Biosynthesis Of Phosphatidylserine Research Articles

Propranolol increases the biosynthesis of phosphatidic acid, phosphatidylinositol and phosphatidylserine in the toad retina: Studies in the entire retina and subcellular fractions

1. Intact toad retinas incubated for short periods of time with [2- 3H]glycerol were subject to subcellular fractionation. 2. The composition and labeling of glycerolipids were studied in the following subcellular fractions: rod outer segment (ROS), nuclear-photoreceptor inner segment synaptic body (P 1, synaptosomal-mitochondrial (P 2), microsomal and cytosolic. 3. It was concluded that the biosynthetic de novo route was followed by [2- 3H]glycerol in the toad retina since radioactivity was located solely in the glycerol backbone of lipids and phosphatidic acid specific activity was the highest. 4. Propranolol produces an increase in the biosynthesis of acidic phospholipids and inhibition in the biosynthesis of zwitterionic lipids in the entire toad retina. The effect was mainly located in microsomes and in the soluble fraction during the first minutes of incubation, being spread afterwards to other fractions. 5. These data are consistent with the view that enzymes of the biosynthesis of glycerolipids are modified in the retinal endoplasmic reticulum by propranolol, providing a useful tool to evaluate the regulation of the pathway.

Biosynthesis of phosphatidylserine in rat brain microsomes

1. 1. Rat brain microsomes incorporated L-serine into phosphatidylserine in the presence of 2 mM ATP. This reaction was stimulated 2-fold by the addition of phosphatidic acid (0.2 mM) and 5-fold by the addition of nickel (0.5 mM). 2. 2. This phosphatidylserine synthesis was inhibited completely by p-hydroxy-mercuribenzoate (0.1 mM) and N-ethylmaleimide (1 mM), whereas the Ca 2+-dependent phosphatidylserine synthesis was unaffected by these sulfhydryl reagents. 3. 3. The specific activity of the ATP-Ni 2+-dependent phosphatidylserine was increased more than 2-fold during active myelination, whereas the Ca 2+-dependent system remained unchanged. 4. 4. Preliminary data indicate that pyrophosphatidic acid ( p, P'-bis'(1,2-diacyl- sn-glycero-3-) pyrophosphate) is the immediate precursor of phosphatidylserine synthesis.

Membrane mutants: a yeast mutant with a lesion in phosphatidylserine biosynthesis.

A single-gene nuclear choline-requiring mutant of Saccharomyces cerevisiae was studied. Choline as a growth supplement to synthetic media could be substituted by low concentrations of dimethylethanolamine, monomethylethanolamine or ethanolamine. DL-Serine also supported growth, but only at high concentrations: on a molar basis it was approximately one hundred times less effective than choline. When cultured in unsupplemented medium the mutant cells soon ceased to grow. The growth-arrested cells contained less than one fifth of the phosphatidylethanolamine present in wild-type cells and only traces of phosphatidylserine. The relative content of the two phospholipid species was raised by growing the mutant cells in the presence of choline of the other supplements but still remained lower than in wild-type cells. The mutant cells depleted of phosphatidylethanolamine and phosphatidylserine had greatly diminished ability to fuse with other cells in mating and their protoplasts showed increased resistance to hypotonic lysis. Respiration was not substantially affected by the deficit of the two phospholipid species in the mutant. In cell-free preparations, the affinity of the phosphatidylserine synthesizing system for serine was found to be almost two orders of magnitude lower in the mutant than in the wild-type. The impairment of phosphatidylserine synthesis accounts for growth requirement and the abnormal phospholipid composition of the mutant cells.

MODIFICATION OF THE AGRANOFF-SUOMI METHOD FOR THE SYNTHESIS OF CDP-DIACYLGLYCEROL

Cytidine 5′-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol) is an important liponucleotide intermediate in the biosynthesis of phosphatidyl-inositol, phosphatidylserine and phosphatidylglycerophosphate. The Agranoff-Suomi method (Agranoff, B. W. and Suomi, W. D. (1963) Biochem. Prep. 10, 47–51) for the synthesis of CDP-diacylglycerol was modified by a procedure which eliminates the lyophilization step required for the preparation of reactants. The reaction was run in chloroform instead of pyridine using the catalyst 4-dimethylaminopyridine. The product of the reaction was purified by silica gel column chromatography and is enzymatically active with the enzymes phosphatidylinositol synthase, phosphatidylserine synthase and phosphatidylglycerophosphate synthase.

