Phospholipase D and phosphatidate phosphohydrolase activities in rat cerebellum during aging

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAG are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAG by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4-(adult),28-, and 33-mon-old (aged) rats. Pl-4,5-bisphosphonate (PIP₂)-dependent PLD, PAP1, and DGL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP₂-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DGL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP₂-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms.     

Difficulties in the assay of phosphatidate phosphohydrolase activity. Influence of ionic strength,detergent, and selection of substrate

In the present paper, problems in connection with assay of the activity of magnesium-dependent rat liver phosphatidate phosphohydrolase (PAP) are discussed. PAP activity is usually measured by following the production of diacylglycerol or inorganic phosphate from the substrate phosphatidate. These two methods may give widely different results due to a number of factors that may affect the assay. One such factor is the composition of the substrate. Higher apparent enzyme activity was observed with dioleoyl-phosphatidate than with dipalmitoyl-phosphatidate. This substrate-dependent difference in apparent PAP activity was 2-2.5-fold in the absence and 10-fold in the presence of Triton X-100, respectively. Triton X-100 reduced the activity as measured with the dipalmitoyl-phosphatidate substrate. In contrast, the activity of PAP as measured with dioleoyl-phosphatidate was stimulated by Triton X-100 The stimulatory effect of Triton was reduced or abolished when the ionic strength in the assay mixture was increased. Assays based on³²P-labeled substrate are rapid and sensitive. It is shown here that³³P can be used as an alternative. This radionuclide has a longer half-life and also emits particles with lower energy, thus posing less potential health hazards for the user.     

Dietary sunflower oil reduces plasma and liver triacylglycerols in fasting rats and is associated with decreased liver microsomal phosphatidate phosphohydrolase activity

Plasma and liver lipids were studied in male weanling rats fed diets containing moderate levels of fat (6% by weight) as sunflower oil (SF diet, rich in linoleic acid), salmon oil (SM diet, rich in long-chain n-3 fatty acids), or a blend of peanut and rapeseed oil (PR diet, rich in oleic acid). After nine weeks of feeding, the fasting plasma cholesterol concentrations were 49 and 24% lower in groups SM and SF, respectively, as compared to group PR. Both dietary salmon oil and sunflower oil lowered the tricylglycerol concentration of plasma and liver but, unexpectedly, the response was higher with sunflower oil. Indeed, in group SM the values were 15 and 30% lower in plasma and liver, whereas in group SF, they were 24 and 53% lower, respectively. As compared to group PR, liver triacylglycerols and microsomes contained 2.5- and 2.3-fold less oleic acid, respectively, in group SF, and they were 9.2- and 3.2-fold enriched in n-3 fatty acids, respectively, in group SM. The liver triacylglycerol concentrations were correlated with changes in the microsomal Mg²⁺-dependent phosphatidate phosphohydrolase activity (r=0.47,P<0.01). As oleic acid, unlike long-chain n-3 fatty acids, is considered to promote the triacylglycerol synthesis and secretion, our findings suggest that changes in the membrane fatty acid composition could affect the triacylglycerol content of liver and plasma. Moreover, the availability within the liver, of oleic acid, predominantly incorporated into triacylglycerols, might limit the triacylglycerol production in SF-fed rats.     

Effect of high fat/high erucic acid diet on phosphatidate synthesis and phosphatidate phosphatase in the subcellular fractions of rat heart and liver

