The Binding of Luxol Fast Blue Arn by Various Biological Lipids

A determination of the selectivity and approximate stoichiometry of Luxol Fast Blue ARN by known chemical compounds showed that phosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl inositol bound the dye with an apparently stoichiometric ratio of 1 dye molecule to 2 molecules of lipid. Phosphatidyl choline, sphingomyelin, and palmitic acid showed a much weaker reaction. Of these, phosphatidyl choline bound the least amount of dye; about 1 dye molecule per 13-20 lipid molecules. Glycerides, methyl and cholesteryl esters of fatty acids, cholesterol, cerebrosides, and oleic acid gave negative results, as did a variety of low molecular weight substances, including ethanolamine, choline, inositol, and serine. Such negative results indicate that no isopropanol-insoluble complexes were formed with the dye. The behavior of the dye toward several phospholipids suggests that the phosphate groups are essential to the binding reaction and that the quaternary amine of phosphatidyl choline may interfere with it. The selectivity of the dye-binding reaction and the properties of the dye-phospholipid complexes suggest that this reaction will be useful for cytochemical studies of phospholipids, particularly those of the cell membrane     

Carbon-13 nuclear-magnetic-resonance spectroscopy of whole cells and of cytochrome C from Neurospora crass grown with (S-Me-13C)methionine.

Neurospora crassa cytochrome C biosynthetically labelled with [S-Me-13C]methionine was prepared and analysed by 13C nuclear-magnetic-resonance spectroscopy. The methyl group of methionine is extensively incorporated into an N-trimethyl-lysine-72 residue arise from S-adenosylmethionine transmethylation, and that the methyl carbons of methionine residues are sufficiently close to the haem centre to experience chemical shifts from the ring currents of the tetrapyrrole pi electrons and broadening due to binding of methionine-80 with the haem, as well as interaction of the S-E113C]methyl groups with the paramagnetic iron centre. Although whole cells of the labelled Neurospora produced a 13C resonance at the expected position for the methionyl methyl group most of the methyl label was diverted into N-tetra-alkyl ammonium compounds. After an active state of growth these labelled N-methyl compounds appear, in the main, to be low-molecular-weight derivatives of choline which, if associated with membrane, are in a sufficiently fluid environment to have short rotational correlation times. During a subsequent dormant growth period these compounds become associated to some extent with relatively more immobile phases as a result of membrane binding or an increase in membrane rigidity.     

Effects of lipids on the activity of soluble mitochondrial pyrophosphatase and its conversion to the membrane-bound form

The effects of lipids on the activity of soluble and membrane-bound pyrophosphatase from beef heart mitochondria were studied. An addition of total mitochondrial lipid, phosphatidyl choline, phosphatidyl ethanolamine or cardiolipin resulted in stimulation of the enzymatic activity and an increase in thermal stability of the soluble enzyme. The maximal activating effect was exerted by the total mitochondrial lipid and phosphatidyl choline. The electrophoretic data suggest that phosphatidyl choline is a component of membrane pyrophosphatase. Preincubation of the soluble enzyme with phosphatidyl choline converted the enzyme into a membrane form, which is capable to carry out the energy-dependent synthesis of PPi in submitochondrial particles.     

Studies on the Origin of the Methyl Groups of Choline in Ochromonas malhamensis*

SYNOPSIS. Five- to 6-day-old resting cells of Ochromonas malhamensis were incubated at pH 6.5 with glucose and appropriate C¹⁴ precursors of the methyl groups of phospholipid-choline. Under the experimental conditions L-methionine-C¹⁴H₃ was the most efficient source of choline-methyl groups, followed by formate-C¹⁴, formaldehyde-C¹⁴ and DL-serine-3-C¹⁴, respectively. Glycine-2-C¹⁴ was not incorporated into choline. Both L-methionine-C¹⁴H₃ and formate-C¹⁴ served as precursors for the methyl groups of monomethylethanolamine, dimethylethanolamine and choline. Addition of non-radio-active L-methionine depressed the incorporation of formate-C¹⁴ into choline-methyl groups by 50%. The results support the hypothesis that methionine can be the source of all 3 methyl groups of choline, and that formate is probably converted to the methyl group of methionine before transmethylation to choline. However, an alternate pathway from single-carbon sources cannot be excluded.     

