Factors affecting the adenosine triphosphate induced release of iron from transferrin.

The release of iron from transferrin was investigated by incubating the diferric protein in the presence of potential iron-releasing agents. The effective chemical group appears to be pyrophosphate, which is present in blood cells as nucleoside di- and triphosphates, notably adenosine triphosphate (ATP). An alternative structure with comparable activity is represented by 2,3-diphosphoglycerate. Neither 1 mM adenosine monophosphate (AMP) nor 1 mM orthophosphate released iron from transferrin. The ATP-induced iron-releasing activity was dependent on weak acidic conditions and was sensitive to temperature and sodium chloride concentration. The rate of iron release rapidly increased as transferrin was titrated with HCl from pH 6.8 to 6.1 in the presence of 1 mM ATP and 160 mM NaCl at 20 degrees C. Iron release from transferrin without ATP was observed below pH 5.5. Ascorbate (10(-4) M) reduced Fe(III), but only after iron release from transferrin by a physiological concentration of ATP. A proposal for the mechanism of iron release from transferrin by ATP and the utilization of reduced iron by erythroid cells is described.     

L-Pyroglutamic Acid Inhibits Energy Production and Lipid Synthesis in Cerebral Cortex of Young Rats In Vitro

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and -glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO₂ production and lipid synthesis from [U-¹⁴C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO₂ production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.     

Anandamide inhibits oxidative phosphorylation in isolated liver mitochondria

A study on the effect of anandamide (AEA) in energy coupling of rat liver mitochondria is presented. Micromolar concentrations of AEA, while almost ineffective on substrate supported oxygen consumption rate and on uncoupler stimulated respiration, strongly inhibited the respiratory state III. AEA did not change the rate and the extent of substrate generated membrane potential, but markedly delayed rebuilding by respiration of the potential collapsed by ADP addition. Overall, these data suggest that anandamide inhibits the oxidative phosphorylation process. Direct measurement of the F_oF₁ ATP synthase activity showed that the oligomycin sensitive ATP synthesis was inhibited by AEA, (IC₅₀, 2.5 μM), while the ATP hydrolase activity was unaffected. Consistently, AEA did not change the membrane potential generated by ATP hydrolysis.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

The binding of thyrotropin to liposomes containing gangliosides.

The relationship between uptake of Ca⁺⁺ and incorporation of sn-[¹⁴C]-glycerol-3-phosphate into phosphatidate, diglyceride, and triglyceride was evaluated in microsomes isolated from livers of normal fed male rats. Uptake of Ca⁺⁺ was dependent on concentration of Ca⁺⁺ (0.1 – 2.5 mM), and accompanied by a decrease in the rate of glycerolipid synthesis. The quantity of Ca⁺⁺ ion taken up at 20 μM CaCl₂ in the presence of ATP was equivalent to that observed with 2.5 mM CaCl₂ in the absence of ATP. The ATP dependent uptake of Ca⁺⁺, like the passive uptake at higher concentrations of Ca⁺⁺, was correlated with inhibition of incorporation of sn-glycerol-3-phosphate into phosphatidate. Accumulation of Ca⁺⁺ in hepatic microsomes, therefore, appears to result in a calcium-dependent decrease in biosynthesis of phosphatidate and other glycerolipids.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions

Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl₃ was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 μM; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 μM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 μm-1 mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 μM to 100 μM.     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.     

CHLORIDE UPTAKE BY ANACYSTIS NIDULANS (CYANOPHYCEAE)1

Anacystis nidulans (Richt.) Drouet & Daily (UTEX 625), grown in batch culture with 0.5% CO₂ in air, was supplied with chloride labelled with ³⁶Cl in light and dark. Uptake in light was stimulated relative to uptake in darkness. A single transport system for Cl^? with an apparent K_m for Cl^? of 0.14 mM was identified. Chloride in the cells reached a maximum value after 30–50 min at 25 C. At this point the internal Cl^? concentration was calculated to be 60-fold the external (0.1 mM) in light and 37-fold in darkness. DCMU (3-[3,4-dichlorophenyl]–1, 1-dime-thylurea), at concentrations which abolished photosynthetic O₂ evolution did not inhibit Cl^? uptake in light. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), at uncoupling concentrations for photosynthesis and dark respiration, strongly inhibited Cl^? uptake in light and darkness. N,N'-dicyclohexyl carbodiimide (DCCD), an energy transfer inhibitor, inhibited light Cl^? uptake more slowly than photosynthesis but had no effect on dark Cl^? uptake. It is concluded that Cl^? uptake in A. nidulans was active in light and darkness, and that ATP was the probable energy source for transport.     

