilized cells of Kluyveromyces lactis.

Kluyveromyces lactis cells permeabilized with nystatin, though no longer viable, were able to incorporate 3H-dATP into DNA. Maximum rate of synthesis was obtained when all four deoxyribonucleoside triphosphates were present. For prolonged incorporation of 3H-dATP into DNA rATP or phosphoenolpyruvate were of absolute requirement. DNA synthesis was inhibited by p-chloromercuribenzoate, N-ethylmaleimide, nalidixate, ethidium bromide and distamycin A. The density of DNA synthesized in permeabilized cells grown on non-fermentable and fermentable carbon sources was analyzed on CsCl gradients in the presence or absence of distamycin A. The DNA synthesized by permeabilized cells previously grown on glycerol was essentially mitochondrial DNA; nuclear DNA (30% of total) was also synthesized by cells previously grown on glucose.     

Synthesis of Protein, Ribonucleic Acid, and Ribosomes by Individual Bacterial Cells in Balanced Growth

Relative rates of protein synthesis in individual cells were determined by allowing random populations to incorporate tritiated leucine for very short periods (pulses) and then examining autoradiographs of these cells to assess the amount of incorporation (grains per cell) as a function of cell size. Relative rates of ribonucleic acid (RNA) synthesis were determined in the same way by using tritiated uracil. Unless the uracil pulse was very short (less than 1/20 generation), the RNA labeled during the pulse was predominantly ribosomal. The rate of protein synthesis in individual cells is directly proportional to cell size. The rate of RNA synthesis also increases linearly with size in larger cells, but there appears to be a slight delay in RNA synthesis immediately after cell division. Total cellular content of protein, RNA, and ribosomes is directly proportional to cell size. Thus, we conclude that, in individual cells during the cell cycle (i) the average rate of protein synthesis per ribosome is constant and (ii) the increase in macromolecular mass of the cell is exponential with age.     

Phospholipid Metabolism in the Absence of Net Phospholipid Synthesis in a Glycerol-Requiring Mutant of Bacillus subtilis

A glycerol-requiring auxotroph of Bacillus subtilis showed no net synthesis of phospholipid when deprived of glycerol. Although there was no net synthesis of phospholipid, we found that: (i) fatty acids and (32)P were slowly incorporated into phospholipid; (ii) in pulse-chase experiments, both (32)P and (14)C in the glycerol portion of the phospholipids were lost from phosphatidlyglycerol (PG) and lysylphosphatidylglycerol and accumulated in cardiolipin (CL); (iii) the proportions of the phospholipids in the membrane changed with a loss of PG and an accumulation of CL. The addition of glycerol to the glycerol-deprived cells resulted in a rapid incorporation of glycerol and restoration to the predeprivation metabolism and PG to CL ratio.     

Consequences of Glycerol Deprivation on the Synthesis of Membrane Components in a Glycerol Auxotroph of Staphylococcus aureus

In a glycerol auxotroph of Staphylococcus aureus, the deprivation of glycerol affected the formation of certain membrane components. (i) There was synthesis of fatty acids at the predeprivation rate even though the fatty acids synthesized accumulated as free fatty acids rather than as esterified fatty acids; (ii) there was a complete cessation of phospholipid and vitamin K isoprenologue biosynthesis; (iii) there was conservation of the glycerol esters of the complex phospholipids and glucolipids; (iv) there was an immediate decrease in the rate of synthesis of monoglucoslydiglyceride (30%) and diglucosyldiglyceride (60%); (v) there was a 50% decrease in the rate of synthesis of the polar and nonpolar carotenoids; (vi) there was synthesis of protoheme, heme a, and nonspecific membrane protein at the predeprivation rate; and (vii) there was an abrupt cessation in the formation of new, functional glycine transport activity.     

Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis.

Minicells produced by Bacillus subtilis CU403 (divIVB1) are capable of mucopeptide biosynthesis as shown by the incorporation of L-alanine, D-alanine, and N-acetylglucosamine into trichloroacetic acid-precipitable material, which can be degraded to trichloroacetic acid-soluble material by lysozyme digestion. Incorporation of the precursors is sensitive to vancomycin and D-cycloserine and insensitive to chloramphenicol. Penicillin inhibits the incorporation of D- and L-alanine N-acetylglucosamine at concentrations in excess of 10 mug of penicillin per ml; however, minicells are insensitive to penicillin-induced lysis. The material synthesized in minicells from N-acetylglucosamine is not subject to turnover during a subsequent 6-h incubation period. [2-3H]glycerol is converted to a cold trichloroacetic acid-precipitable form by minicells. This synthesis is not inhibited by vancomycin, penicillin, D-cycloserine, or chloramphenicol. Fractionation of the material synthesized from glycerol into hot trichloroacetic acid-soluble material and chloroform/methanol-extractable material indicates that minicells convert glycerol into teichoic acid and lipid.     

