Effect of salvin on the incorporation of labeled precursors into macromolecular compounds of Staphylococcus aureus 209P

Salvin is a preparation of Salvia officinalis L. Its effect on synthesis of macromolecules in cells of Staphylococcus aureus 209P was studied with labeled precursors in a system used for investigation of peptidoglycan synthesis. At a concentration of 10 micrograms/ml salvin inhibited incorporation of 14C-lysine into the cell wall polymer and protein fraction by 42.9 and 8.9 per cent respectively and stimulated incorporation of 3H-thymidine and 3H-uridine into the nucleic acid fraction. In the presence of salvin in a quantity of 120 micrograms/ml there was observed inhibition of 3H-uridine incorporation into the nucleic acid fraction by 53.3 per cent and 14C-lysine into the protein fraction by 74.5 per cent along with inhibition of peptidoglycan synthesis by 95.5 per cent. The results conformed to the findings of electron microscopic investigation of the solving effect on ultrastructure of S. aureus 209P. They confirmed the previous assumption that salvin had the primary effect on the processes directly associated with synthesis of the cell wall polymer.     

Protein and Nucleic Acid Metabolism in Germinating Peas

Protease, peptidase, and ribonuclease activities were demonstratedin germinating pea cotyledons and axis tissues. These activitiesindicate that the enzymatic machinery for the hydrolysis ofprotein and nucleic acid reserves are present in the germinatingcotyledon. The fate of hydrolytic products was determined byinjecting leucine-¹⁴C or adenine-8-¹⁴C into the cotyledons.At most, 20 per cent of the leucine-¹⁴C and 10 per cent of theadenine-8-¹⁴C administered were transported from the cotyledonto axis tissues. Both compounds were extensively metabolizedand the labelling patterns suggest that different metabolicpathways are in operation in the two organs. The amounts ofadenine incorporated into nucleic acids and of leucine incorporatedinto protein in the cotyledons suggest that synthesis and turnoverwere occurring at a rapid rate. The adenine transported fromthe cotyledon was not readily available for nucleic acid synthesisin the axis whereas transported leucine was readily incorporatedinto axis protein.     

Acetate is the preferred substrate for long-chain fatty acid synthesis in isolated spinach chloroplasts.

1. Commercially available [2-14C]pyruvate and [2-14C]malonate were found to contain 3-6% (w/w) of [14C]acetate. 2. The contaminating [14C]acetate was efficiently utilized for fatty acid synthesis by isolated chloroplasts, whereas the parent materials were poorer substrates. 3. Maximum incorporation rates of the different substrates examined were (ng-atoms of C/h per mg of chlorophyll): [1-14C]acetate, 2676; [2-14C]pyruvate, 810; H14CO3-, 355; [2-14C]malonate, 19. 4. Products of CO2 fixation were probably not a significant carbon source for fatty acid synthesis in the presence of exogenous acetate.     

Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

Introduction and metabolism of pentose and hexose phosphates in permeabilized Morris hepatoma 5123TC cells

Metabolism of arabinose 5-P, ribose 5-P and glucose 6-P in permeabilized and resealed Morris hepatoma 5123TC cells was investigated by measuring the contribution of these compounds to nucleic acid biosynthesis. The level of [14C]-arabinose (non-phosphorylated) incorporation into nucleic acids was slight, presumably due to the low activity of the transport system or the absence or low activity of a specific 'kinase' enzyme. The permeabilizing procedure involved the brief treatment of Morris hepatoma 5123TC cells with lysolecithin and resulted in a cell population which was permeable to charged compounds i.e. sugar phosphates and nucleotides, that otherwise could not cross the plasma membrane. The permeabilized (and resealed cells) retained normal cellular morphology and intactness of specific organelles as judged by the maintenance of functional properties. Following permeabilization, these cells resealed when transferred back to normal growth medium, and continued to divide and increase at the same rates as control non-permeabilized cell cultures. The permeabilized cells incorporated deoxyribonucleotides ([methyl -3H]-TTP) into DNA at a linear rate of 0.047 nmol per 10(7) cells min-1, representing 90-100 per cent of the DNA synthesis rate in vivo. The permeabilization technique, when coupled with procedures to establish cell synchrony, permitted the comparative estimate of the contributions of [14C]-labelled arabinose 5-P, ribose 5-P and glucose 6-P to RNA, DNA, amino acids, CO2, lactate and sugar mono- and bisphosphates. The percentage of [14C]-isotope incorporated into total nucleic acids by these three labelled sugar phosphates were 2.3, 4.9 and 6.3 respectively. Possible reasons for the lower incorporation of 14C from arabinose 5-P are given. The results are consistent with the proposal that arabinose 5-P, an intermediate of the L-type pentose pathway activity of 5123TC cells, was incorporated into nucleic acids by its interconversion with ribulose 5-P and ribose 5-P and thus into PRPP. This study represents the first report of sugar phosphate as opposed to free sugar metabolism by tumour cells in culture.     