Evaluation of Fe(III) reduction by mitochondria induced with a respiratory substrate NADH or succinate,using a Fe(II)-specific chelator bathophenanthroline disulfonate in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mitochondria are the centers of the cellular iron metabolism. Iron utilization by mitochondria is deeply related to their respiratory chain activity. We isolated mitochondria from Saccharomyces cerevisiae and examined Fe(III) reduction induced by a respiratory substrate (NADH or succinate), using a Fe(II)-specific chelator (bathophenanthroline disulfonate). In the presence of either 50 μM NADH or 5 mM succinate, the amount of reduced Fe(III) was linearly correlated with the amount of mitochondria. As the concentration of the substrate increased, the rate of the mitochondrial Fe(III) reduction reached a plateau. In the presence of 1 mM ADP or 1 mM ATP, the extramitochondrial Fe(III) reduction was repressed when succinate was used as the substrate, but not when NADH was used. ADP had an inhibitory effect even under low concentration of succinate, suggesting that ADP and ATP acted in a manner of both competitive and uncompetitive inhibition.     

Biomass production and fatty acid profile of a Scenedesmus rubescens-like microalga

This investigation examined the effects of nitrogen–phosphate combined deficiency on the biomass yield, fatty acid methyl esters (FAME) production and composition from Scenedesmus rubescens-like microalga. A 15-day indoor culture was performed as a 3 × 3 factorial design (NaNO₃ levels: 3, 10 and 20 mM; KH₂PO₄ levels: 20, 50 and 150 μM). The algae grown under medium nitrogen concentration (10 mM) and high phosphate concentration (150 μM) reached the highest biomass (1223.5 ± 152.5 mg/L). Both nitrogen and phosphate had a significant influence on the FAME yield (P < 0.05 and P < 0.0001, respectively). The FAME yield from algae grown under low nitrogen (3 mM) and phosphate concentration (20 μM) increased throughout the experiment and the highest FAME yield (42.2 ± 2.5% of AFDW) as well as C16 and C18 content (95.8 ± 1.6% of AFDW) was achieved under these conditions. Algae grown under medium nitrogen concentration (10 mM) and low phosphate concentration (20 μM) had the highest FAME productivity (426.0 mg/L ± 135.0 mg/L). Thus, the lower nitrogen concentration (3 mM–10 mM) and low phosphate concentration (20 μM) would be an optimal combination tested to produce the most FAME from S. rubescens-like algae.     

The gelation kinetics of platelet extracts

The kinetics of the gelation process that occurs upon warming cold platelet extracts were studied using a sensitive rheometer. At micromolar or less free Ca²⁺ concentrations and in the presence of 1 mM ATP, the gel rigidity curves showed several peaks, indicating that platelet extract proteins went through network assembling/disassembling cycles during gelation. The gelation kinetics were accelerated by increasing the free Ca²⁺ concentration up to about 2 μM. At 4–15 μM free Ca²⁺, the gelation cycles were completely abolished except for the first peak. The gelation process became one of monotonically increasing elastic modulus at millimolar free Ca²⁺ concentrations. Trifluoperazine (50 μM), a calmodulin inhibitor, did not affect gelation at micromolar free Ca²⁺ concentrations. Except for the first gelation step, which was completed within 5 min after warming, the rest of the gelation process was found to be affected by K⁺, ATP, cytochalasin E and colchicine. K⁺ at concentrations higher than 10 mM retarded the gelation kinetics. Extracts prepared with low (0.1 mM) ATP content showed impaired gelations, and this was partially reversed by adding 1 mM ATP, but not 1 mM adenylylimidodiphosphate (p[NH]ppA). Both cytochalasin E (1 μM) and colchicine (1 mM) interfered with the gelation process.     