Rats of weaning age were fed for a period of 1,3 or 6 weeks either a control diet (laboratory stock diet) or a semisynthetic diet containing 20% by weight of either mustard seed oil (1/3 of the total fatty acids were comprised of erucic acid) or corn oil (2/3 of the total fatty acids consisted of linoleic acid). Mitochondrial and microsomal fractions were isolated from the hearts and livers of these rats, and the rate of acylation ofsn-[U-¹⁴C] glycerol 3-phosphate (P) was examined using palmitoyl-CoA or erucoyl-CoA as the acyl donor. In addition, activities of phosphatidate phosphatase of the mitochondrial, microsomal and soluble fractions were assayed. Studies on the acylation of glycerol 3-P with palmitoyl-CoA demonstrated that feeding of the high fat/high erucic acid diet for 1,3 or 6 weeks significantly increased the rate of formation of monoacylglycerol 3-P by the cardiac subcellular fractions as compared to the control. The rate of formation of diacylglycerol 3-P also increased but to a lesser degree. Feeding the high fat/high linoleic acid diet tended to increase acylation of glycerol 3-P by cardiac subcellular fractions. However, neither high fat diet influenced acyltransferase activities of the hepatic subcellular fractions or phosphatase activities of the cardiac and hepatic fractions. Studies on the acylation of glycerol 3-P with erucoyl-CoA demonstrated that the rate of acylation was ca. 1/10 that measured using palmitoyl-CoA in all experiments; in particular, the formation of diacylglycerol 3-P was extremely slow, suggesting that erucoyl-CoA is an unsuitable substrate for the position-2 of the monoacylglycerol 3-P. The rate of acylation by the cardiac and hepatic subcellular fractions was not influenced by the feeding of the high-fat diets. The rate of glycerol 3-P acylation by both cardiac and hepatic mitochondrial fraction was ca. 2/3 of the rate of acylation by the respective microsomal fraction. In addition, the ratio of monoacyl-to diacylglycerol 3-P synthesized by the mitochondrial fraction was smaller than that by the microsomal fraction. These results suggest that acylation of glycerol 3-P by the mitochondrial cannot be attributed to the action of the contaminating microsomal enzymes.     

Relationship between translocation of long-chain acyl-CoA hydrolase,phosphatidate phosphohydrolase and CTP:Phosphocholine cytidylyltransferase and the synthesis of triglycerides and phosphatidylcholine in rat liver

Translocation of long-chain acyl-coenzyme A hydrolase from the microsomal fraction to the cytosolic fraction was promoted in cell-free extracts of rat liver by palmitic acid, oleic acid, tetradecylthioacetic acid, and tetradecylthiopropionic acid, and by their CoA esters. The CoA esters were more effective than the non-esterified acids in the translocation of the enzyme. Treatment of normolipidemic rats with sulfur-substituted non-β-oxidizable fatty acid analogues resulted in a transitory increase in hepatic concentration of long-chain acyl-CoA. Longer feeding times almost normalized the hepatic long-chain acyl-CoA content. Microsomal long-chain acyl-CoA hydrolase activity was inhibited, whereas the activity of the cytosolic form was stimulated. The rise in enzyme activity coincided with a reduction in liver content of triglyceride and an increase in hepatic phospholipid content. The results suggest that the activity of long-chain acyl-CoA hydrolase in the cytosol may control the amount of acyl-CoA thioesters in the liver. Esterified and non-esterified fatty acids causedin vitro translocation of phosphatidate phosphohydrolase and cytidine 5′-triphosphate (CTP):phosphocholine cytidylyltransferase from the cytosolic fraction to the microsomal fraction. However, the translocation of these two enzyme systems was not obtainedin vivo. The activity of phosphatidate phosphohydrolase decreased in microsomal and cytosolic fractions while the activity of cytidylyltransferase in these fractions increased. The activities of soluble phosphatidate phosphohydrolase and long-chain acyl-CoA hydrolase appeared to be inversely correlated. The results imply that in cytoplasm, long-chain acyl-CoA hydrolase may compete with the biosynthetic enzymes for the acyl-CoA substrate, thus influencing the rate of lipid synthesis. The reduced hepatic triglyceride content observed in tetradecylthioacetic acid-treated rats is probably due to reduced triglyceride synthesis, which is mediated by an inhibition of phosphatidate phosphohydrolase accompanied with translocation and stimulation of long-chain acyl-CoA hydrolase. Development of fatty liver as an effect of tetradecylthiopropionic acid is probably due to accelerated triglyceride biosynthesis, which is mediated by a stimulation of phosphatidate phosphohydrolase and a decrease in cytosolic palmitoyl-CoA hydrolase activity.     

