Toxic effects of fatty acids on yeast cells: dependence of inhibitory effects on fatty acid concentration.

The yeasts Saccharomyces cerevisiae, Candida utilis, and Candida lipolytica were used to investigate the action of different concentrations of fatty acids (from acetic to myristic acid) on cell growth, division, uptake of inorganic phosphate, and substrate oxidation. The former two yeasts were found to undergo an inhibition of growth, cell division, and phosphate uptake at lower acid concentrations and to experience the inhibition of substrate oxidation at higher acid concentrations. The concentration dependence of the action of fatty acids can be classified into four categories: 1) subthreshold concentrations which do not inhibit growth and have either no effect on, or stimulate, oxygen consumption; 2) threshold concentrations which lower the rate of growth, cell division, and phosphate uptake but do not inhibit the oxidation of carbon substrate; 3) above-threshold concentrations which inhibit partially even substrate oxidation, and 4) microbicide concentrations. Candida lipolytica displays the same sensitivity toward the action of fatty acids as the above yeast species; however, the threshold concentrations are higher and can be quickly lowered owing to oxidation by the yeast. The concentrations of fatty acids found in the medium after cultivations of yeast with n-alkanes are of the same order as limiting concentrations; the formation of acids with twelve and less carbons in the molecule can thus be assumed to be one of the basic reasons for lowering of biomass yields during cultivations on these hydrocarbons.     

The effect of SH group inhibitors on phosphate transport in Chlorella pyrenoidosa]

The effect of Sulphydryl reagents have been investigated. pCMB inhibits the transport of phosphate in Chlorella pyrenoidosa. This inhibition is immediate and does not change as a function of time of incubation. This inhibition affects non starved and starved cells (phosphate omitted). pCMPS and Mersalyl act in the same manner as pCMB. When these compounds are used at low concentrations, inhibition of phosphate uptake is observed only in starved cells. The substrate (phosphate) cannot provide protection against this inhibition. NEM inhibits phosphate uptake and this inhibition increases as a function of time of incubation. When the time of incubation is very short (about 15 seconds) the effects seems to be superficial and NEM reacts with SH groups involved in the transport system. When phosphate is present (for 15 seconds of incubation with NEM) the inhibition is less important than when phosphate is omitted. The substrate protects against NEM, but this protection disappears as the incubation with NEM is prolonged. NEM inhibits phosphate uptake in non starved and starved cells, however, it is observed that the inhibition is less important in starved cells than in non starved cells. The authors conclude that two kinds of SH groups might exist in the phosphate transport system, one reacting with pCMB and the other with NEM.     

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

Removal by bovine serum albumin of fatty acids from membrane vesicles and its effect on proline transport activity in Escherichia coli.

Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.     

Significance of non-esterified fatty acids in iron uptake by intestinal brush-border membrane vesicles

Iron uptake from Fe/ascorbate by mouse brush-border membrane vesicles is not greatly inhibited by prior treatment with a variety of protein-modification reagents or heat. Non-esterified fatty acid levels in mouse proximal small intestine brush-border membrane vesicles show a close positive correlation with initial Fe uptake rates. Loading of rabbit duodenal brush-border membrane vesicles with oleic acid increases Fe uptake. Depletion of mouse brush-border membrane vesicle fatty acids by incubation with bovine serum albumin reduces Fe uptake. Iron uptake by vesicles from Fe/ascorbate is enhanced in an O2-free atmosphere. Iron uptake from Fe/ascorbate and Fe3+-nitrilotriacetate (Fe3+-NTA) were closely correlated. Incorporation of oleic acid into phosphatidylcholine/cholesterol (4:1) liposomes leads to greatly increased permeability to Yb3+, Tb3+, Fe2+/Fe3+ and Co2+. Ca2+ and Mg2+ are also transported by oleic acid-containing liposomes, but at much lower rates than transition and lanthanide metal ions. Fe3+ transport by various non-esterified fatty acids was highest with unsaturated acids. The maximal transport rate by saturated fatty acids was noted with chain length C14-16. It is suggested that Fe transport can be mediated by formation of Fe3+ (fatty acid)3 complexes.     

