Kinetic studies of alkyl-dihydroxyacetone-phosphate (alkyl-glycerone-phosphate) synthase in peroxisomes of rat liver.

The properties of peroxisomal enzyme alkylglycerone-phosphate synthase were studied in highly purified peroxisome fractions of rat liver. The requirements for optimal enzyme activity: pH and composition of the reaction mixture, incubation time, and enzyme concentration were investigated, and kinetic studies performed employing both different long-chain fatty alcohols and acyl dihydroxyacetone phosphates as substrates. Activities of the synthase considerably higher as reported before were found in the peroxisome preparation, with alkylglycerone (alkyldihydroxyacetone) phosphate as the sole product of the exchange reaction. The kinetic studies revealed divergent properties of peroxisomal synthase with respect to the substrates involved. Whereas the substrate concentration versus reaction velocity plot for the fatty alcohols reflects Michaelis-Menten kinetic behavior, it displays a maximum followed by inhibition with regard to the acylglycerone phosphate. The enzyme accepts different acylglycerone phosphates without much specificity but it is most active with 9-cis-octadecenol.

Buoyant density and lipid composition of purified myelin of aging human brain.

Purified myelin of human brain from 15 young adult (below 50 years of age) and old (above 70 years of age) autopsy cases each was examined by isopycnic centrifugation in continuous sucrose gradients, and for lipid composition. The mean buoyant density of myelin was the same in both groups. Apparent features of old age were a wide range of density values, less compact myelin bands, and the dissociation of myelin into two bands in six of 15 old cases. Lipid analyses of randomly selected myelin samples of each group revealed an inverse relationship between the total lipid to protein ratio and density of myelin. In old age total lipids decreased by an average 10 mol lipid per mol protein. This decrease was accounted for by cholesterol, phosphatidylserine and cerebrosides. Changes in fatty acid moieties mainly affected sphingolipids. C20:0 and C24:0 of sphingomyelin increased, as did even more markedly the more hydrophilic OH-fatty acids of cerebrosides. Correlations with buoyant density existed for the ratios of cholesterol to protein in young adult cases, and those of galactolipids to protein in old cases. The results suggest that old age is associated with impaired stability and altered lipid composition of myelin.

Lipid changes of astrocytes from mouse cerebellum cultured in lipid-free chemically defined medium and in serum-supplemented medium.

Astrocytes of mouse cerebellum were grown in chemically defined medium for 14 days, subcultivated and grown in serum-containing medium for up to 14 days. After 14 days in defined medium the cells showed fatty degeneration from which they recovered histologically and chemically after one week in serum-supplemented medium. At this point 99% of the cells were glial fibrillary acidic protein-positive and synthesized phosphatidylinositol, phosphatidylserine and bis(monoacylglycero)phosphate besides other phospholipids.

Distribution of alkylglycerone-phosphate synthase in subcellular fractions of rat liver.

Subcellular fractions of rat liver were isolated by density-gradient centrifugation on a linear Metrizamide gradient and were assayed for marker enzymes of peroxisomes, lysosomes, microsomes and mitochondria. Alkylglycerone-phosphate synthase catalysing the formation of the ether bond in glycerolipids was also determined along the gradient. The enzyme was found to be enriched in the peroxisomal and the microsomal fractions thus, displaying a bimodal distribution pattern. Two reaction-products each, alkylglycerone phosphate and alkylglycerone were obtained in the enzymic assays performed, the ratio of which was clearly dependent upon the fraction employed. Alkylglycerone phosphate was mainly synthesized by the 'peroxisomal synthase', whereas an inverse proportion was observed assaying the microsomal counterpart. Furthermore, comparing the mean specific activities of both the enzymes the microsomal one was shown to be roughly twice as active in metabolizing 1-O-palmitoylglycerone 3-phosphate, simultaneously displaying a somewhat different sensitivity to NaF. These findings provide a first line of evidence, that two separate synthases, one in microsomes and another one in peroxisomes might be engaged in the biosynthesis of 1-O-alkyl-glycerolipids in rat liver.

Studies on the phase transitions of lysoderivatives of ethanolamine glycerophospholipids from human brain.

