A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species

Carbon monoxide (CO), produced during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator in mammalian cells. Here we show that precise delivery of CO to isolated heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) uncouples respiration. Addition of low-micromolar concentrations of CORM-3 (1–20 μM), but not an inactive compound that does not release CO, significantly increased mitochondrial oxygen consumption rate (State 2 respiration) in a concentration-dependent manner. In contrast, higher concentrations of CORM-3 (100 μM) suppressed ADP-dependent respiration through inhibition of cytochrome c oxidase. The uncoupling effect mediated by CORM-3 was inhibited in the presence of the CO scavenger myoglobin. Moreover, this effect was associated with a gradual decrease in membrane potential (ψ) over time and was partially reversed by malonate, an inhibitor of complex II activity. Similarly, inhibition of uncoupling proteins or blockade of adenine nucleotide transporter attenuated the effect of CORM-3 on both State 2 respiration and Δψ. Hydrogen peroxide (H₂O₂) produced by mitochondria respiring from complex I-linked substrates (pyruvate/malate) was increased by CORM-3. However, respiration initiated via complex II using succinate resulted in a fivefold increase in H₂O₂ production and this effect was significantly inhibited by CORM-3. These findings disclose a counterintuitive action of CORM-3 suggesting that CO at low levels acts as an important regulator of mitochondrial respiration.     

GRAFT INCOMPATIBILITY BETWEEN PEAR AND QUINCE: THE INFLUENCE OF METABOLITES OF CYDONIA OBLONGA ON SUSPENSION CULTURES OF PYRUS COMMUNIS

The fresh weights of suspension cultures of pear (Pyrus communis) and quince (Cydonia oblonga) increased exponentially for 30 to 40 days after subculturing. Transferring pear cultures to media in which quince cultures had grown for 10 days resulted in a 70% inhibition of callus growth. Transferring quince cultures to media in which pear cultures had grown for 10 days resulted in less than a 20% inhibition of growth. Addition of the cyanogenic glycosides amygdalin and prunasin (as 50 ppm CN ^_) killed pear cultures, while growth of quince cultures was inhibited by only approximately 50%. Addition of 50 ppm CN- severely inhibited growth of both cultures. These results indicate that 1) suspension cultures of quince release factor(s) that significantly inhibit growth of pear cultures, 2) quince cultures are relatively unaffected by metabolites released by pear cultures, 3) the severe inhibition of pear growth by quince metabolites is mimicked by the addition of cyanogenic glycosides ubiquitous to vegetative portions of quince, 4) direct cellular contact is not necessary to elicit incompatibility between pear and quince, and 5) incompatibility between pear and quince need not be associated with any particular stage of graft development.     

Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii

1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (K(m)[unk]15mum), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.     

Differential Inhibition by Allylsulfide of Nitrification and Methane Oxidation in Freshwater Sediment

Addition of nitrapyrin, allylthiourea, C(inf2)H(inf2), and CH(inf3)F to freshwater sediment slurries inhibited CH(inf4) oxidation and nitrification to similar extents. Dicyandiamide and allylsulfide were less inhibitory for CH(inf4) oxidation than for nitrification. Allylsulfide was the most potent inhibitor of nitrification, and the estimated 50% inhibitory concentrations for this process and CH(inf4) oxidation were 0.2 and 121 (mu)M, respectively. At a concentration of 2 (mu)M allylsulfide, growth and CH(inf4) oxidation activity of Methylosinus trichosporium OB3b were not inhibited. Allylsulfide at 200 (mu)M inhibited the growth of M. trichosporium by approximately 50% but did not inhibit CH(inf4) oxidation activity. Nitrite production by cells of M. trichosporium was not significantly affected by allylsulfide, except at a concentration of 2 mM, when growth and CH(inf4) oxidation were also inhibited by about 50%. Methane monooxygenase activity present in soluble fractions of M. trichosporium was not inhibited significantly by allylsulfide at either 200 (mu)M or 2 mM. These results suggest that the partial inhibition of CH(inf4) oxidation in sediment slurries by high allylsulfide concentrations may be caused by an inhibition of the growth of methanotrophs rather than an inhibition of methane monooxygenase activity specifically. We conclude that allylsulfide is a promising tool for the study of interactions of methanotrophs and nitrifiers in N cycling and CH(inf4) turnover in natural systems.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Respiratory oscillations and heat evolution in synchronous cultures of Candida utilis