The uptake of inorganic sulphate by a brewer’s yeast

The uptake of³⁵S-labelled inorganic sulphate by a brewer’s yeast has been examined. Optimum uptake by cell suspensions required the presence in the medium of glucose, ammonium ions and citrate. The omission of phosphate produced little or no effect. Ammonium ions could be replaced almost completely byL-glutamine but not by a number of amino acids. After one hour approximately 60% of the sulphate-sulphur accumulated appeared in protein. This was comparable to the rate of entry of methionine-sulphur into yeast protein. Sulphate uptake was inhibited by azide, 2,4-dinitrophenol, iodoacetate and mercuric ions. Arsenate was inhibitory at high concentrations but stimulated uptake at low concentrations. Selenate inhibited uptake competitively and appeared to have an affinity for the sites of uptake comparable with that of sulphate. Uptake was also partly suppressed byL-methionine,L-ethionine,L-cysteine andDL-homocysteine.     

Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine.

1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.     

Interaction of 2-deoxy-d-glucose and adenine with phosphate anion uptake in yeast

The transport of inorganic phosphate anions into yeast cells (after preincubation with glucose; fructose or another metabolizable sugar, and in the presence of glucose) shows two kinetic components with half-saturation constants of 40 μmol/L and 2.4 mmol/L. The uptake was strikingly stimulated by 2-deoxy-d-glucose (2-dGle) at lower concentrations but inhibited above, 100 mmol/L. A similar stimulation was caused by adenine (0.01–1 mmol/L) and a very small one by uracil and inorganic sulfate. It is suggested that either a phosphorylation reaction accompanies the transport (2-dGlc) or that some compounds stimulate the H⁺-ATPase more than inorganic phosphate itself and thus increase its rate of transport.     

Effects of physiological manipulation on the kinetics of mitochondrial phosphate transport in Saccharomyces cerevisiae

The kinetics of [³²P]phosphate uptake has been studied in different types of Saccharomyces cerevisiae mitochondria. Mitochondria were isolated from yeast grown aerobically on 2% lactate (Lac-mitochondria), 2% galactose (Gal-mitochondria), 5.4% glucose (Glu-mitochondria) or from yeast grown anaerobically on 2% galactose (Promitochondria). The effect of chloramphenicol was also studied by adding it to the growth medium of yeast grown aerobically on 2% galactose (chloramphenicol-mitochondria).[³²P]Phosphate uptake followed an oscillatory pattern in Lac, Gal-mitochondria and Promitochondria.Saturation kinetics were detected in fully differenciated mitochondria and in Promitochondria, but not in chloramphenicol-mitochondria.Glu-mitochondria did not translocate phosphate as shown both by lack of [³²P]phosphate uptake and lack of swelling in isoosmotic potassium solution.Repressed yeast cells were incubated in a resting cell medium and mitochondria were isolated at different times of incubation. The rate of respiration and the oligomycin-sensitive ATPase increased during the course of the incubation. After 2h, a mitochondrial mersalyl-sensitive swelling in an isoosmotic potassium phosphate solution was detected.As expected, no increase of the rate of respiration was observed when chloramphenicol was added in the derepression medium. But the oligomycin-sensitive ATPase decreased. Chloramphenicol did not affect the phosphate transport activity as measured by the swelling of mitochondria, but the [³²P]phosphate uptake did not follow saturation kinetics. A complete derepression of the inorganic phosphate-carrier activity was achieved by a 4 h incubation of the repressed cells in the presence of chloramphenicol, followed by a 6 h incubation in presence of cycloheximide.These data strongly suggest that the mitochondrial protein-synthesis system is required for the normal function of the inorganic phosphate-carrier.     

