Utilization of 35S-Thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans

Differentially labelled ³⁵S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5–4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated K _m value of 0.5 mM for ³⁵S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50°C. In the absence of thiosulphate, the cells bound ³⁵S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-³⁵S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-³⁵S]thiosulphate or ³⁵S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP_sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.Abbreviation APS adenosine-5-sulphatophosphate     

Characterization of cell surface material removed by water and NaCl washing ofParacoccus denitrificans grown under conditions of divalent cation deficiency and sufficiency

Paracoccus denitrificans grown on complex medium deficient in Mg²⁺ and Ca²⁺ are rendered lysozyme susceptible by washing with NaCl, whereas cells grown in a succinate-salts medium (Mg²⁺ and Ca²⁺ sufficient) or complex medium supplemented with Mg²⁺+Ca²⁺ are not. The material released by water washing of cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ was characterized by a high protein content. There was a high lipid: protein ratio and an appreciable amount of 3-deoxyoctulosonic acid in the material released by NaCl washing of cells grown under all conditions, indicating release of outer membrane material. The lipid ornithine: lipid phosphorous ratios of NaCl wash from cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ were 0.54 and 0.34, respectively. Although NaCl washing removed outer membrane material from cells grown under all conditions, only divalent cation deficient cells were rendered lysozyme susceptible. This might be explained by the increased outer membrane ornithine-containing lipid to phospholipid ratio in these cells yielding a more permeable outer membrane.     

The effect of temperature on the continuous ferrous-iron oxidation kinetics of a predominantly Leptospirillum ferrooxidans culture.

The ferrous-iron oxidation kinetics of a bacterial culture consisting predominantly of Leptospirillum ferrooxidans were studied in continuous-flow bioreactors. The bacterial culture was fed with a salts solution containing 12 g/L ferrous-iron, at dilution rates ranging from 0.01 to 0.06 l/h, and temperatures ranging from 30 to 40 degrees C, at a pH of 1.75. The growth rate, and the oxygen and ferrous-iron utilization rates of the bacteria, were monitored by means of off-gas analysis and redox-potential measurement. The degree-of-reduction balance was used to compare the theoretical and experimental values of r(CO(2)), -r(O(2)) and -r(Fe(+2)), and the correlation found to be good. The maximum bacterial yield on ferrous-iron and the maintenance coefficient on ferrous-iron, were determined using the Pirt equation. An increase in the temperature from 30 to 40 degrees C did not appear to have an effect on either the maximum yield or maintenance coefficient on ferrous-iron. The average maximum bacterial yield and maintenance coefficient on ferrous-iron were found to be 0.0059 mmol C/mmol Fe(2+) and 0.7970 mmol Fe(2+)/mmol C)/h, respectively. The maximum specific growth rate was found to be 0.077 l/h. The maximum specific ferrous-iron utilization rate increased from 8.65 to 13.58 mmol Fe(2+)/mmol C/h across the range from 30 to 40 degrees C, and could be described using the Arrhenius equation. The kinetic constant in bacterial ferrous-iron oxidation increased linearly with increasing temperature. The ferrous-iron kinetics could be accurately described in terms of the ferric/ferrous-iron ratio by means of a Michaelis-Menten-based model modified to account for the effect of temperature. A threshold ferrous-iron level, below which no further ferrous-iron utilization occurred, was found at a ferric/ferrous-iron ratio of about 2500. At an overall iron concentration of 12 g/L, this value corresponds to a threshold ferrous-iron concentration of 78.5 x10(-3) mM.     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Effect of control of pH on the excretion of acid phosphatase by Rhodotorula glutinis

Summary The excretion of an acid phosphatase by Rhodotorula glutinis is related to the pH of the medium. During growth, the phosphatase excretion into the medium at a constant pH of 4.5 was 5 times higher than that observed at variable pH. After cultivation at a constant pH of 4.5 or at variable pH, cells were incubated at various pH values between pH 2 and 7. During this second incubation acid phosphatase release occured at pH 4.5 to 6.5 only. There was no release at pH 3.0; but when resting cells incubated at this pH were placed in a buffer solution at pH 5.5 a high activity was released. Extensive washing did not eliminate residual intrinsic acid phosphatase activity. These two types of acid phosphatase were phosphomonoesterases with an identical specificity for different substrates.     

