Inhibition of nuclear T3 binding by fatty acids liberated from nuclear membranes via phospholipase C.

1. We have investigated whether phospholipase C (PLC) might act on phospholipids in the nuclear membrane of rat liver, resulting in inhibition of nuclear T3 binding (INB activity). 2. Incubation of intact nuclei with PLC caused a time- and dose-dependent increase of INB activity; this was correlated with a rise of the free fatty acid concentration in the nuclear ether extract. 3. Removal of the nuclear membrane resulted in a loss of INB activity of the nuclei. 4. Other compounds liberated by the action of PLC (such as diacylglycerols, IP3 and phosphoalcohols), had no INB activity. 5. We conclude that PLC can liberate fatty acids from the rat liver-nuclear membrane via further degradation of the direct product diacylglycerol. 6. These fatty acids display inhibition of nuclear T3 binding in vitro.     

Inhibition of nuclear T3 binding by fatty acids: dependence on chain length, unsaturated bonds, cis-trans configuration and esterification

1. Fatty acids have the capacity for inhibition of nuclear T3 binding (INB). The present studies were undertaken to describe the INB-activity of fatty acids as a function of chain length, unsaturated bonds, cis-trans configuration, and esterification. 2. Isolated rat liver nuclei were incubated with [125I]T3 in the absence or presence of fatty acids in concentrations of 0.011, 0.033, 0.1 and 0.3 mM respectively. 3. INB-activity depended on the chain length, being greatest at 14 carbon atoms. 4. INB by unsaturated fatty acids was greater than that of saturated fatty acids, and increased with increasing number of double bonds. 5. Fatty acids in the cis configuration had greater INB-activity than those in trans configuration. 6. Esterification of fatty acids decreased INB-activity: monoglycerides still had some effect, but di- and triglycerides had no effect.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Reversible intramitochondrial release of protein related to unsaturated fatty acids of membranes.

The influence of temperature on intramitochondrial protein and enzyme release was studied in control and “lipid-deficient” rat liver mitochondria and in synaptosomal and “cell body” mitochondria of rat brain. (i) The fatty acid composition of the phospolipid fraction was shown to be different in control and lipid-deficient preparations. (ii) Arrhenius curves for temperature-dependent release of protein showed breaks. (iii) When comparing control to lipid-free rat liver mitochondria and cell body to synaptosomal rat brain mitochondria, shifts in the breaks in the Arrhenius plots were observed for release of aspartate aminotransferase, protein and malate dehydrogenase. (iv) Intramitochondrial temperature-dependent, succinate-induced protein release was also studied in rat liver mitochondria which had previously undergone a succinate-induced release and rebinding cycle. These mitochondria showed a temperature-dependent protein release identical to that of untreated mitochondria.     

Choline acetyltransferase binding to and release from membranes

1. The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium. 2. The distribution of soluble enzyme bound to synaptosome membranes was studied by density-gradient centrifuging. 3. Choline acetyltransferase shows enzyme activity both in the free and in the membrane-bound form. 4. Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme. 5. The release of choline acetyltransferase from membranes by different anions, thiols, adenosine nucleotides and enzyme substrates was studied.     

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.     

Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport

Summary Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.     

Activation of nuclear chromatin and the release from contact-inhibition of 3T3 cells

A rapid cytophotometric method was developed to measure binding of acridine orange (AO) to the nuclear chromatin of mouse fibroblasts. Contact-inhibited 3T3 cells bound 50–70% less dye than did growing cells in G1 phase of the cell cycle. Release from contact inhibition by fresh serum resulted in stimulated binding of AO within 30 min. No such changes were observed in SV40 virus transformed cells. Differences in AO-binding disappeared at high dye concentration, suggesting that activated cells have a greater affinity to the dye rather than an increased number of binding sites.     

Participation of endogenous fatty acids in Ca2+ release activation from mitochondria

A correlation between the rate of H+/Ca2+ exchange and the content of free fatty acids in mitochondria has been found. Fatty acids were isolated from mitochondria with different activities of H+/Ca2+ exchange. It has been shown that these free fatty acids are able to induce Ca2+ release in exchange to protons after being added to freshly isolated mitochondria.     

