The effect of non-esterified fatty acids on the proton-pumping cytochrome c oxidase reconstituted into liposomes.

Bovine heart cytochrome c oxidase was reconstituted in phospholipid vesicles, and the effect of different non-esterified fatty acids (NEFA) was studied on its proton pump and on the proton permeability of the vesicles. Neither parameter appeared to be affected by concentrations of NEFA known to uncouple oxidative phosphorylation (10 microM). Also the permeability for K+ was not affected by them. The fatty acids caused an increase in the rate of electron transfer in the absence, but not in the presence, of uncoupler and/or valinomycin [diminution of the respiratory-control index (RCI)]. The RCI of 8.7-7.5 was decreased to about 4.5 in the presence of 0.27-10 microM-NEFA. Oleic acid was not effective at the above concentrations. Subunit III-depleted enzyme preparations gave vesicles with an RCI of about 5.5, which was decreased to 4.5 in the presence of NEFA. With both native and subunit III-depleted oxidase the RCI was never decreased to the value of 1 by NEFA, as happens with classical protonophores.     

Specific effects of ATP on the kinetics of reconstituted bovine heart cytochrome-c oxidase

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and the kinetics of cytochrome c oxidation were measured by the polarographic and photometric method under uncoupled conditions in the presence of various polyvalent anions. In order to distinguish between specific and unspecific ionic effects of ATP, the photolabelling reagent 8-azido-ATP was applied. Covalently bound ATP at the enzyme complex caused the same increase of Km for cytochrome c as free ATP, if measured by the photometric assay. The increase of Km by photolabelling with 8-azido-ATP was completely prevented by ATP, but not by ADP. The data indicate the occurrence of a specific binding site for ATP at the cytosolic side of cytochrome-c oxidase, which, after binding of ATP, changes the kinetics of cytochrome c oxidation.     

Influence of albumin and non-esterified fatty acids on serum prostacyclin binding in pregnancy

We investigated the etiology of the previously documented decrease in serum prostocyclin binding during pregnancy. Addition of albumin to the serum of pregnant women failed to raise binding to non-pregnant levels. Pregnancy serum bound significantly more prostacyclin following the removal of non-esterified fatty acids and the addition of fatty acid free albumin resulted in a rise in binding to non-pregnant levels. We conclude that serum protein prostacyclin binding is affected by both albumin concentration and non-esterified fatty acids.     

Modulation of T-cell signalling by non-esterified fatty acids

Polyunsaturated fatty acids (PUFAs) have been shown to be immunosuppressive. In particular, they can decrease important T-cell functions that may have a profound impact on the acquired immune response. Several mechanisms may explain the immunosuppressive properties of PUFAs. Here we review the mechanisms by which they interfere with T-cell activation. PUFAs affect lipid rafts composition and function that play an essential role in T-cell signalling. The possible physiological and pathological significances of this immunomodulation by PUFAs are discussed. Further mechanistic studies and randomized controlled clinical trials are needed to assess more accurately their effects in healthy and pathological states.     

Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart

Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.     

Effects of non-esterified fatty acids on the gluconeogenesis in bovine hepatocytes

Dairy cows experience an increased demand for glucose to support milk production. However, negative energy balance is a common condition in peripartum cows. In response, fat mobilization provides non-esterified fatty acids (NEFAs) for oxidation in the liver to generate ATP. To investigate the effects of NEFAs on gluconeogenesis, the expression and enzyme activity of pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPCK) in cultured bovine hepatocytes were evaluated by quantitative polymerase chain reaction and spectrophotometry, respectively. The results showed that PC and PEPCK mRNA levels were marked decreased when the NEFAs concentrations exceeded 0.5 and 1.5?mmol/l, respectively. The PC and PEPCK enzyme activity showed significantly decreased when the NEFAs concentrations exceeded 1.5 and 0.5?mmol/l, respectively. These findings indicate that high circulating levels of NEFAs inhibit hepatocyte gluconeogenesis, thereby promoting negative energy balance.     

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.     

Influence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline modification on proton translocation and membrane potential of reconstituted cytochrome-c oxidase support "proton slippage".

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and modified with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ reacted mainly with subunits II and III and to a lower extent with subunit I, as shown by difference labeling with [14C]dicyclohexylcarbodiimide. EEDQ treatment of cytochrome-c oxidase vesicles influenced ferrocytochrome c-induced proton pumping by reducing maximally the H+/e- stoichiometry from 0.84 (control) to 0.24, but had only small effects on respiration, respiratory control ratio, and proton conductivity of the proteoliposomes. By titrating the reaction rate of the control and the modified cytochrome-c oxidase vesicles versus the membrane potential, as measured with a Ph3MeP+ electrode, saturation curves are obtained, which in both cases approach 225 mV. The ratios of electron transport rates of the two proton pumps at various membrane potentials decrease between 160 and 225 mV from about 2.2 to 1, indicating that the nonlinear flow/force relationship of these proton pumps is at least partly due to "slippage" of proton pumping.     

Inactivation of cytochrome-c with glucose oxidase

A novel reaction of cytochrome-c from the horse heart with the enzyme glucose oxidase from Aspergillus niger (EC 1.1.3.4), in acidic media is described. Glucose oxidase is able to induce a rapid, profound and irreversible physico-chemical change in cytochrome-c, under anaerobic conditions and in the presence of glucose. The initial rate of reaction is almost independent of the concentration of enzyme and glucose. The striking feature of this reaction is the fact that the reaction proceeds efficiently even below a concentration of 10 nM enzyme.     

Gas-liquid chromatography of non-esterified fatty acids of the rat myocardium]

Non-esterified fatty acids in cardiac cell cultures of postnatal rat, in postnatal rat heart and in ventricle muscle of adult rat were analyzed by gas-liquid chromatography. Among cultured heart cells, three different types were found. Type A0 appeared when arachidonic acid was lacking in tissue culture medium; it had the highest percentage in saturated fatty acids, palmitoleic and linolenic acids. Type A1 and type A2 appeared when arachidonic acid was proved to be present in culture medium. Non esterified fatty acid composition of type A1 was closely similar to that of postnatal rat heart, while type A2 ranged between that of postnatal heart and that of adult ventricular muscle.     

Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro

Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.     

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.     

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.