Inhibition of yeast phosphatidic-acid synthesis by free fatty acids

Particulate preparations obtained from cells of yeast Saccharomyces sake have been shown to possess glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase activities. Glycerolphosphate acyltransferase exhibits a high specificity for saturated and monoenoic fatty acyl-CoA thioesters. When palmitoyl-CoA is employed as sole acyl group donor, the major lipid product is lysophosphatidic acid. 1-Acylglycerolphosphate acyltransferase of this yeast species has a rather strict specificity for monoenoic fatty acyl-CoA thioesters as acyl donor. These two acyltransferases are strongly inhibited in vitro by low concentrations of free fatty acids. 1-Acylglycerolphosphate acyltransferase is much more susceptible to fatty acid inhibition than glycerolphosphate acyltransferase. The inhibition is dependent not only on the concentration of fatty acid, but also on the length of exposure to fatty acid. Both saturated and unsaturated fatty acids inhibit the acyltransferase activities. The inhibitory effects of fatty acids cannot be ascribed to a nonspecific surfactant action of fatty acids. The present results support the view that free fatty acid serves as a regulator of glycerolipid synthesis.     

Inhibition of the glycerol 3-phosphate oxidation by free fatty acids

Oleate inhibits oxidation of glycerol 3-phosphate, but has no effect on glycerol 3-phosphate dehydrogenase. The inhibitory effect may be completely reversed by bovine serum albumin or menadione. Lysophosphatidylcholine has a quite similar inhibitory effect. In this case, however, the inhibitory effect is reversed rather by menadione only than by serum albumin. The results presented indicate that free fatty acids reversibly block transport of hydrogen between glycerol 3-phosphate dehydrogenase and CoQ and may be considered as physiological regulators of the glycerolphosphate cycle.     

Inhibition of topoisomerases by fatty acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure activity relationships and mechanism of action were studied. Saturated fatty acids (C6:0 to C22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C16:1 to C22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Inhibition of free fatty acid mobilization by colchicine

Segments of epididymal adipose tissue from normal male rats were incubated with micromolar concentrations of colchicine for different periods of time up to 4 hr, and the mobilization of free fatty acids (FFA) was measured during a subsequent reincubation. Although pretreatment with colchicine did not alter basal unstimulated FFA release, mobilization of FFA in the presence of epinephrine or theophylline was reduced. However, neither lipolysis, as judged by glycerol production, nor cyclic AMP accumulation was impaired under the same conditions. To assess the possibility that colchicine might limit production of fatty acids by accelerating the entry and metabolism of glucose into adipocytes, the metabolism of glucose by adipose tissue was studied. Pretreatment with colchicine did not affect uptake of glucose nor its oxidation to CO(2), although colchicine-treated tissues did have slightly more [(14)C]glucose incorporated into the glyceride moiety of triglyceride. When adipose tissues pretreated with colchicine were incubated in an albumin-free medium, no reduction in FFA production by colchicine was observed. Because no FFA release occurs in albumin-free media, this experiment suggests that colchicine-induced inhibition of FFA mobilization results from impaired extrusion of FFA from adipose cells.     

Inhibition of hyaluronidases and chondroitinases by fatty acids

The inhibitory effects of various fatty acids on three hyaluronidases (h-ST, h-SH and h-SD) and four chondroitinases (c-ABC, c-B, c-ACI and c-ACII) were examined, and their structure-activity relationships and mechanism of action were studied. The fatty acids used in this experiment showed various inhibitory activities against the enzymes. None of the fatty acids did not inhibit h-ST and h-SH. The saturated fatty acids (C10:0 to C22:0) showed very weak or no inhibition against h-SD, c-ABC, c-B, c-ACI and c-ACII but the unsaturated fatty acids (C14:1 to C24:1) with one double bond strongly inhibited the enzymes, and the inhibitory potency increased with increase in carbon chain length of the fatty acids. In contrast, the increase in number of double bonds caused a decrease in inhibitory potency against the enzymes. The position of the double bond and the stereochemistry of the cis-trans form of oleic acid (C18:1) did not influence the inhibitory potency against the enzymes. Carboxyl and hydroxyl groups in the fatty acid molecule were concerned in the inhibition of c-ACI. Among the fatty acids, eicosatrienoic acid (C20:3) generally inhibited h-SD, c-ABC, c-B and c-ACI, and nervonic acid (C24:1) was a potent inhibitor of c-ACII, and the fatty acids inhibited the enzymes in a noncompetitive manner.     

Inhibition of key glycolytic enzymes from rabbit kidney medulla by free fatty acids in vitro.

In vitro incubation of key glycolytic enzymes in supernatant fluids from rabbit kidney medulla with increasing concentrations of sodium laurate resulted in progressive inhibition of hexokinase, phosphofructokinase and pyruvate kinase. A corresponding reduction in the production of lactate from glucose was also observed. The possible effects of these enzyme inhibitions on the naturesis observed during fasting are discussed.     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Inhibition of phospholipase A2 by cis-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme.