Serine ethanolamine phosphate in avian muscular dystrophy: Mechanism of accumulation in dystrophic muscle and relationship to phospholipid synthesis

The cause of the elevated level of serine ethanolamine phosphate (SEP) in dystrophic chicken pectoralis muscle was investigated. It was established that dystrophic muscle has the ability to synthesize SEP, whereas normal pectoralis muscle does not. The SEP synthase activity was localized in the microsomal fraction. Both dystrophic and normal muscle microsomes also contain the opposing enzyme, SEP diesterase, the activity of which is much higher in the dystrophic than in the normal muscle. In intact cells SEP may not be accessible to SEP diesterase. Experiments with 31P NMR revealed that in kidney slices the intracellular SEP concentration was maintained although SEP added to the incubation medium was hydrolyzed. Serine [ 14C]ethanolamine phosphate was not taken up by dystrophic or normal posterior latissimus dorsi and anterior latissimus dorsi muscles. The impermeability of muscle to SEP as well as the virtual absence of SEP in the blood of dystrophic chickens eliminates the possibility that the SEP found in dystrophic muscle is derived from another tissue. The synthesis of SEP in dystrophic posterior latissimus dorsi and in both dystrophic and normal anterior latissimus dorsi was demonstrated using [ 3H]serine as a precursor. The turnover of SEP in these muscles was much slower than the turnover of the serine moiety of serine phospholipids indicating that SEP is not a precursor to phosphatidylserine biosynthesis.

Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.

Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into phospholipid by a Ca(2+)-dependent base-exchange reaction. This reaction is virtually absent in normal liver mitochondria. The finding cannot be attributed to microsomal contamination of the sucrose gradient-purified 7777 hepatoma mitochondria. The reaction is also absent in the rapid-growth controls, fetal rat liver and regenerating rat liver. [(14)C]Serine incorporation into 7777 hepatoma mitochondrial phospholipid by base-exchange requires Ca(2+) and is inhibited by EDTA. Ca(2+) cannot be replaced by Mg(2+), Mn(2+), or Co(2+). The reaction is inhibited by a sulfhydryl reagent and by detergents and is abolished by heating to 70 degrees C for 10 min. Product analysis indicates that phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, are formed by 7777 hepatoma mitochondria, while phosphatidylserine is the sole product with microsomes. The conversion of phosphatidylserine into phosphatidylethanolamine in 7777 hepatoma mitochondria is inhibited by KCN. This study provides further evidence of abnormal mitochondrial biogenesis in the 7777 hepatoma. Our earlier study indicated a greatly increased mitochondrial activity of CTP:phosphatidate cytidylyltransferase in the 7777 hepatoma (Hostetler, Zenner, and Morris. 1978. J. Lipid Res. 19: 553-560). The presence in mitochondria of these two enzymes, which are primarily microsomal in normal liver, does not appear to be due to rapid growth alone, since their intracellular distribution was not altered in fetal or regenerating rat liver.-Hostetler, K. Y., B. D. Zenner, and H. P. Morris. Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.