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent K_m=22 μM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 μM), dibutyryl cyclic AMP (0.1 mM) and the β-adrenergic agonist ritodrine (10 μM) also had no effect, however the α₁-adrenergic agonist, methoxamine (10 μM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the α-adrenergic agonist methoxamine.     

Anionen-Influx,ATP-Spiegel und CO2-Fixierung in Limnophila gratioloides und Chara foetida

Summary Influx of anions (5x10^–4 M Cl^– or SO₄ ^2–) across the plasmalemma, ATP levels and CO₂ fixation in Limnophila and Chara have been measured in a comparative study.In Limnophila, influx, ATP level and CO₂ fixation were progressively reduced by increasing concentrations of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the light (4000 lux) as well as in the dark. In Chara, not only influx but also ATP levels were much less reduced in the light than in the dark.At 5x10^–4 M external salt concentration the action of light or dark is to change active influx of anions. Thus this study provides strong evidence to support the view that active anion uptake is directly dependent on ATP rather than on electron flow or NADPH. The possible significance of differences in the photophosphorylation systems of various plants is stressed.     

Effects of inhibitor,competitors and temperature on transport of carbohydrate in Crithidia luciliae

Effects of KCN (10^?4 M), simultaneous presence of varying concentrations of D-glucose and L-sorbose, and temperature on transport of carbohydrate in C. luciliae have been studied. The rate of carbohydrate entrance is inhibited, in all sugars used, ranging from 19% to 70% inhibition at 0.5 mM external concentrations. However, this inhibitor does not affect transport from external concentrations of the order of 0.02 M. At 20 mM external concentration, the rate of L-sorbose entrance is greatly inhibited by the simultaneous presence of D-glucose, and the transport mechanism shows enormously greater affinity for glucose than for other monosaccharides. However, at 0.5 mM external concentration, the rate of sorbose entrance is not inhibited at all by the simultaneous presence of D-glucose. In the temperature interval 15°–25°C, the Q₁₀ for rate of entrance when the external concentration is 0.5 mM is 2.8 times larger than the Q₁₀ when the external concentration is 20 mM. These data are interpreted as strongly suggesting two mechanisms for carbohydrate entrance: (a) facilitated diffusion, of importance only at high external concentrations; (b) an active transport mechanism, active at low external concentrations and dependent upon a supply of metabolic energy. These results are compared with those reported in the literature for other types of cells.     

Reduced glutathione esters—antidotes to toxicity. Cytotoxicity induced by hydrogen peroxide, 1-chloro-2,4-dinitrobenzene,and menadione in murine P388D1 macrophages in vitro

Repletion of depleted cellular reduced glutathione (GSH) levels in oxidative stress and exposure to arylating agents is a strategy for the development of antidotes to chemical toxicity. The effect of GSH, reduced glutathione ethyl monoester (GSHEt), and reduced glutathione ethyl diester (GSHEt₂) on the cytotoxicity of hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), and menadione to P388D₁ macrophages in vitro was investigated. The median toxic concentration TC₅₀ values of the toxicants were hydrogen peroxide 24 ± 2 mM (N = 19), CDNB 63 ± 6 μM (N = 18), and menadione 30 ± 4 μM (N = 22). Reduced glutathione, GSHEt, and GSHEt₂ were poor antidotes to hydrogen peroxide toxicity. Indeed, the observed antidote effects were attributed to the nonenzymatic reaction of the GSH derivatives with hydrogen peroxide in the extracellular medium. Reduced glutathione ethyl diester was a more potent antidote of CDNB- and menadione-mediated toxicity than GSHEt and GSH. For cell incubations with the approximate median toxic concentration TC₅₀ values of hydrogen peroxide, CDNB, and menadione, the respective median effective antidote concentration EC₅₀ values were GSHEt 23.8 ± 4.1 mM (N = 9), 3.6 ± 0.6 mM (N = 11), and 226 ± 93 μM (N = 12); and GSHEt₂ 20.4 ± 1.9 mM (N = 6), 603 ± 2 μM (N = 9), and 7.6 ± 2.3 μM (N = 12). Reduced glutathione ethyl diester was a potent antidote to CDNB- and menadione-induced toxicities but not to hydrogen peroxide-induced toxicity under acute intoxication conditions. © 1996 John Wiley & Sons, Inc.     

