Metabolism of mevalonic acid to long chain fatty alcohols in an insect

A mixture of long chain fatty alcohols (LCFA) with chain lengths of 24 to 30 carbon atoms, as free and esterified components, have been isolated from the aphid, $\text{Schizaphis}\text{graminum}$. Both radiolabelled mevalonic acid and palmitic acid served as effective precursors in the synthesis of LCFA. This is the first time that the $\text{trans}$-methylglutaconate shunt has been shown to be operational for fatty alcohol synthesis and to occur in a lower-evolved animal.     

Effects of free fatty acids as membrane components on permeability of drugs across bilayer lipid membranes. A mechanism for intestinal absorption of acidic drugs

Studies of the influence of fatty acids, which were the component of intestinal mucosal lipids, on the permeability of several drugs across bilayer lipid membranes generated from egg phosphatidylcholine and intestinal lipid have been pursued. The permeability coefficients of $\text{p-}\text{aminobenzoic}$ acid, salicylic acid and $\text{p-}\text{aminosalicylic}$ acid (anionic-charged drug) increased when fatty acids such as lauric, stearic, oleic, linoleic and linolenic acid were incorporated into the bilayer lipid membranes generated from phosphatidylcholine. In the presence of methyl linoleate and oleyl alcohol, no enhancing effect on $\text{p-}\text{aminobenzoic}$ acid transfer was obtained. The effect of fatty acids was more marked at pH 6.5 than at pH 4.5. In contrast, upon the addition of fatty acids to intestinal lipid membranes which originally contained fatty acids, the permeability coefficient of $\text{p-}\text{aminobenzoic}$ acid tended to decrease, though the permeability through intestinal lipid membranes was larger than that of phosphatidylcholine membranes. The permeability of $\text{p-}\text{aminobenzoic}$ acid across bilayer lipid membranes from intestinal phospholipids was significantly decreased to about equal that of phosphatidylcholine membranes, and reverted to the value of intestinal lipid membranes when fatty acids were added to intestinal phospholipids. It seemed reasonable to assume that free fatty acids in the intestinal neutral lipid fraction could contribute to the increase in the permeability of $\text{p-}\text{aminobenzoic}$ acid. On the basis of above results, possible mechanisms for good absorbability of weakly acidic drugs from the intestine are discussed.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

Drug-induced changes in lipid composition of E. coli and of mammalian cells in culture: ethanol, pentobarbital, and chlorpromazine.

Both Chinese hamster ovary cells in culture and $\text{E.}$$\text{coli}$ cells change their lipid composition when grown in the presence of ethanol, pentobarbital, and chlorpromazine. The effects of ethanol and the cross-tolerant drug, pentobarbital, are similar. Both cause a shift from 18:0 fatty acid to 16:0 fatty acids in CHO cells and a decrease in the proportion of saturated fatty acids in $\text{E.}$$\text{coli}$. Chlorpromazine, a non-cross-tolerant drug, causes the opposite effect in $\text{E.}$$\text{coli}$, a decrease in the proportion of unsaturated fatty acids. Chlorpromazine has little effect on the fatty acid composition of CHO cells. These changes in lipid composition are proposed as an adaptive response and a part of the mechanism for the development of drug tolerance.     

Repair of membrane hyperpermeability in L. plantarum during partial reversal of lipid deficiency

Previous studies suggest that the reduced amino acid accumulation capacity of pantothenate-deficient $\text{L. plantarum}$ is caused by a lipid deficiency which results in membrane hyperpermeability. The accumulation defect can be reversed by supplying such cells with saturated or unsaturated fatty acids which are incorporated into the major lipid constituents. Simultaneous measurement of ³H-amino acid uptake and ¹⁴C-fatty acid incorporation revealed that some unsaturated fatty acids promote an 80% reversal of the amino acid accumulation deficit when the cells have taken up only enough fatty acid to replace 12 to 20% of the lipid deficit. Apparently, only a small fraction of the absent lipid plays a decisive role in membrane permeability.     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Inhibition of porphobilinogen synthase by heme and an enol-ether amino acid

