N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.     

Influence of dietary fatty acids on endocannabinoid and N-acylethanolamine levels in rat brain, liver and small intestine

Endocannabinoids and N-acylethanolamines are lipid mediators regulating a wide range of biological functions including food intake. We investigated short-term effects of feeding rats five different dietary fats (palm oil (PO), olive oil (OA), safflower oil (LA), fish oil (FO) and arachidonic acid (AA)) on tissue levels of 2-arachidonoylglycerol, anandamide, oleoylethanolamide, palmitoylethanolamide, stearoylethanolamide, linoleoylethanolamide, eicosapentaenoylethanolamide, docosahexaenoylethanolamide and tissue fatty acid composition. The LA-diet increased linoleoylethanolamide and linoleic acid in brain, jejunum and liver. The OA-diet increased brain levels of anandamide and oleoylethanolamide (not 2-arachidonoylglycerol) without changing tissue fatty acid composition. The same diet increased oleoylethanolamide in liver. All five dietary fats decreased oleoylethanolamide in jejunum without changing levels of anandamide, suggesting that dietary fat may have an orexigenic effect. The AA-diet increased anandamide and 2-arachidonoylglycerol in jejunum without effect on liver. The FO-diet decreased liver levels of all N-acylethanolamines (except eicosapentaenoylethanolamide and docosahexaenoylethanolamide) with similar changes in precursor lipids. The AA-diet and FO-diet had no effect on N-acylethanolamines, endocannabinoids or precursor lipids in brain. All N-acylethanolamines activated PPAR-alpha. In conclusion, short-term feeding of diets resembling human diets (Mediterranean diet high in monounsaturated fat, diet high in saturated fat, or diet high in polyunsaturated fat) can affect tissue levels of endocannabinoids and N-acylethanolamines.     

Purification and properties of 5,6-dihydropyrimidine amidohydrolase from calf liver

5,6-Dihydropyrimidine amidohydrolase was isolated from an acetone powder of calf liver and purified to homogeneity. Purification made use of heat treatment, ammonium sulfate fractionation and chromatography on Chelating Sepharose and DEAE-Sepharose with 44% recovery of total activity. The native enzyme has a molecular mass of 217 kDa consisting of four subunits with a molecular mass of 54 kDa each. The amidohydrolase is a metalloenzyme containing one zinc atom/subunit. The enzyme can slowly be inactivated by chelating agents. The kinetic parameters for substrates, 5,6-dihydrouracil, 5,6-dihydrothymine and glutarimide were determined. From log Vmax/KM data, a pKa of 7.6 could be calculated suggesting the formation of a zinc-bound hydroxyl ion which carries out the nucleophilic attack on the C-4 of dihydrouracil.     

Phase behavior of synthetic N-acylethanolamine phospholipids

Both saturated and unsaturated N-acylethanolamine phospholipids form lamellar structures when dispersed in buffer. The addition of excess Ca2+ (Ca2+/N-acylphosphatidylethanolamine greater than 0.5) results in precipitation. Freeze-fracture replicas indicate that the addition of Ca2+ to the unsaturated lipid results in a non-bilayer structure while the Ca2+-complex of the saturated lipid is lamellar. Since unsaturated phosphatidylethanolamine (PE) is a non-bilayer lipid, its N-acylation with a saturated fatty acid converts a non-bilayer lipid into an acidic bilayer lipid capable of interacting with Ca2+ to return to a non-bilayer structure. Ca2+ may thereby exert an influence on membrane phenomena by regulating phase behavior within certain membrane domains. Differential scanning calorimetry (DSC) indicates that N-acylation of unsaturated PE with a saturated fatty acid also results in changes in thermotropic phase behavior. Therefore, N-acylation may affect fluidity within certain membrane domains.     

Properties of phosphoprotein phosphatase from the rat liver

Prosphoproteid phosphatase, an enzyme highly specific to lysyl-tRNA-synthetase and proteins of the high-molecular-multienzymic complex of aminoacyl-tRNA-synthetases, was isolated from the rat liver. The data of electrophoresis in 4-30% PAAG with the presence of DS-Na have shown that phosphoproteid phosphatase is homogeneous and its molecular mass is 56 kDa. The isolated phosphoproteid phosphatase is activated by 2.5 mM Mg2+, Mn2+ and is inhibited by ions of univalent metals ions--200 mM Na+, 5 mM K+ as well as by 1 mM ATP, ADP, AMP.     

