COMMUNICATION
Occurrence of an Unusual Phospholipid,
Phosphatidyl-L-threonine, in Cultured Hippocampal
Neurons
EXOGENOUS L-SERINE IS REQUIRED FOR THE
SYNTHESIS OF NEURONAL PHOSPHATIDYL-L-SERINE AND
SPHINGOLIPIDS*
Junya
Mitoma
§,
Takeshi
Kasama¶,
Shigeki
Furuya
, and
Yoshio
Hirabayashi
From the
Laboratory for Cellular Glycobiology,
Frontier Research Program and the § Special Postdoctoral
Researchers Program, The Institute of Physical and Chemical Research
(RIKEN), Wako, Saitama 351-0198, Japan and the ¶ Instrumental
Analysis Research Center for Life Science, Tokyo Medical and Dental
University, Bunkyo-ku, Tokyo 113-8510, Japan
 |
ABSTRACT |
We have recently reported that
L-serine released from astroglial cells supports the
survival and neuritogenesis of hippocampal neurons under a serum- and
glia-free culture condition (Mitoma, J., Furuya, S., and Hirabayashi,
Y. (1998) Neurosci. Res. 30, 195-199). In this study, we
show that exogenous L-serine is required for the synthesis
of phosphatidyl-L-serine (PS) and sphingolipids in
hippocampal neurons. When hippocampal neurons were maintained under an
astroglial cell-free condition, the levels of sphingolipids and
phosphatidyl-L-serine in the neurons were greatly reduced in the absence of external L-serine or glycine. Instead, a
novel phospholipid appeared just ahead of PS on TLC. This novel lipid was determined to be phosphatidyl-L-threonine by TLC
blotting/negative secondary ion mass spectrometry and amino acid
analysis. Biochemical studies on rat brain microsomes have indicated
that phosphatidyl-L-threonine is synthesized by the base
exchange enzyme that is involved in PS synthesis with much lower
affinity, that is, approximately
of L-serine.
Addition of L-serine or glycine to the culture medium
restored the synthesis of PS and sphingolipids in the neurons. These
observations show that hippocampal neurons require exogenous
L-serine for the synthesis of PS and sphingolipids in the
absence of astroglial cells and suggested that astroglial cells
contribute to neuronal lipid synthesis through the supply of
L-serine.
 |
INTRODUCTION |
L-Serine is indispensable for the biosynthesis of
phosphatidyl-L-serine
(PS)1 and sphingolipids in
cells. These serine-derived lipids play important roles in cellular
functions. For example, PS is essential for cell growth (1), whereas
the mutations that reduce sphingolipid synthesis also inhibit the
growth of Chinese hamster ovary cells (2) and budding yeast (3).
Furthermore, these lipids have been shown to be involved in
intracellular signal transduction. A certain member of the protein
kinase C family interacts with PS (for review see Ref. 4). Recent
biochemical studies have demonstrated that sphingolipid metabolites
such as ceramide and sphingosine-1-phosphate function as signaling
molecules involved in many cellular processes including apoptosis,
proliferation, differentiation, and response to stresses (for reviews
see Refs. 5 and 6). In the case of neurons of the central nervous system, our studies using neuronal cultures have demonstrated that
ceramide plays distinct and significant roles in survival and
phenotypic growth of cerebellar Purkinje cells (7, 8), hippocampal
neurons (9), and spinal motoneurons (10).
Although L-serine is known to be a nonessential amino acid
and to be metabolically formed from 3-phosphoglycerate, a metabolite of
glycolysis, in cells (for review see Ref. 11), we have recently demonstrated that hippocampal astroglial cells release a significant amount of L-serine with rapid kinetics and that the amino
acid is trophic for hippocampal neurons (12). These observations suggest the possibility that L-serine released by
astrocytes is taken up by neurons and utilized for the synthesis of
various cellular compounds, which might underlie the trophic action of L-serine on hippocampal neurons. In this study, we
investigated whether externally supplied L-serine
contributed to the biosynthesis of L-serine-derived
membrane lipids in hippocampal neurons. Sphingolipids and PS
disappeared rapidly when the neurons were maintained in the absence of
exogenous L-serine. In parallel with the disappearance of
these serine-derived lipids, a novel aminophospholipid appeared in the
neurons. We identified the structure of this lipid as
phosphatidyl-L-threonine (PT). Biochemical analysis
indicated that PT was synthesized by a base exchange enzyme that
catalyzed the synthesis of PS with a different affinity. The biological
significance of the glial cell-derived L-serine in the
lipid biosynthesis and in the survival of the neurons is discussed.
