Occurrence of phosphatidyl-d-serine in the rat cerebrum

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Abstract

Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-l-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-d-serine (d-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of d-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of d-PS. Free d-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes d-PS by a base-exchange reaction.

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)

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    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].

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