Modification of cell membrane lipids inMicrococcus lysodeikticus induced by pantoyl lactone

Summary Growth ofMicroccoccus lysodeikticus in the presence of pantoyl lactone brings about both qualitative and quantitative changes in cell membrane lipids. Significant amounts of the two major phospholipids (phosphatidylglycerol and diphosphatidylglycerol) are converted to lyso forms; the largest conversion occurs in the phosphatidylglycerol. In addition, amounts of several phospholipid fatty acids are changed. Physical alteration of the cell membrane can be demonstrated using differential scanning calorimetry. Although growth and transport are significantly inhibited when pantoyl lactone is present, cells possessing altered cell membrane phospholipids and phospholipid fatty acids, brought about by growth in the presence of pantoyl lactone, transportd-alanine,l-glutamic andl-aspartic acid normally when washed free of the pantoyl lactone.     

The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size.     

Molecular cloning and functional analysis of a H+-dependent phosphate transporter gene from the ectomycorrhizal fungus Boletus edulis in southwest China

Phosphate transporters (PTs), as entry points for phosphorus (P) in organisms, are involved in a number of P nutrition processes such as phosphate uptake, transport, and transfer. In the study, a PT gene 1632 bp long (named BePT) was cloned, identified, and functionally characterized from Boletus edulis. BePT was expected to encode a polypeptide with 543 amino acid residues. The BePT polypeptide belonged to the major facilitator superfamily and showed a high degree of sequence identity to the Pht1 family. A topology model revealed that BePT exhibited 12 transmembrane helices, divided into two halves, and connected by a large hydrophilic loop in the middle. A yeast mutant complementation analysis suggested that BePT was a functional PT which mediated orthophosphate uptake of yeast at micromolar concentrations. Green fluorescent protein–BePT fusion proteins expressed were extensively restricted to the plasma membrane in BePT transformed yeast, and its activity was dependent on electrochemical membrane potential. In vitro, quantitative PCR confirmed that the expression of BePT was significantly upregulated at lower phosphorus availability, which may enhance phosphate uptake and transport under phosphate starvation. Our results suggest that BePT plays a key role in phosphate acquisition in the ectomycorrhizal fungus B. edulis.     

Relation between Ca2+ uptake and fluidity of brush-border membranes isolated from rabbit small intestine and incubated with fatty acids and methyl oleate

The rate of incorporation of oleic acid into isolated brush-border membranes was found to be considerably faster than methyl oleate incorporation under similar experimental conditions. The effects of fatty acids and methyl oleate incorporation on Ca2+ uptake and fluidity were monitored. Whereas treatment with 0.01-0.05 mM oleic acid corresponding to incorporations smaller than 90 nmol/mg protein enhanced Ca2+ transport, exposures to higher concentrations of this fatty acid corresponding to incorporations larger than 150 nmol/mg protein, decreased uptake of this cation. On the other hand, treatment with 0.01-0.2 mM methyl oleate corresponding to incorporations of up to 220 nmol/mg protein had only a stimulatory effect on the Ca2+ uptake. Oleic acid, linoleic acid and methyl oleate decreased the fluorescence anisotropy of membranes labelled with diphenylhexatriene in a dose-dependent manner. In contrast, palmitic acid had little or no effect on the diphenylhexatriene-reportable order of the membrane within the range of concentrations used. Monitored as a function of temperature, the anisotropy values showed a gradual melting for both the control and lipid-treated membranes. The results support the concept that saturated and cis-unsaturated fatty acids dissolve in different lipid domains and this in itself appears to be an important factor defining whether the biological function of the membrane is affected by the uptake. Incorporation of cis-unsaturated fatty acids in domains harboring the Ca2+ uptake process increases Ca2+ uptake in concert with increased diphenylhexatriene-monitored fluidity. However, when concentrations of such fatty acids in these domains become sufficiently great, the presence of a largely increased number of free carboxyl groups at the membrane surface causes inhibition of Ca2+ uptake.     

