Phospholipids of rat tissues after feeding pure phosphatidyl ethanolamine and lecithin

Pure phosphatidyl ethanolamine and lecithin from egg yolks were fed to rats in saline or in olive oil and the changes in individual phospholipids in the intestinal wall, liver, and plasma of the animals were studied. Ingestion of olive oil alone produced increased levels of all phospholipid fractions in each of the three tissues. Feeding phosphatidyl ethanolamine in saline resulted in slightly increased plasma phospholipids, but levels of liver total phospholipids were greatly reduced; when phosphatidyl ethanolamine was fed with olive oil, liver phospholipids were again reduced but this reduction was confined to the phosphatidyl ethanolamine and phosphatidic acid fractions. Feeding lecithin alone did not produce significant changes in levels of plasma or tissue phospholipids. The results suggest that liver phospholipid synthesis is depressed by feeding phosphatidyl ethanolamine; in the presence of olive oil, hepatic synthesis of phosphatidyl ethanolamine seems to be more selectively inhibited.     

Lipids of ocular tissues: VII. Positional distribution of the fatty acids in the phospholipids of bovine retina rod outer segments

The positional distribution of the fatty acids in the major phospholipids of bovine retina rod outer segments was determined. Phosphatidyl ethanolamine and phosphatidyl serine have mostly saturated acids in the 1-position and docosahexaenoic acid in position 2. These phospholipids contain 94 and 79%, respectively, of polyun-saturated acids in the 2-position. Phosphatidyl choline contains mostly saturated acids in the 1-position, but has significant quantities of palmitic in the 2-position along with docosahexaenoic acid. The levels of docosahexaenoic acid in rod outer segment phospholipids are among the highest yet reported for membrane phospholipids, amounting to 23% in phosphatidyl choline, 39% in phosphatidyl ethanolamine, and 45% in phosphatidyl serine.     

Thin-layer chromatographic analysis of phospholipids in Echinostoma revolutum (Trematoda) adults.

Thin-layer chromatographic analyses were made of phospholipids extracted from adult Echinostoma revolutum (Trematoda) and from the nonnutrient medium in which worms were maintained. Identity of phospholipids was based on comparison with authentic lipid standards and on specific chemical detection tests. The most abundant phospholipids in the extract were phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine; lesser amounts of cerebrosides and lysophosphatidyl choline were detected also. Phospholipids detected in the incubation medium were essentially as described for extracts.     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.     

Phospholipids activate cathepsin D

Total lipids as well as phospholipids extracted from the mitochondrial-lysosomal fraction of porcine adrenal cortex activated the lysosomal cathepsin D of this tissue 30- and 40-fold, respectively, with bovine serum albumin as the substrate. Phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol and cardiolipin were found to activate greatly the cathepsin D. The degree of activation ranged from 6-fold by phosphatidyl ethanolamine to 40-fold by cardiolipin at 1 mM, respectively. These results strongly point to the importance of phospholipids in intracellular protein degradation by lysosomal cathepsin D.     

Phospholipid-enzyme interactions of chitin synthase of Coprinus cinereus

Abstract The effect of phospholipids on chitin synthase activity has been studied with digitonin-solubilized and partially purified preparations from Coprinus cinereus . When cholate was used as detergent, it inhibited enzyme activity, but this inhibition was reversed by increasing concentrations of phospholipids. Preincubation with cholate and phospholipid caused irreversible loss of activity. When sonicated with solubilized enzyme preparation, dimyristoyl phosphatidyl choline strongly stimulated activity, while dioleoyl phosphatidyl choline was inhibitory. The Arrhenius plot of the effect of temperature on enzyme activity contained breaks, characteristic of a membrane-bound enzyme. It is suggested that chitin synthase requires an annulus of phospholipids for activity.     

Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

Hypervitaminosis A and Liver Phospholipids

Effect of daily oral feeding of 33 mg retinol for nine days on the liver phospholipids of rats has been studied. As early as two days after feeding retinol an increase in the amounts of liver triglycerides, proteins, phospholipids, and cholesterol was noted which kept increasing and reached the peak concentration 6 days after daily retinol feeding and thereafter a decrease in their amounts was noted. Hepatic phospholipid fractions viz. phosphatidyl choline, phosphatidyl ethanolamine, phosphatidic acid and polyglycerol phosphatide, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, lysophosphatidyl ethanolamine and lysophos- phatidyl choline showed the same pattern. Labelling of these phospholipids with NaH₂³²PO₄ in rats fed daily 33 mg of retinol for a period of two days also exhibited the pattern which was observed in their amounts two days after daily feeding of retinol. The results suggest a close relationship between the metabolism of hepatic triglycerides and phospholipids of rats fed excessive amounts of retinol.     

Oxygen-induced changes in pulmonary phospholipids in the rat.