Sulphate uptake in the unicellular marine red algaRhodella maculata

Sulphate uptake by the unicellular marine red algaRhodella maculata conforms to Michaelis-Menten kinetics. Two uptake systems have been found: a low affinity system with an apparentK _m of 22 mM, and a high affinity system with an apparentK _m of 63.4 M. Transition from the low to the high affinity system can occur within 2.5 min, in response to a decrease in the ambient sulphate concentration to below 10 mM. Assimilation rates in the dark are about 20% those in the light, although enhancement by light is independent of the quanlity of light supplied above 27 mol m^-2 s^-1. Use of metabolic inhibitors indicates that photophosphorylation provides the main source of energy for sulphate assimilation, through both cyclic and non-cyclic electron flow.Abbreviations used APS-kinase ATP:adenylyl-sulphate 3-phosphotransferase (E.C. 2.7.1.25) - ATP-sulphurylase ATP:sulphate adenylyltransferase (E.C.2.7.74) - DCMU [3-(3,4-dichlorophenyl)]-1,1 dimethylurea - 2,4 DNP 2,4-dinitrophenol - DBMIB Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)     

Three new DNA helicases from<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

At least six DNA helicases have been identified during fractionation of extracts from the yeastSaccharomyces cerevisiae. Three of those, DNA helicases B, C, and D, have been further purified and characterized. DNA helicases B and C co-purified with DNA polymerse δ through several chromatographic steps, but were separated from the polymerase by hydrophobic chromatography. DNA helicase D co-purified with Replication Factor C over seven chromatographic steps, and was only separated from it by glycerol gradient centrifugation in the presence of 0.2 M NaCl. All three helicases are DNA dependent ATPases with K_m values for ATP of 190 μM, 325 μM, and 60 μM for DNA helicases B, C, and D, respectively. Their DNA helicase activities are comparable. They are 5′–3′ helicases and have pH optima of 6.5–7 and Mg²⁺ optima of 1–2 mM. However, they differ in the nucleotide requirement for helicase action. Whereas all three helicases preferred ATP, dATP, UTP, CTP, and dCTP as cofactors, DNA helicase C also used GTP, but not dTTP. On the other hand, DNA helicase D used dTTP, but not GTP, and DNA helicase B used neither nucleotide as cofactor. These studies allowed us to conclude that DNA helicases B, C, and D are not only distinct enzymes, but also different from two previously identified yeast DNA helicases, the RAD3 protein and ATPase III.     

Mechanisms of Bactericidal Action of Cinnamaldehyde against Listeria monocytogenes and of Eugenol against L. monocytogenes and Lactobacillus sakei

The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20°C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes. At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20°C were measured. Treatment of nonenergized L. monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized L. monocytogenes with 40 mM cinnamaldehyde or 10 μM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of L. sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 μM had no significant effect on the cellular ATP of L. sakei. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.     

Fatty Acid Synthesis in Leucoplasts Isolated from Developing Seeds of Brassica campestris

Fatty acid synthesis from Na (1-¹⁴C) acetate in leucoplasts isolated from developing seeds of Brassica compestris was found to be maximum when leucoplasts were supplied with 0.8 mM acetate, 20 mM NaHCO₃, 8 mM ATP, 8 mM MgCl₂, 4 mM MnCl₂, 0.6 mM CoA, 1 mM NADH, 1 mM NADPH and 0.2 M sorbitol and incubated at 30°C for 2 h. The rate of fatty acid synthesis was highest at pH 8.5 In presence of 0.4 M Bistris-propane buffer and linear for upto 4 h at 30°C with 80–110 μg plastid protein. Sorbitol was an essential requirement as it prevented the rupturing of leucoplasts by osmosis. ATP and divalent cations were almost absolute requirements, whereas nucleotides, CoA and bicarbonate improved the rate of fatty acid synthesis by two to ten folds. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. High concentration of dithiothreltol inhibited the incorporation of (¹⁴C) acetate Into fatty acids. The system developed as above could be used for in vitro studies.