 |
EXPERIMENTAL PROCEDURES |
Hippocampal Neuronal Culture--
Primary cultures of
dissociated hippocampal neurons were prepared from fetal rat (Wistar)
of 18 days of gestation as described previously (9). A total of 2 × 106 cells were plated onto a dish 100 mm in diameter
(Falcon 3003, Becton Dickinson and Co., Franklin Lakes, NJ). After
24 h, the medium was changed to a serum-free minimum essential
medium supplemented with 25 mM HEPES, 30 nM
selenium, 500 µM pyruvate, 3.9 mM glutamine, 16.7 mM glucose, 100 µM putrescine, 10 µg/ml gentamicin sulfate, 0.1 mg/ml bovine serum albumin, 10 µg/ml
bovine insulin, 0.1 mg/ml human apo-transferrin, and 20 nM
progesterone. To establish glial cell-free cultures,
cytosine-
-D-arabinoside (1 µM) was added to the serum-free medium.
Lipid Analysis--
Neurons cultured for 6 days were harvested
from the 100-mm dishes with a cell scraper in
Ca2+,Mg2+-free phosphate-buffered saline.
Lipids were then extracted using the Bligh-Dyer method (13). Upper and
lower phases were collected and dried under N2 gas and
dissolved in chloroform/methanol (2/1, v/v). TLC was performed on a
Silica Gel 60 HPTLC plate (Merck, Darmstadt, Germany) in
chloroform/methanol/12 mM MgCl2 (5/4/1, v/v/v)
and chloroform/methanol/formic acid/acetic acid/1 M
MgCl2 (60/30/6.5/4.5/0.1, v/v/v/v/v) for gangliosides and
phospholipids, respectively. The lipids were detected with iodine vapor
(14), ninhydrin (14), primuline reagent (15), and Ryu-MacCoss reagent (16). For sensitive detection of gangliosides, the HPTLC plate treated
with sialidase was stained with horseradish peroxidase-labeled cholera
toxin B-subunit as described previously (17).
Amino Acid Analysis--
The lipid was scraped out from the
HPTLC plate and extracted with chloroform/methanol (1/1, v/v). After
washing using the Bligh-Dyer technique, the lower phase was evaporated
to dryness. The resultant lipids were hydrolyzed in 6 N HCl
for 2 h at 100 °C. The hydrolysate was then applied to a
Hitachi L-8500 amino acid analyzer using a packed column
for physiological fluid analysis (Hitachi, Ltd., Tokyo, Japan).
TLC Blotting/Mass Spectrometry--
Lipids on the HPTLC plate,
visualized with primuline reagent, were transferred onto a
polyvinylidene difluoride membrane (Atto Corporation, Tokyo, Japan) as
described elsewhere (18). The corresponding portion was punched out,
and the lipids on the polyvinylidene difluoride membrane were directly
subjected to mass spectrometric analysis. Negative secondary ion mass
spectrometry (SIMS) spectra were obtained by a TSQ 70 triple stage
quadrupole mass spectrometer (Finningan MAT, CA) equipped with cesium
gun. Triethanolamine was used as SIMS matrix. The lipid on the target
was bombarded with a Cs+ beam accelerated at 20 keV. The
ion multiplier was 1.5 keV, and the conversion dynode was preset at 20 keV.
Assay for Base Exchange Enzyme Activity--
The microsomal
fraction from the adult Wistar rat brain was obtained as follows. Whole
brain was homogenized in a homogenization buffer consisting of 0.25 M sucrose, 20 mM HEPES-KOH (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethanesulfonyl fluoride. The homogenate was
centrifuged at 600 × g for 15 min, followed by
sequential centrifugations of the supernatant at 10,000 × g for 15 min and 150,000 × g for 15 min.
The resultant microsomal pellet was resuspended in the homogenization
buffer and used directly for the base exchange analysis. Protein was
quantified using the BCA reagent (Pierce, Rockford, IL).