The phase transition temperature of 1,2-distearoylglycerophosphocholine is reduced in presence of equimolar amounts of 1-O-(1'-alkenyl)-glycerophosphoethanolamine (ethanolamine lysoplasmalogen) from 53.3 degrees C-54.1 degrees C to 44.0 degrees C-44.9 degrees C at different pH (4.0; 7.2; 9.0; 10.5). 1-Acyl-glycerophosphoethanolamine leads to a smaller reduction of the 1,2-distearoyl-glycerophosphocholine transition temperature: 45.0 degrees C-46.2 degrees C at the same pH-values. 1-Alkyl-glycerophosphoethanolamine (hydrogenated ethanolamine lysoplasmalogen) possesses a transition temperature, which is 3.3 degrees C-4.9 degrees C higher than the hydrogenated 1-acyl-glycerophosphoethanolamine at each pH investigated. At pH 9.0 and, more pronounced, at pH 10.5 we find a reduction of the transition temperature for both these substances, whereas their transition temperature is nearly unchanged at pH 4.0 and 7.2. Our results clearly show that the ether-bonding in the lysoderivative of plasmalogen is responsible for the closer packing compared to the 1-acyl-glycerophosphoethanolamine.

Changes of transition temperatures of phosphatidylcholine and phosphatidylglycerol in presence of amphiphilic drugs.

The transition temperature of phosphatidylglycerol (PG) was reduced to lower temperatures in presence of propranolol, imipramine, amitriptyline and chlorpromazine. This effect was dependent on drug concentration and was smallest with propranolol. The fluidizing effect, however, increased from propranolol to chlorpromazine according to the octanol/H2O partition coefficients. When the two phospholipids PG and phosphatidylcholine (PC) were compared, the presence of drug lead to a more pronounced reduction of the transition temperature in the case of the acidic phospholipid than in the case of the neutral one.

Amphiphilic cationic drugs and phospholipids influence the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fraction of untreated rats.

The influence of amphiphilic drugs and phospholipids on the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fractions of untreated rats, isolated by affinity chromatography using castor bean lectins, was studied in vitro. Chloroquine (93 microM) inhibited beta-galactosidase activity by about 30%, while O,O'-bis(diethylaminoethyl)hexestrol showed no inhibitory effect. Neutral phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) inhibited the enzyme slightly, while the enzyme activity was drastically reduced in the presence of acidic phospholipids (phosphatidylinositol, phosphatidylserine, bis-(monoacylglycero)phosphate). Lysosomal beta-glucosidase was strongly inhibited by chloroquine and O,O'-bis(diethylaminoethyl)-hexestrol. The neutral phospholipids showed only a moderate inhibitory effect, whereas the acidic phospholipids were stimulators. Bis(monoacylglycero)phosphate was by far the best stimulating compound.

Alkenylhydrolase: a microsomal enzyme activity in rat brain.

Rat brain microsomes have the capacity to liberate radioactive free aldehydes from 1-[1-14C]alk-1'-enyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen). Glycerophosphoethanolamine was found using 1-alk-1'-enyl-sn-glycero-3-phospho-[3H]ethanolamine. The ratio of both products released by lysoplasmalogenase action was 1:1. Another enzymic activity could be demonstrated, which hydrolyzes lysoplasmalogen at the hydrophilic part of the molecule, a lysophospholipid phosphodiesterase. Thus, 1-[1-14C]alk-1'-enylglycerol was detected as well as [3H]ethanolamine, again in a molar ratio, from the respective labeled substrates. This enzyme possesses nearly the same affinity toward the substrate as lysoplasmalogenase. Whereas the lysophospholipid phosphodiesterase is totally inhibited in the presence of NaF or EDTA, lysoplasmalogenase activity is not affected by these reagents. 1-[1-14C]Alk-1'-enylglycerol acts also as substrate for lysoplasmalogenase, which liberates radioactive aldehydes at the same rate as from lysoplasmalogen. Because the apparent Km and Vmax values are nearly identical for both substrates, the enzyme activities are inhibited in the same way, and the pH optimum is about 7.2 in both cases, it is concluded that both substrates were attacked by the same enzyme. The enzyme does not differentiate between a substrate substituted at the sn-3 position of glycerol and one that is not. It requires only a free OH group at the sn-2 position. Phosphoethanolamine phosphatase activity was also determined under our experimental conditions.

The isolation of lysosomes from normal rat liver by affinity chromatography.