The β-exotoxins of Bacillus thuringiensis: III. Effects on in vivo synthesis of macromolecules in an insect system

Heat stable β-exotoxin, purified from fermentations of Bacillus thuringiensis, induced malformed mouthparts in adult cabbage loopers, Trichoplusia ni, when mature larvae were injected with 9 ng β-exotoxin per 280 mg larva. Larvae injected with 26 ng β-exotoxin produced 50% adults with malformed mouthparts. Protein and nucleic acid synthesis was inhibited by presence of β-exotoxin. Specifically, β-exotoxin inhibited in vivo incorporation of ¹⁴C-valine, ¹⁴C-uracil, and ¹⁴C-thymidine into protein, RNA, and DNA, respectively. Percent inhibition, 120 min post-injection of 50 μg β-exotoxin per larva, was 53% (protein), 36% (RNA), and 41% (DNA). The asymptote for inhibition of synthesis was reached ∼30-min after injection of β-exotoxin.     

Drugs affecting the synthesis of glycerides and phospholipids in rat liver. The effects of clofibrate, halofenate, fenfluramine, amphetamine, cinchocaine, chlorpromazine, demethylimipramine, mepyramine and some of their derivatives.

The effects on glycerolipid synthesis of a series of compounds including many drugs were investigated in cell-free preparations and slices of rat liver. p-Chlorobenzoate, p-chlorophenoxyisobutyrate, halofenate, D-amphetamine, adrenaline, procaine and N-[2-(4-chloro-3-sulphamoylbenzoyloxy)ethyl]norfenfluramine had little inhibitory effect on any of the systems investigated. Two amphiphilic anions, clofenapate and 2-(p-chlorophenyl)-2-(m-trifluoromethylphenoxy)acetate, both inhibited glycerol phosphate acyltransferase and diacylglycerol acyltransferase at approx. 1.6 and 0.7 mm respectively. Clofenapate (1 mm) also inhibited the incorporation of glycerol into lipids by rat liver slices without altering the relative proportions of the different lipids synthesized. The amphilic amines, mepyramine, fenfluramine, norfenfluramine, hydroxyethylnorfenfluramine, N-(2-benzoyloxyethyl)norfenfluramine, cinchocaine, chlorpromazine and demethylimipramine inhibited phosphatidate phosphohydrolase by 50% at concentrations between 0.2 and 0.9 mm. The last four compounds inhibited glycerol phosphate acyltransferase by 50% at concentrations between 1 and 2.6 mm. None of the amines examined appeared to be an effective inhibitor of diacylglycerol acyltransferase. Norfenfluramine, hydroxyethylnorfenfluramine and N-(2-benzoyloxyethyl)norfenfluramine produced less inhibition of glycerol incorporation into total lipids than was observed with equimolar clofenapate. The major effect of these amines in liver slices was to inhibit triacylglycerol and phosphatidylcholine synthesis and to produce a marked accumulation of phosphatidate. The results are discussed in terms of the control of glycerolipid synthesis. They partly explain the observed effects of the various drugs on lipid metabolism. The possible use of these compounds as biochemical tools with which to investigate the reactions of glycerolipid synthesis is considered.     

DNA synthesis in polyoma virus infection. III. Mechanism of inhibition of viral DNA replication by cycloheximide.

The formation of viral DNA was inhibited in polyoma virus-infected cells in which protein synthesis had been blocked by cycloheximide. The present studies show the following. (i) The pool of replicating viral DNA molecules was reduced in cycloheximide-treated cells by an amount consistent with inhibition of [3-H]thymidine incorporation into viral DNA, whereas the rate of turnover of the replicating population was not affected. (ii) The rate of conversion of replicating molecules into closed-circular DNA was not affected by cycloheximide. (iii) The rate of elongation of nascent viral DNA fragments into strands of unit genome length was unaffected by cycloheximide. It is concluded that viral DNA synthesis is inhibited in the absence of protein synthesis exclusively at the level of initiation of new rounds of genome replication. Replicating molecules already initiated at the time of addition of cycloheximide matured into progeny closed-circular DNA at a normal rate.     

