Effect of fatty acid deficiency on microsomal membrane fluidity and cooperativity of the UDP-glucuronyltransferase

The effect of fat deprivation on microsomal membrane fluidity of guinea-pig livers and the kinetic cooperativity of UDP-glucuronyl transferase towards its natural substrate, the UDP-glucuronic acid, were studied. Fat deprivation in the diet of weanling guinea-pigs evoked a typical essential fatty-acid-deficient pattern in the composition of the microsomal membrane. The unsaturated:saturated fatty acid ratio progressively declined in the membrane during the 21-day period tested. This decline determined a gradual increase in the fluorescence anisotropy (rs) of the membrane labeled with diphenylhexatriene and the apparent microviscosity of the lipid bilayer calculated from these values increased from 1.1 to 1.8 poise. In addition, when the infinitely slow decaying fluorescence anisotropy ((r infinity), which is proportional to the square of the lipid order parameter, was calculated from rs data, a significant increase in these parameters was also obtained. Furthermore, this decrease of the double bond index:saturated acid ratio of the membrane was associated with a parallel increase in Hill coefficients of the UDP-glucuronyl transferase that gradually lost the negative homotropic effect and cooperativity of UDP-glucuronic acid. The Hill coefficient varied from 0.39 to 0.98 during the 21-day period studied. Our observations indicate on one side that changes in the fat composition of the diet are accompanied by modifications in the lipid composition and fluidity of the microsomal membrane, and the apparent cooperativity of the enzyme. On the other side, they suggest that the evaluation of Hill coefficients of UDP-glucuronyl transferase might be used as a sensitive test to investigate conformational changes in the microsomal membrane of the liver.     

Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids

The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.     

Kinetic properties of microsomal UDP-glucuronyltransferase. Regulation by metal ions

Treatment of microsomes with EDTA abolishes the stimulation of glucuronidation produced by UDP-N-acetylglucosamine. Addition of divalent metal ions to EDTA-treated microsomes restores the sensitivity of UDP-glucuronyltransferase to UDP-N-acetylglucosamine. Regulation of the activity of this enzyme by UDP-N-acetylglucosamine depends, therefore, on the presence of divalent metal ions. In addition, divalent metals increase the rate of glucuronidation of p-nitrophenol at V_max. The data indicate that metals are essential for the efficient function of UDP-glucuronyltransferase.     

Correlations between fatty acid distribution in phospholipids and the temperature dependence of membrane physical state

Cytoplasmic membranes of an unsaturated fatty acid auxotroph of Escherichia coli have been studied using spin labeled hydrocarbon probes. These studies reveal that the membrane lipids undergo changes of state at critical temperatures which reflect the physical properties of the fatty acid supplement supplied to the cells during growth. The critical temperatures observed in spin labeled membranes correlate with characteristic temperatures in membrane functions. Lipid analysis reveals that fatty acid composition and distribution in membrane phospholipids are primary determinants of the temperatures at which changes of state are observed in membrane lipids. Fatty acid composition and distribution can also produce unique interactions between certain spin label probes and their lipid environment.     

Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation.

The degree of plasma membrane fatty acid unsaturation and the copper sensitivity of Saccharomyces cerevisiae are closely correlated. Our objective was to determine whether these effects could be accounted for by differential metal induction of lipid peroxidation. S. cerevisiae S150-2B was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Potassium efflux and colony count data indicated that sensitivity to both copper (redox active) and cadmium (redox inactive) was increased in 18:2-supplemented cells and particularly in 18:3-supplemented cells. Copper- and cadmium-induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements with the membrane probe trimethylammonium diphenylhexatriene. Levels of thiobarbituric acid-reactive substances (lipid peroxidation products) were up to twofold higher in 18:2-supplemented cells than in unsupplemented cells following metal addition, although this difference was reduced with prolonged incubation up to 3 h. Conjugated-diene levels in metal-exposed cells also increased with both the concentration of copper or cadmium and the degree of cellular fatty acid unsaturation; maximal levels were evident in 18:3-supplemented cells. The results demonstrate heavy metal-induced lipid peroxidation in a microorganism for the first time and indicate that the metal sensitivity of PUFA-enriched S. cerevisiae may be attributable to elevated levels of lipid peroxidation in these cells.     

Liver microsomal beta-glucuronidase and UDP-glucuronyltransferase.

Both UDP-glucuronyltransferase (GT) and beta-glucuronidase (betaG) were assayed in untreated liver microsomes. Optimum assay conditions were established with rat liver microsomes using p-nitrophenol (pNP) and its glucuronide (pNPGA) at the pH optima of GT (7.5) and betaG (4.5). The activities of the two enzymes were compared using microsomes from rats, mice, pigs, cattle and horses, with pNP, pNPGA, and phenolphthalein as substrate, in the presence of various cofactors and inhibitors at pH 7.5 and 4.5. These data disclose pronounced differences with respect to species, substrate and other experimental conditions, thereby precluding the establishment of general optimum conditions. The two enzymes were also assayed under strictly identical conditions using pNP and pNPGA and rat liver microsomes at pH 7.5 in the presence and absence of UDP-glucuronate disodium (UDPGA), activators (ATP;UDP-N-acetylglucosamine) and inhibitors. When provided with a functional level of UDPGA, both enzymes proved active under those conditions, and a conjugation-deconjugation interplay was indicated. The two processes could be selectively and totally inhibited by Zn2+ and saccharolactone. The results suggest that conjugation-deconjugation-reconjugation cycles may be operative in the metabolism of drugs in vivo, taking place already at the level of the liver endoplasmic reticulum.     

Self-regulation of membrane fluidity. The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition.

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change being very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by membrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.     

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.     

Modification of membrane lipid: physical properties in relation to fatty acid structure.

Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.     

The relationship between plasma membrane lipid composition and physical-chemical properties. II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive (Na+ + K+)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant differences was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane 'fluidity' based on lipid modulation of biological membranes.     

The relatioship between plasma membrane lipid composition and physical-chemical properties II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant difference was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane ‘fluidity’ based on lipid modulation of biological membranes.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

Effect of alpha-tocopherol and phospholipids with omega-3 fatty acids on membrane properties

As a result of the investigations conducted it was displayed, that alpha-tocopherol and phospholipids including into their composition omega-3-acids, differed in their influencing the composition of heart microsomes membranes lipids. The insufficient quantity of vitamin E in the animals ration was defined as leading to the cardiac microsomes lisophospholipids (lisophosphatidylcholin, lisophospatidylethanolamin), diphosphatidylglycerol increase as well as to the tendency to sphingomyeline and phosphatidylethanolamin decrease. While administrating both alpha-tocopherol and the complex of phospholipids with omega-3-fatty acids, the correction of the phospholipids composition microsomes membranes is observed as tending towards their stabilization, however the marine phospholipids complex is more active than alpha-tocopherol. Administration of phospholipids with omega-3-fatty acids during the period of 30 days provided for the increase of relationship: polyunsaturated fatty acids to saturated fatty acids in the cardiac microsomal membranes, evidencing about increasing the unsaturated cellular membranes. While administrating the phospholipids, into the cardiac microsomes the eicozepentaenic acid was identified, failing to be in the norm, docozahexaenic acid content increased. The results obtained testify, that at the pathology there are changes in the quantitative relationship of membrane phospholipids and fatty acids, being a result of changing the biomembranes permeability as well as their functions disturbances. The adverse effect of E-deficiency to the membrane structure was revealed as capable to be regulated by the marine phospholipid complex, including omega-3-fatty acids.