Base exchange assay followed the method of Kanfer (19). The reaction
mixture consisted of 50 mM HEPES-KOH (pH 7.5), 15 mM CaCl2, 0.5 mM
[U-C14]L-serine (37 MBq/mmol, NEN Life
Science Products, Boston, MA) or
[U-C14]L-threonine (148 MBq/mmol, NEN Life
Science Products, Boston, MA), and 200 µg of microsomal protein in a
total volume of 200 µl. After incubation at 37 °C for 15 min, the
reaction was terminated by the addition of chloroform/methanol (1/2,
v/v), followed by partition using the Bligh-Dyer method. The chloroform
phase was removed, dried, and dissolved in chloroform/methanol (2/1,
v/v). After TLC, the radioactivities of PS and PT were quantified using the BAS-2000 imaging analyzer (Fujifilm, Tokyo, Japan).
 |
RESULTS AND DISCUSSION |
Exogenous L-Serine Requirement for the Synthesis of
Gangliosides and Phosphatidyl-L-serine in Cultured
Hippocampal Neurons--
To investigate the role of exogenous
L-serine in the biosynthesis of lipids in hippocampal
neurons, we compared the composition of membrane lipids in neuronal
cultures maintained in the presence or absence of external
L-serine. Because L-serine and glycine are
interconvertible in cells and glycine is also trophic for hippocampal
neurons (12), we examined the effects of glycine also. Hippocampal
neurons were cultured in a serum- and glial cell-free setting for 6 days, and then the total lipids were extracted. We first analyzed the
composition of sphingolipids, particularly that of gangliosides,
because gangliosides but not sphingomyelin constituted the dominant
species of sphingolipids in cultured hippocampal neurons (9). To
visualize complex gangliosides containing the common GM1a structure,
gangliosides recovered in the upper phase of the Bligh-Dyer partition
were separated by TLC and converted to GM1a by clostridial sialidase
treatment, and the resultant GM1a was detected by staining with
horseradish peroxidase-labeled cholera toxin B-subunit (17). As shown
in Fig. 1A, GM1a, GD1a, and
GT1b were the major gangliosides observed when hippocampal neurons were
maintained in the presence of exogenous L-serine or glycine
(lanes 2 and 3). The levels of these neuronal gangliosides were significantly reduced in the absence of external L-serine and glycine (Fig. 1A, compare
lane 1 with lane 2 or 3). A mixture of
other nonessential amino acids, L-alanine,
L-asparagine, L-aspartic acid, and
L-proline, did not restore the amount of gangliosides.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 1.
Appearance of a novel lipid in hippocampal
neurons cultured without L-serine and glycine. Lipids
were extracted from hippocampal neurons cultured for 6 days by the
Bligh-Dyer partition method. A, gangliosides in upper phase
corresponding to 2 µg of protein were separated by TLC and stained
with horseradish peroxidase-cholera toxin B subunit after treatment of
the plate with sialidase. B, the lipids from lower phase
corresponding to 70 µg of protein were separated by TLC and
visualized with ninhydrin reagent. Hippocampal neurons were cultured in
minimum essential medium with the following supplements: lane
1, hippocampal neurons without serine and glycine; lane
2, 100 µM glycine; lane 3, 100 µM serine; lane 4, mixture of 100 µM L-alanine, L-aspartic acid,
and L-proline.
|
|
We next analyzed the phospholipids in the lower phases of the
Bligh-Dyer partition by TLC. Fig. 1B shows hippocampal
neuronal aminophospholipids visualized by ninhydrin staining. When the hippocampal neurons were cultured under L-serine-free
conditions, PS was specifically depleted, and a novel band, designated
as X1, appeared slightly above the position of PS (Fig. 1B,
lanes 1 and 4). This compound was labeled with
Ryu-MacCoss reagent (not shown), indicating that it belongs to the same
class of aminophospholipids as PS and phosphatidylethanolamine (PE).
The absence of external L-serine or glycine did not alter
the amounts of phosphatidylcholine and phosphatidylinositol (not
shown). Although PE is also an L-serine-derived lipid, only
a slight decrease in the level of PE was observed in the absence of
L-serine. Because our culture medium did not contain free
ethanolamine, PE cannot be synthesized by the direct coupling of
CDP-ethanolamine and diacylglycerol (20). Therefore, the subtle
decrease in the level of PE may be explained by the difference in the
turnover rates and/or the size of the metabolic pool between PS and
PE.