Inhibition of phosphate uptake in barley roots by hydroxy-benzoic acids

The influence of 15 hydroxy-benzoic acids upon active inorganic phosphate absorption by barley roots was examined. For each compound an inhibition constant (k_i) was determined, i.e. the concentration of compound required to bring about a 50% inhibition of absorption. The k_i values of the benzoic acids were strongly correlated with their octanol—water partition coefficients and their pK_a values. This suggests that the inhibition of normal membrane functions, brought about by benzoic acids, results from a generalized increase in cell membrane permeability. Salicylate derivatives were generally more inhibitory than would be predicted from their partition coefficients; their pronounced toxicity probably arises from structural impediments to their detoxication.     

Modification of GABA and calcium uptake by lipids in synaptosomes from normoxic and ischemic brain

The effects of membrane lipid disturbances induced by ischemia and exogenously added lipids on the uptake of GABA and Ca²⁺ were investigated in gerbil brain synaptosomes. Ischemia was produced by bilateral ligation of common carotid arteries in Mongolian gerbil for 10 min. The level of the free fatty acids (FFA) increased significantly in ischemic synaptosomes. Incorporation of [1-¹⁴C]arachidonate into membrane phosphatidylinositol and phosphatidylcholine was decreased by about 20–35%. Furthermore ischemia exerted an inhibitory effect on GABA uptake but remained without effect on calcium accumulation. Thiopental application in dose of 100 mg per kg body weight 30 min before ischemia caused a protective effect on membrane lipid disturbances induced by ischemia and enhanced GABA uptake. Unsaturated fatty acids (arachidonate and docosahexanoate) in concentration of 10⁻⁵−10⁻⁴ mol/l and lysocompounds (lysophosphatidylcholine and lysophosphatidylethanolamine) in concentrations higher than 10⁻⁴ mol/l decreased GABA and Ca²⁺ uptake in synaptosomes from normoxic brains. No effect was seen with saturated stearic acid. These results suggest that the inhibition of GABA uptake into ischemic synaptosomes resulted from an action of unsaturated fatty acids, arachidonic and docosahexanoic acids which were liberated during ischemia. Moreover the transient higher local concentration of lysophospholipids close to GABA carrier system may also have contributed to the inhibition observed during ischemia.     

Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin D

Palmitic acid and gramicidin D at low concentrations uncouple photophosphorylation in a mechanism that is inconsistent with classical uncoupling in the following properties: (1) delta pH, H+ uptake, or the transmembrane electric potential is not inhibited. (2) O2 evolution is stimulated under nonphosphorylating conditions but slightly inhibited in the presence of adenosine 5'-diphosphate + inorganic phosphate (Pi). (3) Light-triggered adenosine 5'-triphosphate (ATP)-Pi exchange is hardly affected, and ATPase activity is only slightly stimulated. (4) ATP-induced delta pH formation is selectively inhibited. This characteristic uncoupling is observed only when the native coupling sites of the electron transport system are used for energization such as for methylviologen-coupled phosphorylation. With pyocyanine, which creates an artificial coupling site, 1000-fold higher gramicidin D and higher palmitic acid concentrations are required for inhibition, and the inhibition is accompanied by a decrease in delta pH. Moreover, comparison between photosystem 1 and photosystem 2 electron transport and the effects of membrane unstacking suggest that low gramicidin D preferentially inhibits photosystem 2, while palmitic acid inhibits more effectively photosystem 1 coupling sites. The inhibitory capacity of fatty acids significantly drops when the chain length is reduced below 16 hydrocarbons or upon introduction of a single double bond in the hydrocarbon chain. It is suggested that palmitic acid and gramicidin D interfere with a direct H+ transfer between specific electron transport and the ATP synthase complexes, which provides an alternative coupling mechanism in parallel with bulk to bulk delta microH+. The sites of inhibition seem to be located in chloroplast ATP synthase, photosystem 2, and the cytochrome b6f complexes.