Distribution of individual fatty acids in the crystallization fractions of lard

Distribution of the individual fatty acids in the triglycerides of lard was determined by fractional crystallization, partial enzymatic hydrolysis with steapsin, and fatty acid analyses by GLC. It was found that none of the individual fatty acids corresponded to a random distribution in the crystallization fractions, but that the distribution of the total saturated and total unsaturated acids was very nearly random. The short chain fatty acids, C₁₄ and C₁₆, both saturated and unsaturated, were found to be more predominant in the 2-position than in the 1- and 3-positions of the lard triglycerides. All of the C₁₈ fatty acids were found to be more predominant in the 1- and 3-positions.     

Pulmonary surfactant: Distribution of lipids,proteins and surface activity in ultracentrifugation of rabbit pulmonary washing and derived fractions

Lavage from normal adult rabbit lung and two known derived fractions, Fraction T and Fraction S, were subjected to either differential ultracentrifugation in 1.090 g/ml KBr or sucrose density gradient ultracentrifugation; the surface activity of the lipid extract of selected fractions was measured. In differential ultracentrifugation, the three starting materials yielded a pellicle containing > 85% of the phospholipid with <1% protein. In sucrose density gradient ultracentrifugation: pulmonary washing, containing about equal weights of phospholipid and protein (60% albumin, 20% sIgA, 10% IgG, 10% minor proteins), produced one single band at density 1.040, containing virtually one single protein, namely >80% of the total sIgA (protein T) and up to 60% of the total phospholipid, whereas all the albumin and IgG were found at very low densities, 1.010 and 1.025, respectively; Fraction T, having nearly equal weights of one signle protein, sIgA, and phospholipid, produced two contiguous bands at densities 1.059 and 1.078, totalling >85% of its phospholipid and <25% of its protein, the balance of which was found free of phospholipid at densities 1.020 to 1.050; comprising >80% of the phospholipid and <20% of the protein of pulmonary washing, Fraction S yielded two small bands at densities 1.028 and 1.044 and a major band at d=1.059. In surface activity measurements: when the total lipid extract of the bands from the sucrose density gradient ultracentrifugation was spread as a film, in spite of similarly high dipalmitoyl lecithin contents (about 70% palmitoyl lecithin contents (about 70% palmitoyl residue), the lipid of the band of Fraction T and that of the high density band of Fraction S were very active (γ _min=0); whereas the lipid of the band of pulmonary washing and that of the lowest density band of Fraction S were not active,γ _min being 18 dyne/cm and 21 dyne/cm, respectively. This wokk brings forth three major conclusions. First, under conditions which are used to isolate serum lipoproteins, no lipoprotein was obtained from either of the three surfactant fractions and most of the lipid was found virtually free of protein. Second, the isopicnic equilibrium of a given ultracentrifugation fraction varied with the molecular structure of its constituents and could not be accounted for by the latter’s average densities; instead, major roles must be player by particle geometry and their water contents. Third, although the various lipid samples contained the same quantities of palmitoyl residues (70%), the surface activities varied with the physical state of the lipid, method of assay, and some other undefined factors.     

Ozone-induced alterations of lamellar body lipid and protein during alveolar injury and repair

Alveolar Type II cells in the rat respond to severe, acute ozone injury (3 ppm ozone for eight hours) by increasing their intracellular pool of surfactant; however, the newly stored surfactant is abnormal in composition. Lamellar bodies isolated between 24 and 96 hours after ozone exposure contained significantly more cholesterol in relation to phosphatidylcholine than did controls. By contrast, the cholesterol content of surfactant isolated from alveolar lavage remained unchanged throughout an 8-day post-ozone period. The total protein content of lamellar bodies in relation to phosphatidylcholine was significantly decreased at 24 and 48 hours post-ozone. Analysis of lamellar body proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the amount of a 14 kDa proteolipid was greatly reduced at the end of the eight-hour ozone exposure and remained low for at least 48 hours. This proteolipid appeared to be a specific lamellar body component since it was not detected in extracellular surfactant. The findings indicate that oxidative alveolar stress initiates characteristic alterations in both lipid and protein constituents of stored surfactant, without perturbation in the composition of extracellular surfactant.     