Electrical conductivity in lipid bilayer membranes induced by pentachlorophenol.

Electrical conductivity induced in thin lipid bilayer membranes by pentachlorophenol has been studied. The membranes were formed from phosphatidyl choline, phosphatidyl ethanolamine, or phosphatidyl glycerol and various amounts of cholesterol. The position and the magnitude of the maximum of the conductivity vs. pH curve depend on the type of lipids and cholesterol content. At low pentachlorophenol concentrations and low pH the concentration dependence of conductivity is quadratic and becomes linear at higher pH. Above 10(-5) M of pentachlorophenol the concentration dependence of the membrane conductivity tends to saturate. Presence of pentachlorophenol enhances membrane transport of nonactin-K+ complex. Increase of cholesterol content increases pentachlorophenol induced conductivity in all membranes and shifts the conductivity toward lower pH. For phosphatidyl choline the largest rate of change of membrane conductivity with cholesterol occurs at 1:1 phospholipid to cholesterol molar ratio. Pentachlorophenol is found to be a class II uncoupler and the experimental results are consistent with the hypothesis that the membrane permeable species are dimers formed by combination of neutral and dissociated pentachlorophenol molecules. Several schemes of membrane conduction, including dimer formation in the aqueous phase as well as at the membrane-water interface have been considered. Arguments are given in favor of the formation of dimers within the membrane surface.     

N-Methyl Groups in Bacterial Lipids III. Phospholipids of Hyphomicrobia

The phospholipids of Hyphomicrobium vulgare NQ-521 have been separated by preparative thin-layer chromatography and analyzed by paper chromotography of the water-soluble products of acid and mild alkaline hydrolysis. The principal phospholipids are phosphatidyl ethanolamine (23%), phosphatidyl N,N'-dimethylethanolamine (36%), lecithin (29%), and phosphatidyl glycerol (10%). Three other strains of Hyphomicrobium were found to have similar phospholipid compositions. Growing cells incorporated the methyl group of methionine into lipid-bound N,N'-dimethylethanolamine and choline. Experiments with sonic extracts of H. vulgare NQ-521 and (14)C (methyl) S-adenosylmethionine demonstrated the formation of phosphatidyl N-monomethylethanolamine in addition to the dimethylethanolamine and choline phosphatides.     

Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein

Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid³²P_i-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little³²P_i-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [³²P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation.     

Control of the potassium ion-stimulated adenosine triphosphatase of pig gastric microsomes: effects of lipid environment and the endogenous activator

The K⁺-stimulated ATPase activity associated with the purified gastric microsomes from the pig gastric mucosa can be completely inactivated by treatment with 15% ethanol for 60 s at 37 °C but not at 25 °C. Sequential exposure of the microsomes to 15% ethanol at 25 and 37 °C caused the release of 2.9 and 4.3% of the total membrane phospholipids, respectively, consisting entirely of phosphatidyl choline and phosphatidyl ethanolamine. The ethanol-treated (37 °C) membrane had high basal (with Mg²⁺ as the only cation in the assay mixture) activity, which was further enhanced during reconstitution with phosphatidyl choline or phosphatidyl ethanolamine. The high basal activities could be reduced to the normal control level by assaying the enzyme in presence of the “activator protein,” partially purified from the soluble supernatant of the pig gastric cells. Phosphatidyl choline was somewhat more effective than phosphatidyl ethanolamine in the restoration of the activity of the ethanol-treated enzyme while phosphatidyl serine, phosphatidyl inositol, and sphingomyelin were without any effect. Synthetic phosphatidyl choline with various fatty acid substitutions were tested for their effectiveness in the restoration of the ethanol-inactivated enzyme. The distearoyl (18:0), dioleoyl (18:1), and dilinoleoyl (18:2) derivatives of phosphatidyl choline were almost equally effective while dipalmitoyl (16:0) phosphatidyl choline was somewhat less effective in the reconstitution process. Cholesterol appeared to interfere with phosphatidyl choline in the restoration of the activity of ethanol-treated enzyme. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine extracted by 15% ethanol at 37 °C was clearly different than those of the total microsome. Our data suggest that the phospholipids extracted by 15% ethanol at 37 °C are derived primarily from the immediate lipid environment of the enzyme and ATP together with Mg²⁺ and K⁺ help the partially delipidated enzyme to retain the appropriate conformation for the subsequent reconstitution. Furthermore, ethanol appears to either release or inactivate the membrane-associated activator protein, demonstrated to be essential for the K⁺-stimulated activity of the pig gastric ATPase.     