The enol-ether amino acid, L-2-amino-4-methoxy-$\text{trans}$-butenoic acid (AMTB) is an inhibitor of porphobilinogen synthase (PBG synthase) when added prior to the addition of the substrate δ-aminolevulinic acid. The inhibition of PBG synthase by several stereoisomers and analogues of AMTB was investigated to determine those structural features of AMTB which may be necessary for inhibition. The D-$\text{trans}$ isomer was also an inhibitor after preincubation, whereas the L-$\text{cis}$ isomer inhibited with or without preincubation. The amino acid analogues, DL-vinylglycine, DL-2-aminobutanoic acid, the reduced form of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid, L-2-amino-4-(2-aminoethoxy)-$\text{trans}$-3-butenoic acid and its reduced congener did not inhibit PBG synthase even with preincubation. This structure activity relationship indicates that the $\text{trans}$ double bond and methoxy moiety of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid are probably required for inhibition.Heme, when preincubated with PBG synthase, was an inactivator of the enzyme. However, when both L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid and heme were simulatneously preincubated with PBG synthase, inactivation of the enzyme was greater than with either compound separately. The possibility of multiple catalytic sites was suggested by the use of multiple inhibition kinetics in the presence of heme and L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid.     

Motion of spin-labeled fatty acids in murine macrophages relation to cellular phagocytic activity

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels.Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings ($\text{2T}{}_{\mathrm{|}}$) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives.Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 °C, or by fixing them with 0.25 % glutaraldehyde.No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with $\text{cis-}$ or $\text{trans-}\text{unsaturated}$ fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

Lipid metabolism and in vitro production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

The effects of Cloprostenol administration on porcine luteal lipid and arachidonic acid accumulation were examined in relation to luteal $\text{in vitro}$ progesterone and prostaglandin F synthesis in 18 mature gilts at day 12 of the estrous cycle. Basal and net $\text{in vitro}$ release of progesterone from luteal tissue was depressed at 8 hr after treatment whereas net $\text{in vitro}$ release of prostaglandin F was elevated at 8 hr. Inclusion of copper dithiothreitol or reduced glutathione in the incubation media resulted in minor alterations of $\text{in vitro}$ release of progesterone and prostaglandin F and no changes in composition of luteal lipids or fatty acids. Luteal contents of triglyceride had increased by 8 hr after treatment whereas contents of free and esterified cholesterols had increased by 32 hr after Cloprostenol administration. Luteal contents of phospholipid and free fatty acids were not affected by Cloprostenol administration. At 32 hr after treatment, percentages and content of arachidonic acid had increased in luteal cholesterol esters and triglycerides. Although arachidonic acid percentages increased in luteal free fatty acids and phospholipids, calculated arachidonic acid contents did not change following Cloprostenol administration. Induced luteal regression was associated with decreased $\text{in vitro}$ progesterone release, increased $\text{in vitro}$ prostaglandin F release, and accelerated lipid and arachidonic acid accumulation within the corpus luteum. The effects of altered lipid metabolism on release of prostaglandin F could not be defined. However, availability of arachidonic acid did not appear to be rate-limiting in relation to luteal $\text{in vitro}$ prostaglandin F synthesis.     

Preliminary characterization of a small intestinal binding component for retinol and fatty acids in the rat

A component which can bind retinol and fatty acids was detected in the rat's intestinal cell cytosol following intestinal perfusion $\text{in}$$\text{vivo}$ with ³H-all-trans retinol. Following Sephadex G-100 filtration of the cytosol, the void volume concentrate was treated with 2-mercaptoethanol and SDS. Sephadex G-100 filtration of the concentrate disclosed the presence of a cytosol binder of an approximate molecular weight of 12,000–17,000. The binder contained most of the ³H-retinol eluted off the column. $\text{In}$$\text{vitro}$ incubation experiments disclosed that ³H-retinol could be displaced from its bindinf cytosol fraction by the addition of nonradioactive retinol, retinyl acetate, and the fatty acids octanoic, linoleic, and linolenic. Butyric acid addition did not displace ³H-retinol from its binding fraction. The intestinal cytosol binding fraction may be involved in the trans-cytosol transport of lipid compounds from the lipid cell membrane to the intracellular organelles.     