Structure and Synthesis of a Lipid‐Containing Bacteriophage

Enzymes of glycerophosphatide biosynthesis and degradation have been characterized using membrane fractions of Pseudomonas BAL‐31, the host cell of the lipid‐containing bacteriophage PM2. Inner and outer membrane fractions were investigated. Whereas the outer membrane con‐tained more phosphatidylethanolamine and less phosphatidylglycerol than the inner membrane, the fatty acid composition was almost the same for both membranes. The pathway for glycerophos‐phatide biosynthesis was the same as that for Escherichia coli and Bacillus megaterium. The biosyn‐thesis of phosphatidylserine and phosphatidylglycerol was shown to proceed via CDP‐diglyceride as a common intermediate. Phospholipase A hydrolyzed phosphatidylethanolamine more efficiently than phosphatidylglycerol. All of the enzymatic activities were stimulated by Triton X‐100. Phos‐phatidylserine synthetase was stimulated by Na2SO4 and inhibited by Mg2+. Phosphatidylserine decarboxylase was completely inhibited by NH2OH. Phosphatidylglyceror synthetase was stimulated by Mg2+. Phospholipase A was stimulated by Ca2+. Phosphatidylserine decarboxylase and phosphatidylglycerol synthetase were localized in the inner membrane fraction. Phosphatidylserine synthetase and phospholipase A were found in both outer and inner membrane fractions.Effects of virus infection on these enzymes were studied. Phosphatidylglycerol synthetase and phospholipase A activity against phosphatidylethanolamine were activated early post‐infection. Phosphatidylserine synthetase and decarboxylase remained unaltered. At later times post‐infection, phosphatidylglycerol synthetase and phosphatidylserine decarboxylase were inactivated and phos‐ phatidylserine synthetase was activated. There was little alteration in phospholipase A activity against phosphatidylglycerol after infection.

The effect of external salinity upon lipid synthesis in the blue crab callinectes sapidus rathbun and in the spider crab libinia em arginata leech

1. 1. Callinectes sapidus and Libinia emarginata were injected with 14C acetate and placed in full or half strength sea water. 2. 2. Tissue samples were taken at intervals and lipids were analyzed for radioactivity. 3. 3. There was a significant enhancement of phosphatidyl ethanolamine and phosphatidyl serine biosynthesis in the gill of C. sapidus in the first 24 hr after dilution but not in the gill of L. emarginata. 4. 4. The possible significance of increased phospholipid synthesis in C. sapidus gill following dilution is discussed.

Biosynthesis of lipids in golgi complex and other subcellular fractions from rat liver

1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria and nuclei from rat liver were isolated and their purity assessed using specific marker enzymes. 2. 2. The various subcellular fractions were assayed for the following processes: biosynthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine, the conversion of phosphatidylserine into phosphatidylethanolamine, the formation of lecithin via N-methylation, and the activation of palmitic and octanoic acids. 3. 3. None of these processes were found to be present in Golgi complex. 4. 4. The endoplasmic reticulum appears to be the principal site in the cell for the synthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine and the formation of lecithin. Interestingly, the biosynthesis of phosphatidylserine appears to be four times more active in rough than in smooth microsomes, which might suggest a ribosomal localization of this process. 5. 5. Except for CDPdiglyceride synthesis, mitochondria do not contain any of the synthesizing activities described in 4. Mitochondria are, however, the only site in the cell where phosphatidylserine is decarboxylated. This activity appears to be localized in the inner membrane. 6. 6. The activation of palmitate is localized predominantly in endoplasmic reticulum and mitochondria, though some activity was detected in plasma membranes as well. All other cell organelles, including Golgi and probably nuclei, did not contain significant palmitoyl-CoA synthetase activity. The subcellular distribution of octanoyl-CoA synthetase resembled that of palmitoyl-CoA synthetase except that the former enzyme is more active in mitochondria than in microsomes.

Alternative pathways in the biosynthesis of phosphatidylserine in mouse cells,.

Alternative pathways in the biosynthesis of phosphatidylserine in mouse cells,.

The Ca 2+-dependent biosynthesis of lecithin, phosphatidylethanolamine and phosphatidylserine in rat liver subcellular particles