Methyl group metabolism in sheep

1. Sheep have a very low intake of methyl nutrients in the post-ruminant state, due to the almost complete degradation of dietary choline by rumen microorganisms, the lack of dietary creatine and the relatively low content of methionine in microbial proteins. 2. Methylneogenesis provides a major source of labile methyl groups in post-ruminant sheep and impairment of the methylneogenesis leads to a marked reduction of the labile methyl pool. 3. S-Adenosylmethionine (AdoMet) metabolism via transmethylation is most active in sheep liver and pancreas and is regulated by the availability of methionine and intracellular ratios of AdoMet to S-adenosylhomocysteine (AdoHcy). 4. Adaptive mechanisms which arise as a consequence of the poor methyl nutrition in post-ruminant sheep are a marked reduction of labile methyl catabolism and an increase in the capacity of methylneogenesis.     

The manipulation of polar head group composition of phospholipids in the wheat Miranovskaja 808 affects frost tolerance

Caryopses of the frost-resistant cultivar of the wheat Triticum aestivum L., Miranovskaja 808, were germinated and grown in the presence of various concentrations of choline chloride. Changes in the composition of leaf total phospholipids and leaf total fatty acids at two extreme temperatures (25°C and 2°C) as well as changes in frost resistance were followed. A choline chloride concentration-dependent accumulation of phosphatidyl choline was observed in the leaves. Seedlings grown at 2°C accumulated more phosphatidyl choline at each choline chloride concentration than those grown at 25°C. There was an inverse relationship between the contents of phosphatidyl choline and phosphatidic acid in the leaves. Neither the temperature nor choline chloride seemed to affect fatty-acid composition. Modification of polar-head group composition of phospholipids affected frost tolerance: Seedlings grown in the presence of 15 mM choline chloride at 25°C exhibited a freezing resistance equal to that of hardened controls. The data indicate that the polar-head group composition of membrane phospholipids in plants can be easily manipulated and point to the importance of phosphatidyl choline in cold adaptation processes.     

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

Effect of lipid composition changes on carbocyanine dye fluorescent response

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.     

Phospholipid and ether linked phospholipid content alter with cellular resistance to vinblastine

The phospholipid and ether linked phospholipid content of leukaemic lymphocytes alters when the cells become resistant to low levels of the anti-cancer drug, vinblastine. Sphingomyelin and cardiolipin increase, and phosphatidyl ethanolamine and serine decrease in resistant cells. In addition, increases in 1-alkyl-2-acyl phosphatidyl choline and 1-alkenyl-2-acyl-phosphatidyl ethanolamine are concomitant with decreased 1,2-diacyl phosphatidyl choline and ethanolamine. Changes to the ultrastructure of the inner half of the plasma membrane bilayer, as a consequence of drug resistance, are illustrated by freeze-fracture electron microscopy.     

Heterogeneity in organization of lipids in the bacterial membrane]

The lipids of Micrococcus lysodeikticus membranes were 50%-substituted by phosphatidyl choline using lipid-exchanging proteins isolated from rat liver. The incorporation of phosphatidyl choline into the membrane did not significantly change the malate dehydrogenase activity and the temperature dependence activity in the Arrhenius plots for the enzyme. Gramicidin S--modifier of membrane lipids--had similar effects both on the intact membranes and on the phosphatidyl-enriched membranes. A conclusion is made on structural heterogeneity of the bacterial membrane and on the presence of a boundary lipid fraction, which controls the functioning of malate dehydrogenase and is characterized by a low-rate exchange with other lipids.     

Phosphoinositides of sheep platelets plasma membranes.

Phospholipid composition of sheep blood platelets and its various plasma membrane fractions have been analyzed. Based on their flotation rates in discontinuous sucrose density gradient centrifugation, three membrane fractions were isolated. 5'-Nucelotidase and alkaline phosphatase were distributed nearly equally in all the three membrane fractions. However these membrane fractions showed differences in the distribution of phosphatidyl ethanolamine, phosphatidyl choline and phosphoinositides. Phosphatidyl ethanolamine was predominant in fraction I (11.05 micrograms PLP/mg protein) while phosphatidyl choline was predominant in fractions II and III (110.10 and 68.30 micrograms PLP/mg protein respectively). Phosphatidyl inositol (Ptd-InsP) was equally distributed in all three membrane fractions. However, both Ptd-InsP and phosphatidyl inositol 4,5-bisphosphate were about 4-fold higher in fraction II (73.55 and 89.89 micrograms PLP/mg protein respectively).     