Release of alkaline phosphatase activity by aqueous-ether treatment of whole cells of Serratia marcescens.

The effect of aqueous-ether treatment according to the method of Ribi et al. (1961) on the release of alkaline phosphatase from cells of two strains of Serratia marcescens was studied. By this method, lipopolysaccharide-protein (endotoxin) complexes associated with alkaline phosphatase activities were released from both strain 08 and strain Bizio. SDS-polyacrylamide gel electrophoresis followed by enzymatic assay showed the presence of two active components in each strain. Fractions released from strain 08 contained alkaline phosphatase A (140,000 dalton) and alkaline phosphatase B (110,000) daltons) while those from strain Bizio contained alkaline phosphatase A' (190,000 daltons) and alkaline phosphatase B (110,000 daltons). Although it is known that saline plays a role in the release of alkaline phosphatase activities from cell envelope of Gram-negative bacteria the presence of saline in the extracting medium affects only slightly the chemical composition and not at all on the enzymatic nature of the released components. By comparing the enzymatic profiles of the materials released by other techniques, such as polymyxin B treatment and osmotic shock, it appears that alkaline phosphatase activities released by aqueous-ether treatment of whole cells of S. marcescens originate from the periplasmic space.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Root exudates of plants

Summary The release of substances from wheat roots was found to be directly related to the growth of the root system. Plants whose root system did not grow released almost no exudates.When exudate concentration in the vicinity of the roots was lowered by frequent replacements of the nutrient solution or by a simultaneous cultivation of exudate-utilizing bacteria, the release of exudates was enhanced. In axenic wheat cultures, the amount of exudates during a 12-day cultivation with 2- or 4-day intervals between medium replacements represented 50% of root dry weight and 12% of whole plant dry weight.Wheat plants cultivated in the presence of the bacteriumPseudomonas putida released up to double the amount of exudates compared with axenic variants.     

Adaptation of adhesion test using Caco-2 cells for anaerobic bacterium<Emphasis Type="Italic">Pseudobutyrivibrio xylanivorans</Emphasis>, a probiotic candidate

Pseudobutyrivibrio xylanivorans strain Mz5(T), an anaerobic bacterium (originating from the rumen of a Holstein-Friesian cow), has some attributes that make it a possible probiotic strain (very active hydrolases, bacteriocin and conjugated linoleic acid production). For the estimation of its adhesion ability, the adhesion test on Caco-2 cells was introduced and adapted. The adhesion was performed in an anaerobic glove box in standard 24-well plates at neutral pH for 30 min. The best method for separation of the adhered bacteria from Caco-2 cells appeared to be homogenization with an automatic pipette. The number of adhered bacteria was too small to be determined microscopically, so a new approach, i.e. detection of the apparent lag phase in liquid growth medium was tested. Under the selected assay conditions 1.04 bacterial cells from the late exponential phase adhered to one Caco-2 cell, which confirms the adhesion capability of P. xylanivorans Mz5(T). The adapted adhesion test using Caco-2 cells is suitable for estimation of adhesion capability of anaerobic bacteria.     

Screening for lignin degrading bacteria by means of 14C-labelled lignins

Several Nocardia and Pseudomonas spp., as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release ¹⁴CO₂ from specifically ¹⁴C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stalk lignins. The bacteria were selected according to their ability to degrade phenolic compounds. However, only some of them could release significant amounts of ¹⁴CO₂ from the labelled lignin. The tested Nocardia spp. were more active than the Pseudomonas spp. and the unidentified bacteria. The most active strains belonged to N. autotrophica. These strains released CO₂ significantly from the methoxyl group and transformed the other carbons from the phenylpropane skeleton of lignin also into CO₂. Other less demethylating strains also released little CO₂ from the other carbons of the lignin molecule. From corn stalk materials which were specifically labelled in the lignin part only small amounts of labelled CO₂ were released.Non-Common-Abbreviation Used DHP dehydropolymers of coniferyl alcohol     

Proton release by polymorphonuclear leukocytes during phagocytosis or activation by digitonin or fluoride