Structural Characterization of a Novel Phospholipid X1--
We
determined the structure of X1. Hippocampal neuronal lipids were
developed on TLC plates, and X1 and two other aminophospholipids, X2
and X3, were purified by scraping out the corresponding bands (Fig.
1B). The amino acid compositions of these lipids were
analyzed after acid hydrolysis. Interestingly, X1 possessed only
L-threonine (Fig.
2A). X2 contained both
L-threonine and L-serine (Fig. 2B), and L-serine was the dominant amino acid of X3, indicating
that X3 corresponds to PS (Fig. 2C). These analyses revealed
that the novel lipid X1 is an L-threonine-containing
lipid.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Structural analysis of lipid X1.
A-C, amino acid analysis. Lipids X1 (A), X2
(B), and X3 (C) were purified by preparative TLC,
treated with 6 N HCl, and subjected to amino acid analysis.
D, TLC blotting/SIMS of X1. Asterisks indicate
matrix related ions. PA, phosphatidic acid;
LysoPT, lysophosphatidyl-L-threonine;
LysoPA, lysophosphatidic acid.
|
|
To characterize the structure of X1 in detail, we carried out TLC
blotting/SIMS analysis. The negative SIMS spectrum of X1 is shown in
Fig. 2D. A pair of major molecule-related ions, [M + Na
2H]
, was observed at
m/z 846 and 870, indicative of the structures of
phosphatidyl-L-threonine with C18:0/C20:4 and with
C18:0/C22:6, respectively. The ions at the m/z
888 and 912 peaks are presumably the adduct of CH2=C=O
derived from triethanolamine as reported for the free amino-containing
gangliosides GM3 (21) and GM1 (22). The characteristic fragment ions
generated by sequential elimination of these constituents were at
m/z 723 and 747 for phosphatidic acid
(C18:0/C20:4 and C18:0/C22:6, respectively), at
m/z 560 and 582 for lysophosphatidylthreonine
(
H and +Na
2H, respectively), and at
m/z 459 for lysophosphatidic acid (C18:0). We
therefore conclude that X1 corresponds to PT. The most abundant unsaturated fatty acid on brain PS was reported to be C18:1 followed by
C22:6 (23). In fact, the peak corresponding to PT with C18:0/18:1, [M + Na
2H]
, was detected at
m/z 824 in X2 (data not shown). Thus, the fatty acid composition of PT from cultured hippocampal neurons is almost identical to that of PS in mammalian brain. The existence of PT in
neurons is being reported for the first time, although this lipid has
been shown to exist in tuna muscle (24) and polyoma-transformed hamster
embryo fibroblasts (25, 26). Because of the presence of astroglial
cells, conditions of L-serine and glycine depletion resulting in PT expression may not occur in hippocampal neurons in vivo. Therefore, the physiological functions of this
unusual lipid remain to be elucidated.
In Vitro Synthesis of
Phosphatidyl-L-threonine--
The simultaneous reduction
in the levels of gangliosides and PS implies that L-serine
is not available in sufficient quantities for the synthesis of these
lipids in hippocampal neurons cultured in the absence of
L-serine or glycine. The concomitant appearance of PT
raises the possibility that neo-synthesis of PT is catalyzed by a base
exchange enzyme that is involved in PS biosynthesis. To test this
assumption, we examined whether the synthesis of PS is inhibited by
L-threonine. We used microsomes from rat whole brain as an
enzyme source, because only a small amount of microsomes were obtained
from the cultured neurons. As shown in Fig.