Lysosomes from normal rat liver were isolated by affinity chromatography using Sepharose-bound Ricinus communis agglutinins I + II. Characterization of the lysosomal fraction by marker enzymes showed--compared with the homogenate--an enrichment in: acid phosphatase and arylsulfatase about 30- to 60-fold, the tartrate-sensitive acid phosphatase about 95-fold, whereas beta-D-glucosidase, beta-D-galactosidase and sphingomyelinase showed a much higher enrichment of 170- to 260-fold. Marker enzymes for other cell organelles were not detectable. The phospholipid pattern and optical control with electron microscopy gave further indications that the isolated fractions were very rich in lysosomes. A comparison of the phospholipid compositions of plasma membranes isolated from normal rat liver and membranes from the isolated fractions of lysosomes, showed that they were quite different; in particular bis(monoacylglycero)phosphate, which we found to be a typical lysosomal phospholipid, was absent in plasma membranes.

Studies on lipids from liver and spleen of a child (O.L.) with Niemann-Pick's disease type C.

Large accumulations of sphingomyelin and bis(monoacylglycero)phosphate were found in the spleen of a child, who died from a lipidosis (Morbus Niemann-Pick). The enrichment of both phospholipids in this organ was more extensive than in liver. Moreover: bis(monoacylglycero)phosphate, the lipid characteristic for lysosomes, had a remarkably high content of oleic acid: 76% in spleen and 63% in liver.

Effect of chloroquine and O,O'-bis(diethylaminoethyl)hexestrol on acidic phospholipid membranes.

The amphiphilic drugs chloroquine and O,O'-bis(diethylaminoethyl)hexestrol are able to form complexes with the acidic-phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol. The dissociation constants of the complexes with chloroquine are independent of pH in the range investigated here (4--7) as well as of temperature (4 degrees C--40 degrees C). The phase transition temperature of phospholipid is markedly reduced by both drugs, the effect is reversible by addition of Ca2.

On the phospholipid metabolism of glial cell primary cultures. II. Metabolism of 1-alkyl-glycerophosphoethanolamine during time course.

Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-3H] alkyl-sn-glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1-20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmalogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl-compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.

On the phospholipid metabolism of glial cell primary cultures. III. Utilization of 1-alkenyl-glycerophosphoethanolamine (lysoplasmalogen).

Primary cultures prepared from newborn rat brain, consisted after 16 or 17 days mainly of astrocytes and of oligodendrocytes. 1-Alkenyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) was used as substrate for studies on the metabolism of ethanolamine-glycerophospholipids. After 3 hr incubation two main products were observed: a) 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (= ethanolamine plasmalogen) and b) 1-alkenyl-2-acyl-sn-glycero-3-phosphocholine (= choline plasmalogen). The acylation rate reached saturation at about 10 nmol substrate/mg cell protein with a Vmax of 30 nmol x mg cell protein-1 x 3 hr-1. This acylated compound amounted to almost 60% of all radioactivity internalized, whereas the second product, choline plasmalogen, came to 20%. Unchanged substrate was found within the cells only in small amounts, even at maximum substrate internalization. These results were discussed in comparison with those obtained with 1-alkyl-sn-glycero-3-phosphoethanolamine under the same conditions (25).

Lysoplasmalogenase--a microsomal enzyme from rat brain.

An enzymic activity of rat brain that liberates radioactive free aldehydes from 1-[1-14C]alk-1'-enyl-sn-glycero-3-phosphoethanolamine (lyso-plasmalogen) is described. It was present mainly in microsomal fractions (crude) of brains of rats of different ages. The highest specific enzyme activity was found in 21-day-old animals. The formation of free aldehyde was dependent on the amount of enzyme protein as well as the amount of substrate added, and was linear to the incubation time up to 60 min. The pH optimum was between 7.1 and 7.3. Bivalent cations (Mg2+, Ca2+) and detergents inhibited the reaction. However, the same cell fractions as well as extracts of acetone-dried powder of brain from young or old rats possessed no enzyme activity for liberating the aldehyde from the acylated substrates: 1-[1-14C]alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen) or plasmalogen of ox corpus callosum.

Phospholipid metabolism of glial cell primary cultures, IV. Metabolism of 1-alkenyl-sn-glycero-3-phosphoethanolamine between 1 and 20 hours incubation.