Synchronous cultures of the budding yeast Candida utilis prepared by continuous-flow size selection showed respiratory oscillations when the energy source was either glucose, acetate or glycerol. The period of the oscillations was about one-third of the cell cycle time (i.e. about 0.5 h). No fluctuations in heat evolution could be detected. In organisms growing with acetate or glycerol, the effects of cyanide, N,N'-dicyclohexylcarbodi-imide and carbonyl cyanide m-chlorophenylhydrazone (maximum inhibition of respiration at respiratory maxima, maximum uncoupling of energy conservation at respiratory minima) suggest that the control mechanism responsible for the oscillations is mitochondrial respiratory control in vivo. The effects of cyanide and N,N'-dicyclohexylcarbodi-imide on the respiration of cultures growing synchronously with glucose were different from those for cultures growing with the non-fermentable substrates; this suggests that the mitochondrial respiratory system interacts with the early reactions of glucose utilization.     

Methylmercury Oxidative Degradation Potentials in Contaminated and Pristine Sediments of the Carson River, Nevada

Sediments from mercury-contaminated and uncontaminated reaches of the Carson River, Nevada, were assayed for sulfate reduction, methanogenesis, denitrification, and monomethylmercury (MeHg) degradation. Demethylation of [(sup14)C]MeHg was detected at all sites as indicated by the formation of (sup14)CO(inf2) and (sup14)CH(inf4). Oxidative demethylation was indicated by the formation of (sup14)CO(inf2) and was present at significant levels in all samples. Oxidized/reduced demethylation product ratios (i.e., (sup14)CO(inf2)/(sup14)CH(inf4) ratios) generally ranged from 4.0 in surface layers to as low as 0.5 at depth. Production of (sup14)CO(inf2) was most pronounced at sediment surfaces which were zones of active denitrification and sulfate reduction but was also significant within zones of methanogenesis. In a core taken from an uncontaminated site having a high proportion of oxidized, coarse-grain sediments, sulfate reduction and methanogenic activity levels were very low and (sup14)CO(inf2) accounted for 98% of the product formed from [(sup14)C]MeHg. There was no apparent relationship between the degree of mercury contamination of the sediments and the occurrence of oxidative demethylation. However, sediments from Fort Churchill, the most contaminated site, were most active in terms of demethylation potentials. Inhibition of sulfate reduction with molybdate resulted in significantly depressed oxidized/reduced demethylation product ratios, but overall demethylation rates of inhibited and uninhibited samples were comparable. Addition of sulfate to sediment slurries stimulated production of (sup14)CO(inf2) from [(sup14)C]MeHg, while 2-bromoethanesulfonic acid blocked production of (sup14)CH(inf4). These results reveal the importance of sulfate-reducing and methanogenic bacteria in oxidative demethylation of MeHg in anoxic environments.     

Effect of (+)-pulegone and other oil components of Mentha x Piperita on cucumber respiration

Peppermint (Mentha x piperita L.) essential oil and main components were assessed for their ability to interfere with plant respiratory functions. Tests were conducted on both root segments and mitochondria isolated by etiolated seedlings of cucumber (Cucumis sativus L.). Total essential oil inhibited 50% of root and mitochondrial respiration (IC50) when used at 324 and 593 ppm, respectively. (+)-Pulegone was the most toxic compound, with a 0.08 and 0.12 mM IC50 for root and mitochondrial respiration, respectively. (-)-Menthone. followed (+)-pulegone in its inhibitory action (IC50 values of 1.11 and 2.30 mM for root and mitochondrial respiration respectively), whereas (-)-menthol was the less inhibitory compound (IC50 values of 1.85 and 3.80 mM respectively). A positive correlation was found for (+)-pulegone, (-)-menthone and (-)-menthol between water solubility and respiratory inhibition. The uncoupling agent. carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP), lowered (-)-menthol and (-)menthone inhibition and annulled (+)-pulegone inhibition of mitochondrial respiration, whereas salicyl-hydroxamic acid (SHAM) 2-hydroxybenzohydroxamic acid, the alternative oxidase (AO) inhibitor, increased (-)-menthone inhibition and annulled both (+)-pulegone and (-)-menthol inhibitory activity. The possible interaction of (-)-pulegone and (-)-menthol with AO and the mechanism of action of(+)-pulegone, (-)-menthone and (-)-menthol on mitochondrial respiration are discussed.     