3A, L-threonine
certainly inhibited PS synthesis in vitro, although the
amount of L-threonine required for such inhibition is
rather high. The Lineweaver-Burk plot indicated that
L-threonine is a competitive inhibitor with a
Ki value of 23 mM, whereas the
Km value for L-serine was 0.11 mM. We then examined whether PT was synthesized from
L-threonine instead of L-serine as a substrate
(Fig. 3B). The formation of PT was dependent on L-threonine concentration, and the Km
value for L-threonine was determined to be 16 mM, which is much higher than that for L-serine. The synthesis of PT was greatly inhibited by the
addition of lower concentrations of L-serine. The
Lineweaver-Burk plot indicated that L-serine competitively
inhibited the formation of PT with a Ki value of
0.10 mM. We also observed the synthesis of PT using rat
liver microsomes (data not shown). These results indicate that PT is
synthesized by the base exchange enzyme that catalyzes the synthesis of
PS with much lower affinity.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Synthesis of PT by L-serine base
exchange enzyme in vitro. Lineweaver-Burk plots of the
effect of L-threonine on the incorporation of
[14C]L-serine to PS (A) and of
L-serine on the incorporation of
[14C]L-threonine to PT (B).
[S] indicates the amino acids added (mM), and
V is the amount of amino acids incorporated into PT or PS
(nmol/mg/h).
|
|
Our present study demonstrates that in the absence of exogenous
L-serine, hippocampal neurons synthesize PT instead of PS. The generation of PT implies that the intracellular level of
L-serine is insufficient for the synthesis of PS when the
neurons are maintained in the absence of exogenous
L-serine. This is supported by the observation that
exogenous glycine also maintains the level of PS (Fig. 1B).
Glycine is metabolically converted to L-serine by serine
hydroxymethyltransferase (27). Furthermore, this assumption explains
why ganglioside biosynthesis, which requires L-serine, is
also inhibited when cultured in the absence of external
L-serine or glycine. We have recently reported that
significant amounts of L-serine are released from
hippocampal astrocytes but not neurons and that this amino acid
promotes the survival and morphological development of hippocampal
neurons (12). Thus, we suggest that hippocampal neurons lose the
ability to synthesize sufficient quantities of L-serine and
consequently depend on astroglial cells for this amino acid. PT was not
detected in lipids extracted from hippocampal regions of normal rat
brains (data not shown), suggesting that L-serine was not
depleted in normal brain tissues. Indeed, extracellular free
L-serine has been reported to be present in the central
nervous system (28). L-Serine released by astroglial cells
probably contributes to the synthesis of L-serine-derived membrane lipids as well as other cellular components in the neurons, which are necessary for the survival of the neurons in vitro
and also probably in vivo.
At present, the molecular basis of exogenous L-serine
requirement in hippocampal neurons is unknown. However, our most recent work demonstrated that the expression of mRNA of 3-phosphoglycerate dehydrogenase, a rate-limiting enzyme for L-serine
synthesis, is significantly reduced in hippocampal
neurons.2 This
down-regulation might underlie the dependence of hippocampal neurons on
astroglial cells for L-serine. Neurons have to take up
exogenous L-serine, which is presumably released from
astroglial cells, through a specific transporter system. In this
context, a cell membrane transporter for L-serine must be
regarded as a key factor in terms of the regulation of lipid
biosynthesis in neurons and, eventually, neuronal survival.
 |
ACKNOWLEDGEMENTS |
We thank Dr. K. Takio and M. Chijimatsu
(Division of Biomolecular Characterization, RIKEN) for the amino
acid analysis. We also thank Dr. Y. Ohashi (Laboratory for
Glycotechnology, Frontier Research Program, Riken) for critical reading
of the manuscript. We are grateful to Prof. Y. Nagai (RIKEN and
Mitsubishi Kagaku Institute of Life Science) for encouragement.
 |
FOOTNOTES |
*
This work was supported by the Special Postdoctoral Research
Program of the Institute of Physical and Chemical Research (RIKEN), Grants-in-Aid for Encouragement of Young Scientists 0980671 (to J. M.) and 09780733 (to S. F.), and Grants-in-Aid for Scientific Research on Priority Areas 07278248 (to Y. H.) from the Ministry of Education, Science and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory for
Cellular Glycobiology, Inst. of Physical and Chemical Research (RIKEN),
Wako, Saitama 351-0198, Japan. Tel.: 81-48-467-9613; Fax:
81-48-462-4690; E-mail: hirabaya{at}postman.riken.go.jp.
1
The abbreviations used are: PS,
phosphatidyl-L-serine; PT,
phosphatidyl-L-threonine; PE, phosphatidylethanolamine;
SIMS, secondary ion mass spectrometry; HPTLC, high performance thin
layer chromatography.