The effect of 3-methylindole on the rates of phospholipid and neutral lipid synthesis in cultured fibroblasts

The present study was designed to test the hypothesis that a pneumotoxin, 3-methylindole, alters the basic metabolic pathways involved in phospholipid and neutral lipid synthesis in cultured fibroblasts. Rat skin fibroblasts were obtained from day-old pups. Confluent monolayers were preincubated for up to 24 h with a range of concentrations (0-0.76 mM) of 3-methylindole. Following these treatments, the cell lipids were labelled by incubation for 6 h with [14C]glycerol. The lipids were extracted, separated by thin layer chromatography, and the radioactivity in each fraction was determined. 3-Methylindole had no effect on the total incorporation of [14C]glycerol into lipids, but significantly altered the distribution among lipid fractions. Incubation with 3-methylindole caused a decrease in the incorporation of [14C]glycerol into phosphatidylcholine, while radioactivity accumulated in the neutral lipid fraction. The other lipid fractions responded variably. Similarily, Flow 2000 human diploid lung fibroblasts were incubated for 24 h with 3-methylindole followed by treatment with [14C]glycerol, resulting in a 74% decrease in the incorporation of [14C]glycerol into phosphatidylcholine and a 50% increase in its accumulation in neutral lipid. The results indicate that 3-methylindole inhibits the synthesis of phosphatidylcholine from diacylglycerol precursors on the endoplasmic reticulum in cultured fibroblasts. This is an important observation as it shows that 3-methylindole affects the synthesis of phospholipids required for membrane turnover in cells that are not specialized for the production of phospholipids for surfactant.     

Myxospore coat synthesis in Myxococcus xanthus: in vivo incorporation of acetate and glycine.

Myxospore coat synthesis in Myxococcus xanthus was studied by incorporation of [(14)C]acetate into intermediates in the biosynthesis of coat polysaccharide and into acid-insoluble material during vegetative growth and after glycerol induction of myxospores. During short labeling periods at 27 degrees C, the radioactivity was shown to be located primarily in N-acetyl groups rather than sugar moieties. Two hours after glycerol induction, the pools of N-acetylglucosamine 6-phosphate and uridine 5'-diphosphate-N-acetylgalactosamine (UDPGalNAc) plus uridine 5'-diphosphate-N-glucosamine increased about twofold and were labeled at twice the rate measured for vegetative cells. The increased rate of synthesis of UDPGalNAc and its precursors could be correlated with increased enzyme activities measured in vitro. Controlled acid hydrolysis revealed that the galactosamine portion of the myxospore coat was N-acetylated. After glycerol induction, the incorporation of acetate into acid-insoluble material increased threefold. This enhanced incorporation was sensitive to neither penicillin nor d-cycloserine. In contrast, bacitracin inhibited the incorporation of [(14)C]acetate into acid-insoluble material more effectively 2 h after myxospore induction than during vegetative growth. Chloramphenicol added to cells 90 min after induction blocked further increase in the rate of [(14)C]acetate incorporation. Since the myxospore coat contains glycine, polymer synthesis was also measured by chloramphenicol-insensitive [(14)C]glycine incorporation into acid-insoluble material. Although protein synthesis decreased after glycerol induction, glycine incorporation increased. Two hours after induction, glycine incorporation was only 75% inhibited by chloramphenicol and rifampin. The chloramphenicol-insensitive rate of incorporation of [(14)C]glycine increased during the first hour after myxospore induction and reached a peak rate after 2 to 3 h. The chloramphenicol-resistant incorporation of [(14)C]glycine was resistant to penicillin but sensitive to bacitracin.     

Glycerol metabolism in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozocinin-diabetic rats. With glucose in the incubation medium, incorporation of exogenous [1,3-14C]glycerol into disaturated phosphatidylcholine, total phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) was increased 4-fold in cells from diabetic rats. In the absence of glucose, glycerol incorporation was 5-fold greater than in its presence in cells from normal animals, but was further increased 2.2-fold in cells from diabetic rats. Insulin treatment of diabetic rats returned all incorporation rates to control values. The increased glycerol incorporation rates were not due to differences in either phospholipid turnover or the size of the glycerol 3-phosphate precursor pool. Kinetic analysis of glycerol entry into the acid-soluble cell fraction indicated that glycerol transport occurred largely by simple diffusion, and was not rate limiting for its entry into lipids. Glycerol entry into the total lipid fraction was saturable, reaching a Vmax of 48 pmol/micrograms DNA per h in normal cells and 120 pmol/micrograms DNA per h in cells from diabetic rats, with no change in the Km (0.31 mM). While glycerol oxidation was reduced 23% in cells from diabetic rats in the presence of glucose and by 44% in the absence of glucose, glycerol kinase activity in sonicates of cells from diabetic animals was increased 210% and was reversed by in vivo insulin treatment. These results suggest that glycerol utilization in type II pneumocytes is a hormonally regulated function of both glycerol oxidation and glycerol phosphorylation.     