Inhibition by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell-dependent B cell activation

Anti-CD3 mAb can activate T cells to help in B cell activation as detected by late events, such as maturation of B cells into Ig-secreting cells (IgSC), or by early events, such as B cell surface expression of the activation marker CD23. Two different anti-CD2 mAb each inhibited anti-CD3-induced T cell-dependent B cell activation in a dose-dependent fashion. Neither irradiation of the T cells prior to culture nor depletion of CD8+ cells abrogated the inhibitory effects of anti-CD2 mAb. Despite the ability of these anti-CD2 mAb to inhibit anti-CD3-induced IL2 production, addition of exogenous IL2 to anti-CD2 mAb-containing cultures could not fully reverse the inhibitory effects on IgSC generation. Furthermore, addition of various combinations of IL1, IL2, IL4, and IL6 or crude PBMC or monocyte culture supernatants also could not reverse anti-CD2-driven inhibition. In T cell-depleted cultures, anti-CD2 mAb had no effect on the ability of IL4 to induce B cell CD23 expression, confirming that anti-CD2 mAb had no direct effect on B cells. However, in cultures containing T+ non-T cells, anti-CD2 mAb did partially inhibit IL4-induced B cell CD23 expression. Taken together, these observations demonstrate that certain CD2 ligands can modulate T cell-dependent B cell activation by a mechanism which, at least in part, involves a direct effect by the CD2 ligand on the T cell itself.     

Studies on the mechanism of inhibition of nuclear triiodothyronine binding by fatty acids

Studies were designed to elucidate the mechanism by which unsaturated fatty acids inhibit the binding of triiodothyronine (T3) ro rat liver nuclei. The possibility of a direct interaction between oleic acid and T3 was excluded by dialysis experiments. Oleic acid inhibits nuclear T3 binding in a strictly competitive manner. The Ki value of oleic acid was approx. 10(4) times greater than that of T3. The inhibitory effect of oleic acid could be reversed by bovine serum albumin.     

Effects of fatty acids on activity and calmodulin binding of Ca2+-ATPase of human erythrocyte membranes

Activation and inhibition of Ca2+-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of (Na+ + K+)-ATPase, Mg2+-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.     

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 DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of density-dependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density- dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.     

Insights into binding of fatty acids by fatty acid binding proteins

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.     

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.     

Omega-3 fatty acids in cellular membranes: a unified concept

The Omega-3 fatty acid DHA (docosahexaenoic acid, 22:6) and its sister molecule EPA (eicosapentaenoic acid, 20:5) are highlighted here. These highly unsaturated fatty acids are widespread in nature, especially in the marine environment, and are essential in membranes ranging from deep sea bacteria to human neurons. Studies of DHA/EPA in bacteria have led to a working model on the structural roles of these molecules and are described in this review. The main points are: (a) genomic analysis shows that genes encoding the DHA/EPA pathways are similar, supporting the idea that structural roles in bacteria might be similar, (b) biochemical analysis shows that DHA and EPA are produced in bacteria by a polyketide process distinct from the pathway of plants and animals; this allows DHA and EPA to be produced in anaerobic or oxygen-limited environments, (c) regulatory systems triggered by temperature and pressure have been identified and studied, and add to the understanding of the roles of these molecules, (d) DHA/EPA bacteria are located almost exclusively in the marine environment, raising the prospect of an important linkage between membrane processes and marine conditions, (e) physiological studies of an EPA recombinant of E. coli show that EPA phospholipids contribute essential fluidity to the bilayer and that an EPA-enriched membrane supports a respiratory lifestyle dependent on proton bioenergetics; the EPA recombinant displays other physiological properties likely attributed to high levels of EPA in the bilayer, and (f) chemical studies such as chemical dynamic modeling support the idea that DHA and presumably EPA contribute hyperfluidizing properties to the membrane. We hypothesize that DHA/EPA phospholipids contribute fluidity and other properties to the bilayer which distinguish these highly unsaturated chains from monounsaturates and polyunsaturates such as 18:2 and 18:3. We further hypothesize that the structural properties of DHA/EPA functioning in bacteria are also harnessed by higher organisms for enhancing crucial membrane processes including photosynthesis and energy transduction.