The stimulation of polymorphonuclear leukocytes with bacteria or digitonin causes protons to be released into the reaction medium at the same time as the respiratory burst. Although lactic acid is released from the cells due to the stimulated metabolic activity, proton release was not due to lactic acid accumulation as fluoride markedly inhibited lactic acid accumulation in untreated cells within 5 minutes of incubation and a rapid proton release and superoxide anion production occurred after lactate production had ceased. The proton releasing mechanism, however, is closely linked with the activation of the plasma membrane NAD(P)H oxidase since the proton release is depressed only when the activation mechanism and/or the oxidase is inhibited.     

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

Separation and localization of cell wall layers of a gram-negative bacterium

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.     

Effect of sodium taurodeoxycholate, CaCl2 and albumin on the action of pancreatic lipase on droplets of trioleoylglycerol and the release of lipolytic products into aqueous media

1. Effects of various substances on the activity of pancreatic lipase and on the release of lipolytic products into aqueous media were studied with droplets of trioleoylglycerol suspended from a membrane filter at the top of a flow-through chamber. The droplets were perifused for 7 min with a commercial preparation of pancreatic lipase in 0.15 M NaCl solution at pH 6.5 and then perifused for 60 min with lipase-free media, either 0.15 M NaCl at pH 6.5 or basal medium at pH 7.4 (70 mM sodium barbital) containing different additives. 2. About 6% of the trioleoylglycerol in droplets was hydrolyzed during the perifusion with lipase. Another 15% was hydrolyzed in 30 min, but none thereafter, when the droplets were perifused with 0.15 M NaCl alone. The rate of hydrolysis was doubled and prolonged when droplets were perifused with basal medium at pH 7.4. Lipolytic products formed at pH 7.4 were 62% oleic acid, 20% monooleoylglycerol and 18% dioleoylglycerol, yet only 4% of the lipolytic products were released into the perifusate. 3. Sodium taurodeoxycholate (TDC) (17 mM ) added to basal medium increased 18 x the amount of lipolytic products released into the perifusate but increased lipolysis only 13%. The molar ratio of oleic acid to monooleoylglycerol in the perifusate was 5.7 during the first 30 min and 4.0 during the last 30 min. 4. Ca2+ (3.3 mM) added to basal medium increased lipolysis 87% but did not affect the amount (4%) of lipolytic products released into the perifusing medium. 5. TDC and Ca2+ added to basal medium produced the largest increase in lipolysis, with 59% of trioleoylglycerol hydrolyzed in 15 min and 91% in 60 min. The amount of lipolytic products released into the perifusing medium, however, was not increased above that released into medium containing TDC alone. 6. Serum albumin (0.6 mM) and Ca2+ added to basal medium increased 14 x the amount of lipolytic products released into the perifusate without affecting the basal lipolytic rate. Albumin, however, suppressed by 40% the stimulatory effect of Ca2+ on pancreatic lipase activity.     

Survival and activity of Pseudomonas sp. strain B13(FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton.

Genetically engineered Pseudomonas sp. strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms). Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR. The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe. Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control. The number of Pseudomonas sp. strain B13(FR1) cells decreased rapidly to ca. 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa. Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers. Immediately after the release, the ribosomal content of Pseudomonas sp. strain B13(FR1) corresponded to a generation time of 2 h. The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells. Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state. The release of Pseudomonas sp. strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently. Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate.     

Factors affecting the lytic susceptibility of some marine and terrestrial bacteria