The composition and synthesis of alveolar and lung tissue phospholipids were investigated in normal and oxygen-poisoned rat lungs. Sixty-hour exposure to oxygen increased the total amount of phospholipids in the endobronchial extracts and lung tissue. Phosphatidyl glycerol was identified in both endobronchial extracts and lung tissue. The amount of unsaturated fatty acids in surfactant lecithin and phosphatidyl glycerol was slightly increased in oxygen-poisoned lungs whereas the composition of phospholipids in the endobronchial extracts was not affected by oxygen. After intraperitoneal administration of [32P]phosphate the specific activities of surfactant lecithin and phosphatidyl glycerol were clearly lower in oxygen-treated animals whereas the specific activities of lung tissue lecithin and phosphatidyl glycerol remained unaffected. The synthesis of lecithin from [14C]methionine through N-methyltransferase pathway was markedly depressed in lung slices but increased in liver tissue taken from oxygen-poisoned rats and incubated under oxygen indicating a difference between lung and liver methyltransferase enzymes. In conclusion, the present work suggests impaired synthesis and removal of alveolar phospholipids in oxygen-poisoned rats.     

THE INCORPORATION OF 32P ORTHOPHOSPHATE INTO THE PHOSPHOLIPIDS OF ELONGATING AVENA COLEOPTILES

Four phospholipids of Avena coleoptile tissue were identifiedas phosphatidyl inositol, phosphatidyl glycerol, phosphatidylethanolamine and phosphatidyl choline. IAA caused an increase in total uptake of ³²P and incorporationof ³²P in phospholipids. IAA also caused a shift in the proportionsof identified ³²P phospholipids. Incorporation of ³²P into phosphatidylcholine was greater while incorporation into phosphatidyl glyceroland phosphatidyl ethanolamine was less in IAA-treated tissuecompared with untreated control tissue. ¹Contribution No. 338 from the Department of Botany, PennsylvaniaState University and 3001 from the Pennsylvania AgriculturalExperiment Station. ²Present address: Juniata College, Huntingdon, Pennsylvania,U.S.A.     

Are polyphosphorylated phospholipids involved in the hormonal control of cholesterol side-chain cleavage activity in tumour Leydig cells?

The possible role of LH or dcAMP induced changes in polyphosphorylated phospholipid metabolism in the regulation of cholesterol side-chain cleavage activity has been studied in tumour Leydig cells. Mitochondria isolated from LH-stimulated Leydig cells were 400% more active in pregnenolone production than mitochondria from control cells. Steroid production in isolated mitochondria from control cells could be stimulated only 25% by cytosol fractions from stimulated cells and 100 microM phosphatidyl inositol-4'-phosphate (PtdIns4P). Other polyphosphorylated phospholipids were either inactive or showed aspecific effects. During a preincubation period tumour cells were labelled with [32P]phosphate and steady-state labelling was obtained for the pholyphosphorylated phospholipids after 40-60 min. [32P]Phosphate incorporation in Ptd Ins4P, phosphatidyl inositol (PtdIns), phosphatidyl choline (PtChl), phosphatidyl ethanolamine (PtdEtn) and cardiolipin (CL) was not affected by treatment of the Leydig cells with LH which stimulated (6-fold), or with cycloheximide which suppressed (4-fold) steroid production. A 25% increase of phosphate incorporation by LH was observed only in phosphatidyl inositol-4',5'-biphosphate (PtdIns4,5P2). 32P Incorporation in PtdIns4,5P2, PtdIns,PtdEtn and CL was stimulated by quinacrine 50 microM. Under these conditions the LH-stimulated pregnenolone production but not the 25-hydroxycholesterol dependent pregnenolone production, was completely inhibited. The results obtained with isolated mitochondria and intact cells indicate that increased levels of polyphosphorylated phospholipids are not consistently correlated with increased mitochondrial pregnenolone production. This argues against an important role of polyphosphorylated phospholipids in the hormonal regulation of cholesterol side-chain cleavage activity in tumour Leydig cells.     

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.     

Effect of Feeding Defatted Millet (Sorghum vulgarie) Flour at Different Protein Levels on Phospholipids of Rat Liver

Effect of feeding defatted millet flour at 5, 10 and 14.5 percent protein levels respectively to rats for 6 weeks has been studied on their liver phospholipids. Feeding of millet at these protein levels significantly increased liver total lipids and total glycerides as compared to control rats fed casein at 10 percent level. Liver phospholipids and phosphatidyl choline were significantly increased in rats fed millet at 10 and 14.5 percent protein levels whereas phosphatidyl ethanolamine was increased in all millet protein fed groups. Incorporation of (counts/liver/100 g body weight) into liver total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine was significantly increased in M–5 and M–15% groups and it was decreased in phosphatidyl choline in rats of M–10% groups as compared to control. The effects of feeding millet on liver phospholipids are due to the quantity as well as quality of the millet protein.     