In Phosphorylating <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> Mitochondria the Sensitivity of Uncoupling Protein Activity to GTP Depends on the Redox State of Quinone

In isolated Acanthamoeba castellanii mitochondria respiring in state 3 with external NADH or succinate, the linoleic acid-induced purine nucleotide-sensitive uncoupling protein activity is able to uncouple oxidative phosphorylation. The linoleic acid-induced uncoupling can be inhibited by a purine nucleotide (GTP) when quinone (Q) is sufficiently oxidized, indicating that in A. castellanii mitochondria respiring in state 3, the sensitivity of uncoupling protein activity to GTP depends on the redox state of the membranous Q. Namely, the inhibition of the linoleic acid-induced uncoupling by GTP is not observed in uninhibited state 3 respiration as well as in state 3 respiration progressively inhibited by complex III inhibitors, i.e., when the rate of quinol (QH₂)-oxidizing pathway is decreased. On the contrary, the progressive decrease of state 3 respiration by declining respiratory substrate availability (by succinate uptake limitation or by decreasing external NADH concentration), i.e., when the rate of Q-reducing pathways is decreased, progressively leads to a full inhibitory effect of GTP. Moreover, in A. castellanii mitochondria isolated from cold-treated cells, where a higher uncoupling protein activity is observed, the inhibition of the linoleic acid-induced proton leak by GTP is revealed for the same low values of the Q reduction level.     

Role of the ADP/ATP and aspartate/glutamate antiporters in the uncoupling effect of fatty acids, lauryl sulfate, and 2, 4-dinitrophenol in liver mitochondria.

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 microM tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.     

The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii.

1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture.     

Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV

MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5–10 μM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols.     

Uncoupling effect of lauryl sulfate on mitochondria can be mediated by release of bound endogenous fatty acids

The mechanism of uncoupling by lauryl sulfate (LS) has been studied. The very fact that uncoupling by low concentration of LS (a strong acid) resembles very much that by fatty acids (weak acids) was used as an argument against the fatty acid cycling scheme of uncoupling where protonated fatty acids operate as a protonophore. We have found that rat liver and heart muscle mitochondria can be uncoupled by low (70 microM) LS concentration in a fashion completely arrested by the ATP/ADP antiporter inhibitor carboxyatractylate (CAtr). On the other hand, uncoupling by two-fold higher LS concentration is not sensitive to CAtr. Addition of oleate desensitizes mitochondria to low LS so that addition of bovine serum albumin becomes necessary to recouple mitochondria. The data are accounted for assuming that low LS releases endogenous fatty acids from some mitochondrial depots, and these fatty acids are responsible for uncoupling. As to high LS, it causes a nonspecific (CAtr-insensitive) damage to the mitochondrial membrane.     

Chemiosmotic energy conservation with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by Acetobacterium woodii

Cell suspensions of Acetobacterium woodii prepared from cultures grown on fructose plus caffeate catalyzed caffeate reduction with electrons derived from molecular hydrogen. Hydrogen-dependent caffeate reduction was strictly Na(+) dependent with a K(m) for Na(+) of 0.38 mM; Li(+) could substitute for Na(+). The sodium ionophore ETH2120, but not protonophores, stimulated hydrogen-dependent caffeate reduction by 280%, indicating that caffeate reduction is coupled to the buildup of a membrane potential generated by primary Na(+) extrusion. Caffeate reduction was coupled to the synthesis of ATP, and again, ATP synthesis coupled to hydrogen-dependent caffeate reduction was strictly Na(+) dependent and abolished by ETH2120, but not by protonophores, indicating the involvement of a transmembrane Na(+) gradient in ATP synthesis. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) abolished ATP synthesis, and at the same time, hydrogen-dependent caffeate reduction was inhibited. This inhibition could be relieved by ETH2120. These experiments are fully compatible with a chemiosmotic mechanism of ATP synthesis with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by A. woodii.     

Cyanide-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration during postharvest ripening of tomato fruit

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.     