Primary cell cultures prepared from newborn rat brain were incubated on the 16th or 17th day with the substrate 1-([1-3H]-1-alkenyl)-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) for 1-20 h. The internalization of the substrate into the cells depended on the incubation time as well as on the amount of substrate. At any given time the acylation reaction to 2-acyl-1-alkenyl-sn-glycero-3-phosphoethanolamine (plasmalogen) was the most important event amounting to nearly 50-60% of the total radioactivity incorporated. Unchanged substrate was found in only small amounts within the cells. During incubation, the formation of 2-acyl-1-alkenyl-sn-glycero-3-phosphocholine (choline plasmalogen) increased, reaching saturation after 6 h with nearly 40% of the total radioactivity within the cells. These results were compared with those previously obtained with the substrate 1-([1-3H]alkyl)-sn-glycero-3-phosphoethanolamine under the same conditions. The acylation of this substrate as well as its conversion to the choline-containing analogue had been observed. Furthermore plasmalogen formation was also determined as a slow enzyme reaction. Both series of experiments showed a high acylation rate of 1-alkenylglycerophosphoethanolamine and a slow desaturation rate of the 1-alkyl compound. Thus, the following pathway of plasmalogen formation is proposed: 1-alkyl-sn-glycero-3-phosphoethanolamine leads to 1-alkenyl-sn-glycero-3-phosphoethanolamine leads to 2-acyl-1-alkenyl-sn-glycero-3-phosphoethanolamine.

Effect of chloroquine treatment on the different phospholipid species of rat liver lysosomes.

The lysosomal phospholipids of rat liver were determined after treatment of the animals with chloroquine for varying periods up to 15 days. During this period, all the measured phospholipids increased. After two weeks of treatment the absolute level of each phospholipid had increased 3 to 6-fold, with the exception of bis(monoacylglycero)phosphate which was increased 25-fold, accounting for one third of the total phospholipids. The fatty acid pattern not only differed between the classes of lipids; it changed during treatment within every lipid class. In addition to other changes, the polyenoic fatty acids of the main glycerophospholipids were markedly reduced between the 3rd and 14th day of chloroquine treatment: to one half in phosphatidylcholine, to about one tenth in phosphatidylethanolamine, and to one quarter in phosphatidylinositol. However, the polyenoic fatty acid content of bis(monoacylglycero)phosphate was doubled, reaching 76% of the total at the end of treatment. Whereas docosahexaenoic acid was not detected in this acidic phospholipid in the first 3 days of treatment, it was present to the extent of 55% of the total fatty acids after 15 days of chloroquine application;

Studies on the liberation of fatty acids from 2-lysophosphatidylcholine by a liver lysosomal enzyme activity from chloroquine-treated rats.

A lysophospholipase activity was observed in cell fractions from rat liver without and after treatment with chloroquine following incubation with 1-[1(-14)C]palmitoyl-sn-glycero-3-phosphocholine. The specific activity of the enzyme (optimum pH at 4.2) is highly enriched in lysosomes but decreases after prolonged treatment of the animals with chloroquine. Some of the properties of the enzyme were studied.

On the phospholipid metabolism of glial cell primary cultures: cell characterization and their utilization of 1-alkyl-glycerophosphoethanolamine.

Primary cultures prepared from newborn rat brain were grown for 16 or 17 days in culture. Addition of brain extract from newborn rats to the medium stimulated the maturation of astrocytes and the development of oligodendrocytes. Both cell types were characterized by morphology and by immunohistochemistry. The phospholipid composition of these cells was estimated. Incubations were performed with 1-[3H]alkyl-sn-glycerophosphoethanolamine in varying concentrations for 3 h. About one-third of the substrate supplied was internalized by the cells. Several enzymic reactions were observed. The acylating enzyme system was the most active one--a Km was determined with 5 nmol intracellular 1-alkyl-sn-glycerophosphoethanolamine/mg cell protein. Plasmalogen formation was rather low. 1-Alkyl-sn-glycerol, a hydrolysis product, was found in small amounts. Some radioactivity was also incorporated into the phosphatidylcholine fraction.

Studies on the properties of phospholipase A1 in liver lysosomes of chloroquine-treated rats.

The influence of chloroquine on phospholipase A1 (acid pH optimum) activity was studied in subcellular fractions of rat liver after intraperitoneal application of the drug for 9, 12 and 21 days. In comparison with other cell fractions lysosomes of treated rats contained the highest enzyme activity with a pH optimum of 4.0. The activity of phospholipase A1 in lysosomes showed a direct relationship to the number of days of chloroquine treatment. The effects of the incubation time, Ca2+, Hg2+, enzyme and substrate concentration on phospholipase A1 activity were studied.