Eighteen gram-negative marine bacteria and two terrestrial species, Escherichia coli and Pseudomonas aeruginosa, were examined for their sensitivity to lysis in distilled water after exposure to a salt solution containing a sea water concentration of Mg2+ (0.05 M) or to 0.5 M NaCl. A spectrum of lytic susceptibility was observed among the marine bacteria ranging from those organisms which lysed in distilled water after exposure to the Mg2+-containing solution, through organisms which could be sensitized to lysis by washing with the NaCl solution, to organisms which failed to lyse in distilled water even after having been washed with a solution of 0.5 M NaCl. Pseudomonas aeruginosa and E. coli fell within this spectrum, the former being capable of being induced to lyse in distilled water by washing with 0.5 M NaCl, while the latter failed to lyse in distilled water after this treatment. It was thus concluded that no overall distinction could be made between marine and terrestrial bacteria on the basis of the sensitivity of the two groups of organisms to lysis in freshwater. Quite large decreases in optical density and increases in the release of ultraviolet-absorbing material took place when cells preexposed to the Mg2+-containing solution or to 0.5 M NaCl were subsequently suspended in distilled water even though in some cases no loss of cell numbers could be detected. In most cases two to three times as much K+ as Na+ and 1/10 to 1/100 as much Mg2+ was required to prevent these changes. For three of the marine bacteria and P. aeruginosa grown in a terrestrial type medium little difference in the requirements for Na+ and K+ to prevent the optical density changes was noted. For P. aeruginosa grown in a marine type medium, cells required more K+ than Na+ to prevent these changes.     

Localization of alkaline phosphatase in three gram-negative rumen bacteria

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.     

21-kDa polypeptide,a low-molecular-weight cyclophilin,is released from pollen of higher plants into the extracellular medium in vitro

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca²⁺. This phenomenon was also dependent on pH and on the concentrations of MgCl₂ in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl₂ concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis-trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.Abbreviations CaM Calmodulin - CBB Coomassie-brilliant-blue - CsA Cyclosporin A - CyP Cyclophilin     

Detection and quantification of superoxide formed within the periplasm of Escherichia coli

Many gram-negative bacteria harbor a copper/zinc-containing superoxide dismutase (CuZnSOD) in their periplasms. In pathogenic bacteria, one role of this enzyme may be to protect periplasmic biomolecules from superoxide that is released by host phagocytic cells. However, the enzyme is also present in many nonpathogens and/or free-living bacteria, including Escherichia coli. In this study we were able to detect superoxide being released into the medium from growing cultures of E. coli. Exponential-phase cells do not normally synthesize CuZnSOD, which is specifically induced in stationary phase. However, the engineered expression of CuZnSOD in growing cells eliminated superoxide release, confirming that this superoxide was formed within the periplasm. The rate of periplasmic superoxide production was surprisingly high and approximated the estimated rate of cytoplasmic superoxide formation when both were normalized to the volume of the compartment. The rate increased in proportion to oxygen concentration, suggesting that the superoxide is generated by the adventitious oxidation of an electron carrier. Mutations that eliminated menaquinone synthesis eradicated the superoxide formation, while mutations in genes encoding respiratory complexes affected it only insofar as they are likely to affect the redox state of menaquinone. We infer that the adventitious autoxidation of dihydromenaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm of E. coli. This endogenous superoxide may create oxidative stress in that compartment and be a primary substrate of CuZnSOD.     

Ferrous-iron induces lipid peroxidation with little damage to energy transduction in mitochondria

Addition of ferrous sulfate, but not ferric chloride, in micromolar concentrations to rat liver mitochondria induced high rates of consumption of oxygen. The oxygen consumed was several times in excess of the reducing capacity of ferrous-iron (O: Fe ratios 5–8). This occurred in the absence of NADPH or any exogenous oxidizable substrate. The reaction terminated on oxidation of ferrous ions. Malondialdehyde (MDA), measured as thiobarbituric acid-reacting material, was produced indicating peroxidation of lipids. The ratio of O₂: MDA was about 4: 1. Pretreatment of mitochondria with ferrous sulfate decreased the rate of oxidation (state 3) with glutamate (+malate) as the substrate by about 40% but caused little damage to energy tranduction process as represented by ratios of ADP: O and respiratory control, as well as calcium-stimulated oxygen uptake and energy-dependent uptake of [⁴⁵Ca]-calcium. Addition of succinate or ubiquinone decreased ferrous iron-induced lipid peroxidation in intact mitochondria. In frozen-thawed mitochondria, addition of succinate enhanced lipid peroxidation whereas ubiquinone had little effect. These results suggest that ferrous-iron can cause peroxidation of mitochondrial lipids without affecting the energy transduction systems, and that succinate and ubiquinone can offer protection from damage due to such ferrous-iron released from the stores within the cells.