The Effect of Cycloheximide (Actidione) on Protein and Nucleic Acid Synthesis by Chlorella

Incorporation of ¹⁴C-phenylalanine, ¹⁴C-carbon dioxide, ¹⁴C-glucose,and ¹⁴C-glycine into the protein of Chlorella is inhibited bycycloheximide. A concentration of 2.5 µg per ml inhibitsincorporation by about 80 per cent; increasing the concentrationup to 10 µg per ml does not increase the degree of inhibition.The incorporation of ¹⁴C-adenine into ribonucleic acid (RNA)and deoxyribonucleic acid (DNA), and of ¹⁴C-glucose into polysaccharideis also inhibited. Unlike inhibition of protein synthesis, thatof nucleic acid and polysaccharide synthesis is observed onlyafter some delay. The delay is shortest for DNA synthesis andlongest for polysaccharide synthesis. Inhibition of ¹⁴C-glycineincorporation into DNA and RNA follows a similar pattern tothat obtained with ¹⁴C-adenine but the delay is much shorter.Cycloheximide also inhibits the formation of isocitrate lyasc(isocitrate-glyoxylate lyase, EC 4.1.3.1 [EC] ) when autotrophicallygrown cells are supplied with acetate.     

Effect of Phleomycin on Polyoma Virus Synthesis in Mouse Embryo Cells

The addition of phleomycin (25 mug) to primary mouse embryo cells infected with polyoma virus was found to cause 96% inhibition of the synthesis of infectious virus. When ribonucleic acid and protein synthesis was investigated in these cells by use of isotope incorporation, it was found that neither was inhibited drastically. Immunofluorescent staining studies with the use of antibody directed to the viral structural proteins showed that proteins were synthesized in the presence of the antibiotic. However, when deoxyribonucleic acid (DNA) synthesis was investigated, it was found that DNA synthesis in uninfected cells was completely inhibited within the initial 10 hr of phleomycin addition, whereas DNA synthesis in infected cells proceeded at a reduced rate. Selective DNA extraction (Hirt method) of phleomycin-treated infected cells demonstrated that synthesized viral DNA was salt-extractable, similar to that in infected control cells lacking phleomycin. This extracted DNA was further fractionated by ethidium bromide-cesium chloride density gradient equilibrium centrifugation. The phleomycin-treated preparations revealed twice as much component II (circular nicked and linear) as component I (supercoiled) DNA, whereas the DNA from normally infected control cells showed the reverse picture. It was also demonstrated that viral particles synthesized in the presence of phleomycin did not contain component I DNA. This packaged DNA was found to consist of fragments of both the host and viral types. Cells that were prelabeled with (3)H-thymidine and then treated with phleomycin demonstrated host DNA degradation. However, fragments formed from prelabeled host DNA were not encapsidated into viral particles.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of the Folic Acid Analogue, Trimethoprim, on Growth, Macromolecular Synthesis, and Incorporation of Exogenous Thymine in Escherichia coli

The effect of trimethoprim [2,4-diamino-5(2',4',5'trimethoxybenzyl)-pyrimidine] in the presence of thymine on Escherichia coli B temperature-sensitive and non-temperature-sensitive Thy(') strains and a phosphodeoxyribomutase-negative mutant was studied. The inhibitory effect of 5 mug of trimethoprim per ml on the growth of E. coli B was not overcome by thymine, thymidine, or thymidylate even in the presence of one-carbon metabolites and related metabolites. Deoxyribonucleic acid (DNA) and protein synthesis were more severely inhibited than ribonucleic acid (RNA) synthesis. The inhibition of DNA synthesis was partially reversed by addition of deoxyadenosine to increase the incorporation of exogenous thymine. By contrast, the inhibition of protein was not reversed even with one-carbon metabolites present, in keeping with the requirement for formylmethionyl-transfer RNA(F) for initiation. However, the inhibition of both DNA and protein synthesis in a phosphodeoxyribomutase-negative strain by 1 mug of trimethoprim per ml with thymine present was partially reversed by deoxyadenosine and one-carbon metabolites, and nearly normal growth occurred. 5-Fluorodeoxyuridine added at the time of addition of trimethoprim prevented the inhibition. Sulfadiazine in the presence of thymine inhibited both Thy(+) and Thy(-) strains whereas trimethoprim (with thymine) did not inhibit Thy(-) organisms. The effect of trimethoprim on the incorporation of labeled thymine into DNA was also studied. These experiments support the concept that trimethoprim in conjunction with the action of thymidylate synthetase inhibits the growth of Thy(+) cells because of a depletion of tetrahydrofolate. DNA synthesis is inhibited initially by a limitation of thymine nucleotide precursor, resulting from the indirect inhibition of thymidylate synthetase and the poor incorporation of exogenous thymine.