Choline uptake is increased by Ca++ and liposomes+ Ca++ in ascites tumor cells

Steady-state uptake of choline by Lettre-Ehrlich tumor cells in vitro, resulting in cell-to-medium ratios of 10 or more, is significantly increased by 0.2-1.0 mM Ca++ as well as by dipalmitoyl phosphatidyl choline multilamellar liposomes + Ca++. The increases occur in spite of a decrease in carrier affinity, as indicated by the Km, and therefore result either from increased carrier velocity or utilization of new carriers. About half of the labelled choline which is taken up is firmly bound to cells. That label which freely leaves cells is phosphocholine, thus, these cells utilize choline mainly in phospholipid synthesis. Choline and nitrogen mustard (HN2) share a plasma membrane carrier but the intracellular distribution of HN2 into DNA, RNA and protein, contrasts with that of choline, into phospholipid.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-¹⁴C and glycerol-³H were examined. Choline-¹⁴C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-¹⁴C in the reaction mixture and the choline-¹⁴C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-¹⁴C, the apparent turnover of glycerol-³H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-³H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-³H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (~96%) of the glycerol-³H recovered from microsomal membranes was in phospholipids, whereas only a minor component (~2%) of the glycerol-³H was in the phospholipids isolated from nonmembrane lipids, glycerol-³H was judged to be a specific marker for membrane phospholipids.     

Regulation of Staphylococcus protease using complement, interferon and immunoglobulin as substrates.

The effects of various agents on the cleavage of serum albumin, interferon, immunoglobulin and complement component C1q by the extracellular protease from Staphylococcus aureus were analysed by SDS-polyacrylamide gel electrophoresis. Arachidonic acid moderately stimulated the proteolysis of serum albumin, interferon and complement component. Phosphatidic acid effectively enhanced the proteolysis of serum albumin and IgG, whereas it inhibited the cleavage of IgM. The proteolysis of IgG was appreciably enhanced by sphingosine. In contrast, phosphatidyl choline and phosphatidyl glycerol were shown to have an inhibitory effect on the proteolysis of IgG and IgM. Phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine also inhibited the proteolysis of IgG. The failure of any of these agents to exert a persistent effect on the cleavage of all substrates, revealed the complexity of the interactions among the agent, the substrate and the protease.     

Reduction of various lipid hydroperoxides by rat liver homogenate

Rates of disappearance of hydroperoxy groups of various lipid peroxides added to rat liver homogenate differed from each other. The hydroperoxy group of linoleic acid disappeared rapidly, while those of L-3-phosphatidyl choline dilinoleoyl and trilinolenin disappeared slowly. The hydroperoxy groups of cholesterol linoleate were stable in the homogenate. Most of the conjugated dienes of these lipid peroxides remained. The hydroperoxy groups of the unsaturated fatty acids of the phosphatidyl choline were found to be changed to hydroxy groups as analyzed by high-performance liquid chromatography.     

The action of phospholipase C and lipase on black film bilayer membranes

Phospholipase C(C. perfringens) added to one side of a phosphatidyl choline—phosphatidyl serine—cholesterol (0.7: 0.3: 1.0, w/w/w) bilayer membrane resulted in a rapid decrease in membrane resistance which leveled off after several minutes at about one-half its initial value. Pancreatic lipase added at this time resulted in a rapid increase in membrane resistance to a value somewhat higher than the initial. This effect was independent of the side to which the lipase was added, indicating that diglyceride is rapidly equilibrated across both sides of the membrane. When both phospholipase C and lipase were added at zero time to the same or opposite sides, the resistance decreased slightly and then increased to a value higher than the initial. Replacement of phosphatidyl choline by 2-hexadecoxy-3-octadecoxypropyl-phosphonylcholine, a phosphonate analog, resulted in an inhibition of the phospholipase C reaction which was equal to the percent analog. The analog, thus, seemed to act only as inert lipid and did not inhibit hydrolys of the active lipid.