1. 1. The Ca 2+-dependent incorporation of choline, ethanolamine and l-serine into rat liver phospholipids has been studied in vitro. The incorporation was located exclusively in the microsomal fraction. 2. 2. No incorporation took place in the presence of EDTA. The optimal Ca 2+ concentration was found to increase with decreasing pH, and vice versa. At pH 8.8 and 0.16 mM Ca 2+ the K m values of choline, ethanolamine and l-serine were 2.3 · 10 -5, 9.5 · 10 -6 and 6.2 · 10 -5 M, respectively. At pH 7.5 the K m and V for both choline and ethanolamine increased with increasing Ca 2+ concentration. At pH 8.8 high Ca 2+ concentrations were inhibitory. The K m and V of l-serine showed no significant changes. 3. 3. Choline incorporation was competitively inhibited by ethanolamine and l-serine; incorporation of l-serine was competitively inhibited by choline and ethanolamine. In contrast, ethanolamine incorporation was inhibited noncompetitively by l-serine and uncompetitively by choline. This indicates that the Ca 2+-stimulated incorporation of choline, ethanolamine and l-serine does not occur via a single common phosphatidyl-enzyme complex. 4. 4. Ethanolamine is incorporated preferentially into polyunsaturated phosphatidylethanolamine species in rat liver microsomes. The rate of incorporation into hexa-unsaturated species was twice that into mono- and di-unsaturated species, while choline was incorporated 10–15 times more rapidly into the hexa-unsaturated species. 5. 5. The results show that the incorporation rate of l-serine found in vitro can account for the rate of phosphatidylserine synthesis observed in vivo. The Ca 2+-dependent mechanism thus seems to be the major pathway of phosphatidylserine synthesis in the liver. In contrast, the synthesis of lecithin and phosphatidylethanolamine via this mechanism must be quantitatively negligible. 6. 6. A metabolic scheme is discussed which implies interconversion of the respective phospholipids.

Effects of fatty acids on incorporation of serine into phosphatidylserine in mouse liver microsomes

An acetone-soluble fraction from mouse liver microsomes was found to inhibit the incorporation of serine into phosphatidylserine of microsomes. The active component of the microsomal extract might be identical with free fatty acids. Among the saturated fatty acids, C 12 fatty acid exhibited the greatest activity which diminished progressively as the chain was lengthened or shortened. Introduction of an unsaturated bond, especially of cis-form, enhanced activity. The inhibition by free fatty acids may play a physiological role in the regulation of phosphatidylserine biosynthesis.

Enzymatic synthesis and decarboxylation of phosphatidylserine in Tetrahymena pyriformis

Cell-free extracts of the protozoan Tetrahymena pyriformis have been found to catalyze the following reactions: Phosphatidylethanolamine + L-Serine <==> Phosphatidylserine + Ethanolamine. Phosphatidylserine --> Phosphatidylethanolamine + CO(2) divided by Net: L-Serine --> Ethanolamine + CO(2). The biosynthesis of phosphatidylserine in this organism resembles that found in higher animals. In contrast, phosphatidylserine is formed in bacteria in a reaction between CDP-diglyceride and L-serine. A detailed description is presented of the enzyme that catalyzes the synthesis of phosphatidylserine in Tetrahymena by the exchange reaction, together with some properties of the phosphatidylserine decarboxylase in this organism.

Studies on the biosynthesis of phosphatidyl serine

1. 1. A method is described for the determination of the radioactivity of [ 14C]-phospholipids using a liquid scintillation counter. 2. 2. The incorporation of l-[ 14C]serine into mitochondrial phospholipids was studied. CMP, ATP, CoASH, glutathione, glycerophosphate and magnesium ions were required for optimal incorporation. CTP was less effective than CMP in stimulating the incorporation. 3. 3. Phosphoryl serine did not produce an isotope dilution effect and does not seem to be an intermediate in the biosynthesis of phosphatidyl serine. 4. 4. The effects of pH, magnesium, fluoride and pyrophosphate ions on this system were studied. High concentrations of calcium ions inhibited the incorporation of l-serine, whereas low concentrations brought about a marked stimulation in the rate of incorporation. 5. 5. The radioactive phospholipid was identified as phosphatidyl serine by means of mild alkaline hydrolysis and ion-exchange chromatography of the resulting diesters.

In vitro incorporation of serine labelled with carbon-14 into mitochondrial phospholipids.

LITTLE is known about the biosynthesis of phosphatidyl serine. It has been suggested that the biosynthetic pathway of this phospholipid might be similar to that of phosphatidyl choline and phos-phatidyl ethanolamine1. The present investigation describes the incorporation of uniformly labelled L-serine-14C into the phospholipids of rat liver mitochondria and some of the co-factors required for this process.