Distribution of ubiquinone and ubiquinol homologues in rat tissues and subcellular fractions

The oxidized (UQ_ox) and reduced (UQ_red) forms of ubiquinone (UQ) homologues in rat tissues and subcellular fractions were analyzed to elucidate their distribution and physiological role. UQ-9 and UQ-10 were detected in all tissues studied, and UQ-9 was the predominant homologue. The total amount of UQ_ox-10 and UQ_red-10 was 20–50% that of UQ_ox-9 and UQ_red-9. The levels of these homologues were highest in heart with lesser amounts occurring in kidney, liver and other organs. In liver and blood plasma, the UQ_red homologue amounted to 70–80% of the total UQ (UQ_ox+UQ_red=t-UQ). UQ_red was less than 30% of t-UQ in other tissues and blood cells. t-UQ was much higher in leukocytes and platelets in blood than in erythrocytes. In erythrocytes, t-UQ was exclusively located in the cell membranes. UQ_ox and UQ_red were also found in all subcellular fractions isolated from liver and kidney in about the same ratio as UQ_red/t-UQ was present in the whole organ. The levels of UQ_ox and UQ_red per mg protein in subcellular fractions from liver were highest in mitochondria, with lesser amounts present in plasma membranes, lysosomes, Golgi complex, nuclei, microsomes and cytosol. In the mitochondria, the outer membranes were richer in t-UQ than the inner membranes. In the Golgi complex, the light and intermediate fractions were rich in t-UQ when compared to the heavy fraction. The possible physiological role of UQ_ox and UQ_red in tissues and subcellular fractions is discussed.     

Distribution of cholesteryl esters and other lipid in subcellular fractions of the adrenal gland of the pig

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.     

Analysis of FA contents in individual lipid fractions from human placental tissue

Data on FA contents in the human placenta are limited. Different methods have been used for the FA analysis, and only percentage results have been presented. We developed and evaluated a method for the determination of FA concentrations in placental tissue. Lipids were extracted from placental tissue with a chloroform/methanol mixture; and phospholipids (PL), nonesterified FA (NEFA), TG, and cholesterol esters (CE) were isolated by TLC. Individual lipid fractions were derivatized with methanolic hydrochloric acid, and the FAME, were quantified by GC with FID. The CV of intra-assay (n=8) of absolute concentrations were evaluated for FA showing a, tissue content >0.01 mg/g. CV ranges were 4.6–11.0% for PL, 6.4–9.3% for NEFA, 6.1–8.9% for TG, and 11.4–16.3% for CE. The relative FA composition across a term placenta indicated no differences between samples of central and peripheral locations of maternal and fetal site (CV 0.5–9.9%), whereas the absolute FA concentrations were only reproducible in the PL fraction (CV 7.0–12.8%). The method shows a reasonably high precision that is well suited for physiological and nutritional studies.     

Distribution of aflatoxins in various fractions separated from raw peanuts and defatted peanut meal

The present investigation is the first definitive study of the distribution of aflatoxins in a wet-milling process of raw peanuts. The results show that the majority of the aflatoxins originally present in the peanuts remained in the solid fractions, particularly the protein fraction, during wet-milling. In the protein concentrate preparation, the concentrates carried 81–89% of the total toxin; crude oil, 5–8%; and whey fraction, 3–14%. In the case of protein isolate preparation, 51–56% of the total toxin remained with the isolates, 22–26% with the residue, 11–17% with the whey, and 7–8% with the crude oil. Distribution of aflatoxins in the preparation of protein isolates from defatted peanut meal showed that 55–65% of the total toxin originally present in the meal remained with the protein isolates, 20–28% with the residue, and 10–20% with the whey fraction. Changes in extraction pHs for the preparation of protein isolates either from raw peanuts or defatted meal did not alter the distribution pattern mentioned above. A new approach based upon charge-transfer (electron acceptor-donor) complex formation is suggested to shift this aflatoxin distribution from protein products to disposable whey or residue fraction during the processing of raw peanuts and defatted meal for protein products.     