Heterotrophic Carbon Metabolism by Beggiatoa alba

The assimilation and metabolism of CO(2) and acetate by Beggiatoa alba strain B18LD was investigated. Although B. alba was shown to require CO(2) for growth, the addition of excess CO(2) (as NaHCO(3)) to the medium in a closed system did not stimulate growth. Approximately 24 to 31% of the methyl-labeled acetate and 38 to 46% of the carboxyl-labeled acetate were oxidized to (14)CO(2) by B. alba. The apparent V(max) values for combined assimilation and oxidation of [2-(14)C]acetate by B. alba were 126 to 202 nmol min(-1) mg of protein(-1) under differing growth conditions. The V(max) values for CO(2) assimilation by heterotrophic and mixotrophic cells were 106 and 131 pmol min(-1) mg of protein(-1), respectively. The low V(max) values for CO(2) assimilation, coupled with the high V(max) values for acetate oxidation, suggested that the required CO(2) was endogenously produced from acetate. Moreover, exogenously supplied acetate was required by B. alba for the fixation of CO(2). From 61 to 73% of the [(14)C]acetate assimilated by washed trichomes was incorporated into lipid. Fifty-five percent of the assimilated [2-(14)C]acetate was incorporated into poly-beta-hydroxybutyric acid. This was consistent with chemical data showing that 56% of the heterotrophic cell dry weight was poly-beta-hydroxybutyric acid. Succinate and CO(2) were incorporated into cell wall material, proteins, lipids, nucleic acids, and amino and organic acids, but not into poly-beta-hydroxybutyric acid. Glutamate and succinate were the major stable products after short-term [1-(14)C]acetate assimilation. Glutamate and aspartate were the first stable (14)CO(2) fixation products, whereas glutamate, a phosphorylated compound, succinate, and aspartate were the major stable (14)CO(2) fixation products over a 30-min period. The CO(2) fixation enzymes isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate; reversed) and malate dehydrogenase (nicotinamide adenine dinucleotide phosphate; decarboxylating) were found in cell-free extracts of both mixotrophically grown and heterotrophically grown cells. The data indicate that the typical autotrophic CO(2) fixation mechanisms are absent from B. alba B18LD and that the CO(2) and acetate metabolism pathways are probably linked.     

Photosynthate Partitioning and Fermentation in Hot Spring Microbial Mat Communities

Patterns of (sup14)CO(inf2) incorporation into molecular components of the thermophilic cyanobacterial mat communities inhabiting hot springs located in Yellowstone National Park and Synechococcus sp. strain C1 were investigated. Exponentially growing Synechococcus sp. strain C1 partitioned the majority of incorporated (sup14)CO(inf2) into protein, low-molecular-weight metabolites, and lipid fractions (45, 22, and 18% of total incorporated carbon, respectively). In contrast, mat cores from various hot springs predominantly accumulated polyglucose during periods of illumination (between 77 and 85% of total incorporated (sup14)CO(inf2)). Although photosynthetically active, mat photoautotrophs do not appear to be rapidly growing, since we also detected only limited synthesis of macromolecules associated with growth (i.e., protein and rRNA). To test the hypothesis that polysaccharide reserves are fermented in situ under the dark anaerobic conditions cyanobacterial mats experience at night, mat cores were prelabeled with (sup14)CO(inf2) under illuminated conditions and then transferred to dark anaerobic conditions. Radiolabel in the polysaccharide fraction decreased by 74.7% after 12 h, of which 58.5% was recovered as radiolabeled acetate, CO(inf2), and propionate. These results indicate tightly coupled carbon fixation and fermentative processes and the potential for significant transfer of carbon from primary producers to heterotrophic members of these cyanobacterial mat communities.     

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.     

Nucleic Acid Synthesis in Bacteriophage SPO2c1-Infected Bacillus subtilis

The synthesis of host macromolecules was shut off very slowly and incompletely by bacteriophage SPO2c(1). No change in the rate of incorporation of radioactive precursors into protein and ribonucleic acid (RNA) could be detected after infection, and the rate of incorporation of thymidine was increased only slightly. The relative proportions of phage and host species of nucleic acids at various intervals in the latent period were determined by means of nucleic acid hybridization. Phage-specific RNA populations synthesized early were different from those synthesized late in the latent period. Host deoxyribonucleic acid (DNA) replication continued until 8 to 10 min after SPO2c(1) infection and then decreased markedly as phage-specific DNA synthesis was initiated. Host DNA was not degraded to trichloroacetic acid-soluble fragments, and its nucleotides were not found in either newly synthesized intracellular phage DNA or in progeny phage particles. The average burst size of SPO2c(1) was approximately 200 plaque-forming units per cell.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Kinetics of Glucose Incorporation by Aphanocapsa 6714