Effect of Growth Temperature on the Lipid Composition of Cyanidium caldarium: II. Glycolipid and Phospholipid Components

Cyanidium caldarium was grown at 20 and 55 C and harvested during exponential growth phase. Lipids were extracted and separated by silicic acid column and thin layer chromatography. The major glycolipids were identified as mono- and digalactosyl diglyceride and sulfolipid. Major phospholipids were identified as phosphatidyl choline and phosphatidyl ethanolamine. The cells grown at 20 C contained significantly larger quantities of these glycolipids and phospholipids than cells grown at 55 C.     

Fatty acid composition of glycerolipids from potato leaves

A method for rapid isolation of glyco- and phospholipids from potato leaves by a two-fold separation in a thin layer of silica gel is described. Using gas-liquid chromatography, the fatty acid compositions of monogalactosyldiglyceride, digalactosyldiglyceride, sulfolipid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol, diphosphatidyl glycerol, phosphatidic acid and non-identified lipid from potato leaves were determined. The monogalactosyl diglyceride was found to contain up to 25% of 7,10,13-hexadecatrienic acid. Trans-3-hexadecenic acid as well as phosphatidyl glycerol is a constituent component of phosphatidic acid, diphosphatidyl glycerol and the non-identified lipid.     

The effect of antioxidant-deficiency on phospholipid composition in the chick brain

Abstract— A significant increase in arachidonate was noted in the total phospholipids of brain of chicks with nutritional antioxidant-deficiency and encephalomalacia. After thin-layer chromatography of the brain lipids, this increase in arachidonate was found to be restricted to the phosphatidyl serine fraction. Significant decreases in docosahexaenoate and docosapentaenoate were noted in the phosphatidyl ethanolamine fraction. The changes in fatty acid composition of phospholipids in chick brain are comparable to those previously observed in phospholipids of skeletal muscle, liver and testes of the rat.     

Effect of phospholipids on estrogen receptor of rat uterine cytosol

Phospholipids such as phosphatidyl inositol and phosphatidyl serine inhibit the binding of R5020 and progestin receptors. The effect of phospholipids on the binding of estrogen and estrogen receptors of rat uterine cytosol was studied. Phosphatidyl choline, sphingomyelin, phosphatidyl inositol, phosphatidyl ethanolamine, cardiolipin, and phosphatidic acid inhibited the binding of estradiol and estrogen receptors. This inhibitory effect of phosphatidyl inositol and cardiolipin was dose dependent.     

Effect of phospholipids on the activity of highly-purified liver microsome cytochrome P-450 in a reaction of aniline and naphthalene hydroxylation by hydroperoxides

The effect of different phospholipids on the functional activity of highly purified cytochrome P-450 used as a co-substrate of cumene hydroperoxide was examined. At the molar ratio of phospholipids to cytochrome P-450 that was equal to 30, phosphatidyl serine, phosphatidyl inositol, the total fraction of microsomal phospholipids, and lysophosphatidyl choline increased the hydroxylation rate of aniline and naphthalene. The effect of the above phospholipids on the rate of naphthalene oxidation was much more pronounced. Phosphatidyl choline and sphingomyelin in similar large quantities did not exert a stimulating effect on the reactions studied. The kinetic parameters of aniline oxidation in the systems containing phospholipids that produced an activating effect were investigated.     

Phospholipid composition of oligodendroglial cells during normal development and in 18 day old hyperthyroid and malnourished rats.

The phospholipid composition of isolated oligodendroglial cell perikarya was studied in normal rats during development and in 18 day old malnourished and hyperthyroid rats. Phosphatidyl choline and phosphatidyl ethanolamine were found to be the major phospholipid constituents of oligodendroglial cells. Phospholipid content increased during development, mainly due to an increase of the above mentioned phospholipids. The major changes were observed in sphingomyelin, phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine between 18 and 30 days of age. The phospholipid and protein content per cell was significantly decreased in the oligodendroglial cells isolated from malnourished rats as compared to controls. When data were expressed as a function of total proteins, the composition was similar to that of normal animals. In the hyperthyroid rats on the other hand, there were no changes in the amount of phospholipids per cell, while phospholipids per milligram of total oligodendroglial cell protein were markedly decreased. The changes in myelin composition produced by hyperthyroidism that we have previously described, do not follow closely those produced by this experimental condition in oligodendroglial cells, suggesting that the metabolism of myelin might be to a certain extent, independent of that in the parent cell.     

Interrelations of matrix lipids, vesicles, and calcification.

When calcifying tissues were extracted with hot pyridine or hot benzene and then decalcified and stained with Sudan black B, the areas where mineralization was being initiated stained strongly, the rest of the calcified tissues being unstained. Histochemical methods showed that lipids were responsible for the staining. They were isolated biochemically and found to be phospholipids, very resistant to extraction before decalcification of the tissues and consisting predominantly of phosphatidyl serine and phosphatidyl inositol. It was proposed that these phospholipids were active at nucleating sites in apatite crystal formation, since it is known that phosphatidyl serine binds calcium strongly. It has been shown that phosphatidyl serine is present in matrix vesicles.