Desaturation of endogenous and exogenous palmitate in lung tissue in vitro

Lung slices from rats fed a fat-free diet supplemented with safflower oil (control) or tripalmitoyl-glycerol (essential fatty acid [EFA]-deficient) were incubated with [¹⁴C] acetate, [¹⁴C] palmitate, or [¹⁴C] stearate. Of the¹⁴C recovered in phospholipids after incubation with [¹⁴C] acetate, more than 87% was in 16-carbon fatty acids. Desaturation, as assayed by the percentage of radioactivity in monoenoates in phospholipid fatty acids, was generally double in EFA-deficient slices compared to control slices, regardless of substrate. Desaturation was significantly greater in slices incubated with acetate or octanoate compared to palmitate, indicating that endogenously synthesized palmitate was desaturated more actively than that derived from an exogenous source. Presented in part to the American Physiological Society, Toronto, Canada, October 1980, and published in abstract form inPhysiologist (1980) 23, 135.     

Distribution of mutagenic activity in petroleum and petroleum substitutes

Several petroleum crude oils and shale- and coal-derived petroleum substitutes have been fractionated by chemical class and the fractions have been tested for mutagenic activity. Crude petroleum substitutes tend to exhibit greater mutagenicities than do petroleum crude oils but the mutagenicity is reduced to comparable levels in low-boiling distillates and samples which have been hydrotreated. The mutagenicity is caused by alkaline and neutral constituents of the petroleum substitutes, but only neutral constituents for the petroleum crudes. The mutagenic components constitute less than ten per cent of the mass of the samples.     

Stearoyl-CoA desaturase activity in adipose tissue and liver of the cardiomyopathic hamster

Stearoyl-CoA desaturase activity and the fatty acid composition of lipids of adipose tissue and liver were determined in 35- and 180-day-old cardiomyopathic hamsters and age-matched normal controls. Enzyme activity was unchanged in the adipose tissue of 35-day-old animals but was significantly depressed in the 180-day-old cardiomyopathic hamsters. In the liver, stearoyl-CoA desaturase activity was significantly lower in the 35-day-old disease animals but was unchanged in the 180-day-old animals. The analysis of the fatty acid composition of the lipids isolated from adipose tissue showed an increase in the relative percentage of saturated fatty acids accompanied by a decrease in the relative percentage of unsaturated fatty acids in both age groups of the cardiomyopathic hamsters. However, linoleic acid content was increased in the diseased animals. Similar changes in fatty acid composition of lipids from the livers of 35-day-old cardiomyopathic hamsters were observed, but no significant differences in the fatty acid composition between 180-day-old cardiomyopathic hamsters and normal controls were observed. The changes in the fatty acid composition appear to be related to the observed changes in desaturase activity. In is concluded that such changes in desaturase activity and fatty acid composition could affect the normal structure and functions of membranes and membrane-related processes.     

芪类化合物的分布和生理活性

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Molecular, thermal and morphological characterization of narrowly branched fractions of 1-octene LLDPE: 2. Study of the lamellar morphology by transmission electron microscopy

The lamellar morphology of a linear low density ethylene/1-octene copolymer (LLDPE A), and its fractions with short chain branching contents ranging between 3 and 28 branches per 1000 C atoms, has been investigated using transmission electron microscopy. The weight average molecular weight of the samples studied decreases from 2.7 × 10⁵ at the lowest branching content down to 4.9 × 10⁴ at the highest branching content. The branching content is the major factor determining the lamellar thickness, while the morphological structure of the lamellae depends both on the branching content and the molecular weight. In contrast to the unfractionated copolymer LLDPE A, lamellar stacks were observed in all fractions.