11-Trans leukotriene C: A naturally occurring slow reacting substance

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9,11-$\text{trans}\text{-14-}\text{cis}\text{-eicosatetraenoic acid (11-}\text{trans}$ leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.     

Alteration of the fatty acid composition of Ehrlich ascites tumor cell lipids.

The fatty acid composition of Ehrlich ascites tumor lipids was altered markedly $\text{in vivo}$ by changing the type of fat fed to the tumor-bearing mice. As compared with regular chow, large differences were produced in polar and neutral lipid fatty acyl groups when the tumor cells were grown in mice fed coconut oil, sunflower oil or fat deficient diets. Subcellular membrane fractions obtained from these cells exhibited similar variations in fatty acyl composition. This experimental system provides large quantities of malignant cells for study of the relationships between membrane lipid structure and function.     

Leukotriene A4: Enzymatic conversion to leukotriene C4

An unstable epoxide, leukotriene A₄ (5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C₄. In the present work synthetic leukotriene A₄ was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C₄ and 11-$\text{trans}$-leukotriene C₄ were the main products with SRS activity. It was not established whether the 11-$\text{trans}$-compound was formed by isomerization at the leukotriene A₄ or C₄ stage.     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Hydration of cis- and trans-epoxymethyl stearates by the cytosolic epoxide hydrase of mouse liver.

Man is exposed to epoxides of fatty acids from a number of sources, yet their degradative metabolism is not well understood. In mouse liver the 100,000 g supernatant or the cytosolic fraction is the most active fraction in hydrating $\text{cis}$- and $\text{trans}$-epoxymethyl stearates with the oxirane ring opening in a $\text{trans}$ manner to give the corresponding $\text{threo}$ and $\text{erythro}$ diols, respectively. Hydration was also observed in the microsomal, nuclei and cell debris, and mitochondrial fractions in decreasing order of specific activity.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Cytochrome P-450 and 18-oxygenase system from beef adrenocortical mitochondria - inhibitory effect of puromycin.

$\text{Trans}$-3-dehydro-D, L-ornithine and $\text{trans}$-1, 4-diamino-2-butene have been synthesized and shown to be potent competitive inhibitors of ornithine decarboxylase. The K_I′S for $\text{trans}$-3-dehydro-L-ornithine and $\text{trans}$-1, 4-diamino-2-butene are 2.2 and 2.0 μM respectively. Both analogs bind much more tightly to the enzyme than either ornithine or putrescine. Studies of chick embryo muscle cells in culture show results consistent with reversible inhibition of division and/or fusion by addition of $\text{trans}$-3-dehydro-D, L-ornithine to the culture medium.     

Enzyme secretion in E. coli K12 : studies on alkaline phosphatase synthesis using an unsaturated fatty-acid auxotroph.

The role of unsaturated fatty-acid starvation, and of the substitution of $\text{trans}$ for $\text{cis}$ fatty acids in the membrane phospholipid, on the secretion of alkaline phosphatase, has been investigated. A system in which alkaline phosphatase synthesis was initiated by a temperature shift has been used to obviate possible complications resulting from phosphate depletion. In contrast to earlier reports, we find (a) there is very little effect of unsaturated fatty-acid starvation on the synthesis of alkaline phosphatase; (b) the synthesis of both β-galactosidase and alkaline phosphatase synthesis was severely reduced below 27–30°C in cells grown on $\text{trans}\text{Δ}{}^9$ 16:1 fatty-acid, compared to cells grown on the $\text{cis}\text{Δ}{}^9$ 16:1 analogue. Thus no $\text{preferential}$ effect on alkaline phosphatase synthesis was observed.     

Leukotriene A: stereochemistry and enzymatic conversion to leukotriene B

Leukotriene A was assigned the structure 5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}\text{-11,14-}\text{cis}$-eicosatetraenoic acid by the enzymatic conversion of a synthetic product of known stereochemistry into the naturally occurring isomer of 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid in human polymorphonuclear leukocytes.