Photoautotrophic metabolism of CO(2) was compared with glucose metabolism in the facultative unicellular blue-green alga, Aphanocapsa 6714. Glucose-fed cells incorporated more (14)C into phosphorylated sugar intermediates of the reductive and oxidative pentose phosphate cycles than autotrophic cells. The relative increases were: 140-fold in dark cells; 32-fold in dichlorophenylmethylurea (DCMU)-inhibited cells; and 16-fold in cells assumilating glucose during photosynthetic carbon reduction. On the other hand, incorporation of (14)C from glucose into 3-phosphoglycerate and the amino acid pools of glutamate and aspartate was reduced in dark cells. Rates of protein synthesis in dark and DCMU-inhibited cells were reduced 50 and 80% compared to photoautotrophic cells. In cells assimilating glucose during photosynthesis, rates of (14)C incorporation into the two amino acids and protein were the same as in photoautotrophic cells. Chase experiments, using an excess of (12)C-glucose and CO(2), revealed slow turnover of carbon in dark cells and intermediate turnover rates in DCMU-inhibited cells, when compared to cells assimilating glucose during photosynthesis.     

Studies on the Growth in Culture of Plant Cells: XV. UPTAKE AND UTILIZATION OF URIDINE DURING THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

[2-¹⁴C]-uridine is rapidly taken up by sycamore cells in suspensionculture. A proportion of the radioactivity enters RNA withoutmeasurable delay, whilst the remainder equilibrates with a largepool of phosphorylated compounds, the major radioactive componentof which is 5'-UMP. Both the uracil and cytosine residues ofRNA receive label from [¹⁴C]-uridine and, when the cells aresupplied with high concentrations of uridine, these bases arederived almost exclusively from the nucleoside. [¹⁴C]-uridine is incorporated into RNA at all stages of thegrowth cycle of batch cultures; its continuing incorporation,when the total RNA content of the cells is rapidly decreasing,indicates a high rate of turnover of the total RNA. Long-termlabelling experiments also indicate turnover of RNA during thephase of active cell division and suggest that a large proportionof the degradation products are not re-utilized for RNA synthesis. Sycamore cells degrade [2-¹⁴C]-uridine with release of ¹⁴CO₂.The proportion degraded increases from 25 per cent at an externaluridine concentration of 10^–6M to 75 per cent at 10^–3M. Despite this, nucleic acids are the only macromolecules thatreceive a significant amount of radioactivity from [2-¹⁴]C-uridine.     

Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions.

An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.     

Disparity in the effects of two N-methyl nicotinamides on poly(ADP-ribose) synthetase and macromolecular synthesis in hepatomas

The inhibitory effects of nicotinamide analogs on the activity of poly(ADP-ribose)) synthetase were compared to effects on precursor incorporation into macromolecules in three lines of hepatoma cells (Morris hepatomas 5123C, 7777 and HTC). N'-methylnicotinamide was a less effective inhibitor of poly (ADP-ribose) synthetase than was 1-methylnicotinamide while both these compounds had smaller inhibitory effects on the enzyme than were seen with nicotinamide or 3-aminobenzamide. On the other hand, the incorporation of [3H]thymidine into DNA and of [3H]uridine into RNA were inhibited by N'-methylnicotinamide in the concentration range 2-20 mM but not by 1-methylnicotinamide. Under the conditions examined there were no significant effects on the incorporation of [14C]lysine and [3H]leucine in hepatoma cells. The data indicated that the inhibitory effect of N'-methylnicotinamide on nucleic acid synthesis may be unrelated to action on poly (ADP-ribose) synthetase.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

14CO2 fixation in incubated rat diaphragms

The time course (0-60 min) of label incorporation from NaH14 CO3 into citric-acid-cycle intermediates and amino acids was investigated in incubations of isolated rat diaphragms. On the basis of these results, 14CO2 exchange by isocitrate dehydrogenase and 14CO2 fixation by propionyl-CoA carboxylation and pyruvate carboxylation could be estimated. Apparent rates amounted to about 30-40, 2, and 35 nmol/min per g of muscle, respectively. About 90 percent of C4-carbon compounds originating from 14CO2 fixation were subsequently removed by decarboxylation. 2-Cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, effectively reduced 14CO2 production from [1-14C]pyruvate but did not affect incorporation of radioactive label from NaH14CO3. In cell-free muscle extracts, 14CO2 fixation was demonstrable under assay conditions suitable for NADP -dependent 'malic' enzyme(s). Addition of hydroxymalonate, an inhibitor of the latter enzyme(s), significantly reduced 14CO2 incorporation. The results provide evidence for a continuous cytosolic replenishment and mitochondrial depletion of citric-acid-cycle carbon skeletons in resting skeletal muscle tissue. The functional role of malic (iso)enzyme activities in these processes is discussed.     

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.