Calcium-dependent phospholipases A2 are markedly inhibited in vitro by cis-unsaturated fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids. Thus, CUFAs may function as endogenous suppressors of lipolysis. To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca(2+)-dependent phospholipase A2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [1-14C]oleate-labelled, autoclaved Escherichia coli and [1-14C]linoleate-labelled phosphatidylethanolamine in an apparent competitive manner. When subjected to gel permeation chromatography, [3H]arachidonate, but not [3H]palmitate, comigrated with the enzyme. Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 microM = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50 microM = 122% of control). Concentrations of arachidonate that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A2 to the E. coli substrate. Collectively, these data demonstrate that, while CUFAs selectively bind to the enzyme, they do not influence phospholipase A2-substrate interaction. Inhibition of in vitro phospholipase A2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-substrate-inhibitor complex.     

Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential

Amongst the diverse and potent biological activities of free fatty acids (FFAs) is the ability to kill or inhibit the growth of bacteria. The antibacterial properties of FFAs are used by many organisms to defend against parasitic or pathogenic bacteria. Whilst their antibacterial mode of action is still poorly understood, the prime target of FFA action is the cell membrane, where FFAs disrupt the electron transport chain and oxidative phosphorylation. Besides interfering with cellular energy production, FFA action may also result from the inhibition of enzyme activity, impairment of nutrient uptake, generation of peroxidation and auto-oxidation degradation products or direct lysis of bacterial cells. Their broad spectrum of activity, non-specific mode of action and safety makes them attractive as antibacterial agents for various applications in medicine, agriculture and food preservation, especially where the use of conventional antibiotics is undesirable or prohibited. Moreover, the evolution of inducible FFA-resistant phenotypes is less problematic than with conventional antibiotics. The potential for commercial or biomedical exploitation of antibacterial FFAs, especially for those from natural sources, is discussed.     

Effects of unsaturated fatty acids on the peroxisomal enzyme activities of Tetrahymena pyriformis

The effects of unsaturated fatty acids on the activities of peroxisomal enzymes of Tetrahymena pyriformis were investigated. When saturated fatty acids and the corresponding unsaturated fatty acids (C18) were added to the culture medium at 0.05%, the activities of peroxisomal enzymes [fatty acyl-CoA oxidase (FAO), carnitine acetyltransferase (CAT), isocitrate lyase (ICL), and malate synthase (MS)] were significantly increased. The order of effectiveness was linoleic acid greater than oleic acid greater than stearic acid. However, alpha-linolenic acid and gamma-linolenic acid at the same concentration were lethal to the cells. The inhibitory effect on growth disappeared upon addition of an antioxidant, alpha-tocopherol. Lipid peroxides derived from unsaturated fatty acids induced marked cell lysis. In the presence of a low concentration (0.005%) of linolenic acid the production of lipid peroxide was lower and no inhibitory effect on the growth was observed, while the activities of peroxisomal enzymes participating in lipid metabolism and that of catalase were significantly increased. These results indicate that the peroxisomal enzyme systems related to the beta-oxidations of fatty acids and the glyoxylate cycle are regulated by unsaturated long-chain fatty acids, including linolenic acid, at low concentrations, as well as by saturated fatty acid in the medium.     

Inhibition by fatty acids of the biodegradation of petroleum

The extent of biodegradation of petroleum by two marine bacterial isolates was found to increase when the organisms were grown in dialysis culture. This suggests that inhibitory products are formed during growth on petroleum. Fatty acids were produced by both organisms and were present in the dialyzate (dialyzable material). Fatty acids and crude oil were found to have a synergistic toxic effect. Short-chain acids were more toxic than longer-chain ones.     

Inhibition of chymase activity by long chain fatty acids

The activity of chymase was markedly inhibited by fatty acids with carbon chain lengths of 14-22 at doses greater than 0.02 microM, irrespective of the number of double bonds. Cis acids with a carbon chain length of 18, such as stearic acid, oleic acid, linoleic acid, and linolenic acid were potent inhibitors, whereas the trans isomer of oleic acid, elaidic acid, showed less inhibitory activity. The extent of inhibition by oleyl alcohol was almost the same as that by oleic acid, suggesting that the acid moiety itself was not necessary for the inhibition; but a fatty acid with a terminal functional amide, oleamide, showed little inhibitory activity. The inhibition was noncompetitive and was reversible, and the Ki value of oleic acid was 2.7 microM. Stearic acid and oleic acid inhibited all chymotrypsin-type serine endopeptidases tested. The ID50 values of these fatty acids for atypical mast cell protease were higher than those for the other chymotrypsin-type serine endopeptidases tested. Other proteases, such as papain, trypsin, collagenase, and carboxypeptidase A, except cathespin D, were not affected by stearic or oleic acid.     

Inhibition of methanogens by n- and iso-volatile fatty acids

n-Butyrate, n-valerate and n-caproate were more inhibitory towards Methanobacterium byrantii, Methanobacterium formicicum and Methanosarcina barkeri than the corresponding iso-acids. Butyrate caused maximum inhibition irrespective of isomer. Methanobacterium bryantii was more sensitive to inhibition than Methanobacterium formicicum.The authors are with the Division of Microbial Sciences, Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, India.