Occurrence of phosphatidyl-d-serine in the rat cerebrum
Introduction
Phosphatidylserine (PS) is a component of mammalian cell membranes and plays important roles in biological processes such as apoptosis [1] and cell signaling [2], [3]. PS is a derivative of phosphatidic acid in which the phosphoric acid is bound by ester linkage to the hydroxyl group of a serine moiety. In mammalian cells, PS is synthesized by base-exchange reactions in which the polar head groups of preexisting phospholipids are replaced by serine. Despite the presence of two enantiomers, namely, l- and d-forms of serine, it has long been believed that only phosphatidyl-l-serine (l-PS) occurs in living organisms, according to the pioneering studies by Folch et al. [4], [5], [6]. Meanwhile, recent advances in analytical techniques have enabled the identification of endogenous free d-amino acids, including d-alanine, d-aspartate, and d-serine, in mammalian brain [7], [8], [9], [10], [11]. However, the occurrence of phosphatidyl-d-serine (d-PS) in mammalian tissues has not been investigated in the context of recently developed techniques for sensitive determination of enantiomers of amino acids by HPLC. In this report, we demonstrate for the first time the presence of d-PS in the cerebrum of normal rat brains.
Section snippets
Materials and methods
Purification of PS. Male rats (Japan SLC, Inc., Japan) at postnatal week 7 were sacrificed, and their cerebrum extirpated. Crude lipids were extracted from the cerebrum by Folch’s method [12]. To further purify lipids, 0.2 volumes of 0.9% KCl was added to the crude lipid extract, and the mixture was centrifuged at 100 g for 20 min at ambient temperature. The chloroform phase was collected and dried under a stream of nitrogen gas. The purified lipids were resuspended in chloroform.
PS was isolated
Identification of d-PS in the rat brain by HPLC analysis of acid hydrolysates of PS
To investigate whether d-PS occurs in the rat brain, we analyzed the hydrolysates of PS purified from the cerebrum of rats according to the method developed by Hashimoto et al. [18]. Boc-l-Cys-OPA derivatives of l- and d-serine from the hydrolysates were separated by reversed-phase HPLC with highly sensitive fluorometric detection. Retention times of the derivatives of authentic l- and d-serine were 11.5 and 13.0 min, respectively. As shown in Fig. 1, a peak of the d-serine derivative was
Discussion
In this study, we demonstrated for the first time the occurrence of d-PS in mammalian tissues. The amount of d-PS was determined to be approximately 1% of total PS in the lipid fraction of the rat cerebrum. Our analysis depended on the sensitive and reliable HPLC methods developed by Hashimoto et al. [18] and Goodnough et al. [19]; these methods have been successfully used to identify a considerable amount of free d-serine in brain tissues. By using these methods, we were able to detect d
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 19370040) and Global COE Program “International Center for Integrated Research and Advanced Education in Materials Science” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References (36)
- et al.
Lipid activation of protein kinase C
J. Biol. Chem.
(1991) - et al.
An amino acid constituent of brain cephalin
J. Biol. Chem.
(1941) The isolation of phosphatidyl serine from brain cepharin, and identification of the serine component
J. Biol. Chem.
(1941)- et al.
Free d-aspartate and d-alanine in normal and Alzheimer brain
Brain Res. Bull.
(1991) - et al.
The presence of free d-serine in rat brain
FEBS Lett.
(1992) - et al.
Preparation of lipid extracts from brain tissue
J. Biol. Chem.
(1951) - et al.
The enzymatic synthesis of phosphatidylserine and purification by CM-cellulose column chromatography
Biochim. Biophys. Acta
(1977) - et al.
Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus
Methods Enzymol.
(1974) - et al.
Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert-butyloxycarbonyl-l-cysteine and o-phthaldialdehyde
J. Chromatogr.
(1992) - et al.
Separation and quantification of d- and l-phosphoserine in rat brain using Nα-(2, 4-dinitro-5-fluorophenyl)-l-alaninamide (Marfey’s reagent) by high-performance liquid chromatography with ultraviolet detection
J. Chromatogr. B Biomed. Appl.
(1995)
Phosphatidohydrolase activity in a solubilized preparation from rat brain particulate fraction
Arch. Biochem. Biophys.
Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity
J. Biol. Chem.
Genetic evidence that phosphatidylserine synthase II catalyzes the conversion of phosphatidylethanolamine to phosphatidylserine in Chinese hamster ovary cells
J. Biol. Chem.
Regio- and stereospecific analysis of glycerolipids
Methods
Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles
J. Biol. Chem.
Metabolism and functions of phosphatidylserine
Prog. Lipid Res.
Extracellular concentration of endogenous free d-serine in the rat brain as revealed by in vivo microdialysis
Neuroscience
Serine racemase modulates intracellular d-serine levels through an α,β-elimination activity
J. Biol. Chem.
Cited by (4)
Phosphatidylserine in the brain: Metabolism and function
2014, Progress in Lipid ResearchCitation Excerpt :It is synthesized in a crude homogenate of rat cerebrum in a Ca2+-dependent process, but the enzyme was not identified and the rate of d-serine incorporation was only 1/10th the rate of PS synthesis from l-serine. Phosphatidyl-d-serine also is contained in heart, spleen, lung, testis, liver or kidney, but in much smaller amounts, and it has not been detected in the cerebellum [70,71]. d-serine is an obligatory co-agonist of the NMDA glutamate receptor involved in synaptic plasticity and memory.
Identification of novel mammalian phospholipids containing threonine, aspartate, and glutamate as the base moiety
2011, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :We previously showed that d-PS occurs in rat cerebrum and various other rat tissues. We also showed that free d-serine can be incorporated into PS in an in vitro system by using cerebrum extract, suggesting that d-PS is formed by a base-exchange reaction [12,13]. In mammals, free d-serine is distributed in the cerebrum [27,28] and plays an important role as a coagonist of the glycine site of N-methyl-d-aspartate (NMDA) receptors [29,30].
I know? D-amino acid
2019, Seibutsu-kogaku KaishiThe distribution of phosphatidyl-D-serine in the rat
2010, Bioscience, Biotechnology and Biochemistry