Kinetic Analysis of Competition Between Sulfate Reducers and Methanogens for Hydrogen in Sediments

The competition between sulfate-reducing and methanogenic bacteria for hydrogen was investigated in eutrophic lake sediments that contained low in situ sulfate concentrations and in sulfate-amended sediments. Sulfate reduction and methane production coexisted in situ in lake surface sediments (0 to 2 cm), but methane production was the dominant terminal process. Addition of 10 to 20 mM sulfate to sediments resulted in a decrease in the hydrogen partial pressure and a concomitant inhibition of methane production over time. Molybdate inhibition of sulfate reduction in sulfate-amended sediments was followed by an increase in the hydrogen partial pressure and the methane production rate to values comparable to those in sediments not amended with sulfate. The sulfate reducer population had a half-saturation constant for hydrogen uptake of 141 pascals versus 597 pascals for the methanogen population. Thus, when sulfate was not limiting, the lower half-saturation constant of sulfate reducers enabled them to inhibit methane production by lowering the hydrogen partial pressure below levels that methanogens could effectively utilize. However, methanogens coexisted with sulfate reducers in the presence of sulfate, and the outcome of competition at any time was a function of the rate of hydrogen production, the relative population sizes, and sulfate availability.     

Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua

AIMS: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. METHODS AND RESULTS: In laboratory media, MICs of 15, 75 and 35 mmol l(-1) vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10-40 mmol l(-1) vanillin inhibited respiration of E. coli and L. innocua. Addition of 50-70 mmol l(-1) vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l(-1) vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l(-1) vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l(-1) vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. CONCLUSIONS: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.     

Response of atmospheric methane consumption by maine forest soils to exogenous aluminum salts

Atmospheric methane consumption by Maine forest soils was inhibited by additions of environmentally relevant levels of aluminum. Aluminum chloride was more inhibitory than nitrate or sulfate salts, but its effect was comparable to that of a chelated form of aluminum. Inhibition could be explained in part by the lower soil pH values which resulted from aluminum addition. However, significantly greater inhibition by aluminum than by mineral acids at equivalent soil pH values indicated that inhibition also resulted from direct effects of aluminum per se. The extent of inhibition by exogenous aluminum increased with increasing methane concentration for soils incubated in vitro. At methane concentrations of >10 ppm, inhibition could be observed when aluminum chloride was added at concentrations as low as 10 nmol g (fresh weight) of soil(-1). These results suggest that widespread acidification of soils and aluminum mobilization due to acid precipitation may exacerbate inhibition of atmospheric methane consumption due to changes in other parameters and increase the contribution of methane to global warming.     

Inhibition of topoisomerase I by heparin

DNA topoisomerase I isolated from mouse mammary cacinoma cells was shown to be inhibited by heparin, the dose giving 50% inhibition (IC₅₀) being 0.20 μg/ml. Other chemically related acid mucopolysaccharides including heparan sulfate, dermatan sulfate etc. were more than 500 times less active than heparin. When the amount of enzyme was doubled relative to the substrate the inhibition was reversed. Addition of heparin to assay mixtures after the initiation of the reaction immediately inhibited the enzyme reaction.     

Inhibition of palmitoyl CoA deacylase by chlorophenoxyisobutyrate and betabenzalbutyrate

Palmitoyl CoA deacylase activity was measured in preparations of rat liver microsomes. Two hypolipidemic agents, chlorophenoxyisobutyrate and betabenzalbutyrate caused inhibition of palmitoyl CoA deacylase in vitro. The I(50) values for chlorophenoxyisobutyrate and betabenzalbutyrate were 5.0 and 7.5 mm, respectively. The inhibition by both agents was reversible, and both drugs lowered the palmitoyl CoA-binding capacity of microsomes. The deacylase reaction rate was maximum when the binding of palmitoyl CoA to soluble or microsomal protein was low. Addition of albumin to the reaction mixture resulted in greater binding of palmitoyl CoA to protein and a lower reaction rate. Inhibition of the deacylase by chlorophenoxyisobutyrate and betabenzalbutyrate also was greater when palmitoyl CoA was not protein bound. The inhibition of palmitoyl CoA deacylase by these hypolipidemic agents may contribute to their effects on lipid metabolism.