Properties of tyrosine aminotransferase from rat liver.

A series of sequential chromatographic procedures which yield essentially homogeneous tyrosine aminotransferase (l-tyrosine:2-oxglutarate aminotransferase, EC 2.6.1.5) from rat livers is described. Analysis of the purified enzyme indicates that its molecular weight is about 100,000, and that it consists of two subunits of identical mass and charge, each bearing one functional site for reaction with pyridoxal phosphate.     

Properties of rat liver nuclear protein kinases.

Two cyclic nucleotide-independent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) have been purified to homogeneity from rat liver nuclei. While these enzymes have many similar catalytic properties (preference for acid rather than basic proteins), they differ in molecular weight and subunit composition. Protein kinase NII will utilize ATP and GTP as phosphate donors while protein kinase NI will only effectively use ATP. Both enzymes reveal an unusual activation by Fe2+.     

Purification and some properties of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

Human liver 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was purified 17 500-fold to apparent homogeneity as judged from polyacrylamide-gel disc electrophoresis. A pH optimum of 7.7-9.0 was found. The Km value was pH- and temperature-dependent. At 37 degrees C and pH 7.7, Km was 0.16 mM and it increased to 0.29 at pH 6.0 and 0.23 at pH 9.0. At 25 degrees C and pH 7.7, a Km value of 0.99 mM was obtained. When the substrate concentration was varied, apparent Michaelis-Menten kinetics were obtained. p-Hydroxymercuribenzoate, glutathione or cysteine had no effect on the enzyme activity; 5 mM-N-acetylcysteine inhibited about 47% of the total enzyme activity. Apart from Cu2+, other bivalent ions were virtually ineffective at 1 mM. The kinetic study differentiates this enzyme from aspartylglucosylaminase from other sources.     

Effect of different compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

The effect of varous compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was studied. N-Acetylcysteine inhibited the nezyme non-competitively (Ki 3.2 mM), whereas 3-hydroxybutanone inhibited competitively (Ki 4.1 mM). Methionine, isoleucine and cystathionine apparently enhanced the enzyme activity. The enzyme had a mol. wt. of 63000 as determined by gel filtration. The present studies differentiate between the aspartylglucosylaminase from human liver and that obtained from various other sources.     

Concurrent disappearance of N-acylethanolamine glycerophospholipids and phagolysosomes enriched in N-acylethanolamine glycerophospholipids as Dictyostelium discoideum cells aggregate

As the cellular slime mold, Dictyostelium discoideum, undergoes development, a phospholipid fraction containing 80% N-acylethanolamine glycerophospholipids (NAEGPs) and 20% acylphosphatidylglycerol (APG) disappears during the aggregation stage. In this study, the subcellular distribution of that NAEGP phospholipid fraction and the precise time period of disappearance of the fraction were determined. The content of the NAEGP fraction was determined in aggregating cells at 2-h intervals from the beginning of the developmental phase through 14 h, when the cells were completely aggregated. The NAEGP fraction comprised about 8% of the phospholipids in amoebae just starting the development cycle and about 12% in cells between 2 and 6 h of development; then its level decreased until it could not be detected at 12 and 14 h of development. The mole percentage of the total lipid phosphate in the NAEGP fraction was determined in isolated subcellular organelles. The phagolysosomes were enriched in the NAEGP fraction 1.7-2-fold over the level found in the amoebae and about 8-fold over the level in fractions highly enriched in the plasma membrane, mitochondria or peroxisomes. The content of phagolysosomes was determined by electron microscopy of aggregating cells. The amoebae contained large amounts of phagolysosomes up to 6 h of development, and then they gradually disappeared between 6 and 12 h of development. This combination of quantitative phospholipid analysis, subcellular organelle isolation and electron microscopy has revealed that in D. discoideum amoebae, the phagolysosomes were selectively enriched in the NAEGP fraction and both the NAEGP-enriched phagolysosomes and the NAEGPs disappeared concurrently between 6 and 12 h of development.     

Properties of rat liver signal peptidase reconstituted into liposomes

EDTA/KCl- or pyrophosphate-treated rough microsomes of rat liver clearly showed the co-translational cleavage of pre-human placental lactogen and translocation of the product into membrane vesicles. The signal peptidase fraction was isolated by chromatography on Sephacryl S-300 of deoxycholate-treated membranes and reconstituted into liposomes by dialysis or by the Biobeads SM-2 method. Assay of the signal peptidase activity was performed with pre-human placental lactogen synthesized by the reticulocyte lysate system programmed with human placental lactogen mRNA. The signal peptidase reconstituted into liposomes showed stable activity over the temperature range of 0 to 45 degrees C; in contrast, the detergent-solubilized signal peptidase of dog pancreatic membranes was completely inactivated at the unusually low temperature of 37 degrees C. It was shown that this inactivation was due to the presence of detergent. Signal peptidase from rat liver was insensitive to a variety of protease inhibitors, like the enzyme from dog pancreas, but differed from the latter in being inhibited by chymostatin and TPCK.     

Properties of NADH-dependent estradiol 2-hydroxylase in rat liver

The NADH-supported cytochrome P-450-dependent 2-hydroxylation of estradiol in rat liver microsomes has been investigated. Estradiol 2-hydroxylation proceeded well with NADH instead of NADPH as a cofactor. Dimethyltetrahydropterine was incapable of serving as a hydrogen donor for this biotrans-formation. When both NADH and NADPH were used, the 2-hydroxylation increased additively. Molecular oxygen dissolved in the incubation medium was enough for the occurrence of the NADH-dependent 2-hydroxylation. The presence of carbon monooxide suppressed the formation of catechol estrogen where the CO/O₂ ratio needed for 50% inhibition of the bioconversion was 7.7. The inhibitory effect was reversed completely by illumination with white light. p-Chloromercuribenzoate inhibited almost completely the 2-hydroxylase activity, and the enzyme activity was also inhibited by SK.F-525A. These results strongly imply the possible involvement of a cytochrome P-450 system in the NADH-dependent 2-hydroxylation of estradiol with rat liver microsomes.     

Properties of peroxisomal 3-ketoacyl-coA thiolase from rat liver

Peroxisomal 3-ketoacyl-CoA thiolase has a molecular weight of 89,000 and consists of 2 polypeptide chains of identical size. The enzyme has no interchain disulfide bonds and is reversibly dissociated to an inactive monomer in the cold. Mitochondrial 3-ketoacyl-CoA thiolase and acetoacetyl-CoA specific thiolase have molecular weights of 154,000 and 149,000, respectively. They each consist of 4 polypeptide chains of identical size. Peroxisomal thiolase and mitochondrial 3-ketoacyl-CoA thiolase operate by a ping-pong mechanism. The catalytic properties, including substrate specificity, of the peroxisomal enzyme were compared to those of mitochondrial 3-ketoacyl-CoA thiolase.     

Properties of rat liver microsomal stearoyl-coenzyme A desaturase.

1. Rat liver microsomal stearoyl-CoA desaturase activity was shown to be stimulated by both bovine serum albumin and a basic cytoplasmic protein from rat liver. 2. Partially purified desaturase is unaffected by either of these two proteins. 3. Bovine serum albumin appears to exert its effect on the crude system by protecting the desaturase substrate, stearoly-CoA, from the action of endogenous thiolesterases. 4. By using partially purified enzyme preparations, it was possible to establish the substate specificity of the delta9-fatty acyl-CoA desaturase with the C14, C15, C16, C17, C18 and C19 fatty acyl-CoA substrates. Maximum enzyme activity was shown with stearoyl-CoA decreasing with both palmitoyl-CoA and nonadecanoyl-CoA, as reported previously for free fatty acids. 5. Both cytochrome b5 and NADH-cytochrome b5 reductase (EC 1.6.2.2) are required for these studies and a method is described for the purification of homogeneous preparations of detergent-isolated cytochrome b5 from rat liver. 6. From amino acid analyses, a comparison was made of the hydrophobicity of the membrane portion of cytochrome b5 with the hydrophobicity reported for stearoyl-CoA desaturase. The close resemblance of the two values suggested that unlike cytochrome b5 and its reductase, the stearoyl-CoA desaturase may be largely buried in the endoplasmic reticulum.