2
J. Mitoma, S. Furuya, and Y. Hirabayashi,
manuscript in preparation.
 |
REFERENCES |
-
Kuge, O.,
Nishijima, M.,
and Akamatsu, Y.
(1986)
J. Biol. Chem.
261,
5795-5798[Abstract/Free Full Text]
-
Hanada, K.,
Nishijima, M.,
Kiso, M.,
Hasegawa, A.,
Fujita, S.,
Ogawa, T.,
and Akamatsu, Y.
(1992)
J. Biol. Chem.
267,
23527-23533[Abstract/Free Full Text]
-
Pint, W. J.,
Srinivasan, B.,
Shephered, S.,
Schmidt, A.,
Dickson, R. C.,
and Lester, R. L.
(1992)
J. Bacteriol.
174,
2565-2574[Abstract]
-
Bell, R. M.,
and Burns, D. J.
(1991)
J. Biol. Chem.
266,
4661-4664[Free Full Text]
-
Hannun, Y. A.
(1997)
Science
274,
1855-1859[Abstract/Free Full Text]
-
Spiegel, S.,
and Merrill, A. H., Jr.
(1996)
FASEB J.
10,
1388-1397[Abstract/Free Full Text]
-
Furuya, S.,
Ono, K.,
and Hirabayashi, Y.
(1995)
J. Neurochem.
65,
1551-1561[Medline]
[Order article via Infotrieve]
-
Furuya, S.,
Mitoma, J.,
Makino, A,
and Hirabayashi, Y.
(1998)
J. Neurochem.
71,
366-377[Medline]
[Order article via Infotrieve]
-
Mitoma, J.,
Ito, M.,
Furuya, S.,
and Hirabayashi, Y.
(1998)
J. Neurosci. Res.
51,
712-722[CrossRef][Medline]
[Order article via Infotrieve]
-
Irie, F.,
and Hirabayashi, Y.
(1997)
Soc. Neurosci. Abstr.
23,
1156 (abstr. 461.6)
-
Snell, K.
(1986)
Trends Biochem. Sci.
11,
241-243[CrossRef]
-
Mitoma, J.,
Furuya, S.,
and Hirabayashi, Y.
(1998)
Neurosci. Res.
30,
195-199[CrossRef][Medline]
[Order article via Infotrieve]
-
Bligh, E. G.,
and Dyer, W. J.
(1959)
Can. J. Biochem.
37,
911-917
-
Skipski, V. P.,
and Barclay, M.
(1969)
Methods Enzymol.
14,
530-612
-
Wright, R. S.
(1971)
J. Chromatogr.
59,
220-221[CrossRef][Medline]
[Order article via Infotrieve]
-
Ryu, E. K.,
and MacCoss, M.
(1979)
J. Lipid Res.
20,
561-563[Abstract]
-
Hirabayashi, Y.,
Koketsu, K.,
Higashi, H.,
Suzuki, Y.,
Matsumoto, M.,
Sugimoto, M.,
and Ogawa, T.
(1986)
Biochim. Biophys. Acta
876,
178-182[Medline]
[Order article via Infotrieve]
-
Taki, T.,
Kasama, T.,
Handa, S.,
and Ishikawa, D.
(1994)
Anal. Biochem.
223,
232-238[CrossRef][Medline]
[Order article via Infotrieve]
-
Kanfer, J. N.
(1992)
Methods Enzymol.
209,
341-348[Medline]
[Order article via Infotrieve]
-
Tijburg, L. B. M.,
Geelen, M. J.,
and van Golde, L. M.
(1989)
Biochim. Biophys. Acta
1004,
1-19[Medline]
[Order article via Infotrieve]
-
Nores, G. A.,
Hanai, N.,
Levery, S. B.,
Eaton, H. L.,
Salyan, M. E. K.,
and Hakomori, S.
(1988)
Carbohydr. Res.
179,
393-410[CrossRef][Medline]
[Order article via Infotrieve]
-
Hidari, K. I.,
Irie, F.,
Suzuki, M.,
Kon, K.,
Ando, S.,
and Hirabayashi, Y.
(1993)
Biochem. J.
296,
259-263[Medline]
[Order article via Infotrieve]
-
Sastry, P. S.
(1985)
Prog. Lipid Res.
24,
69-176[CrossRef][Medline]
[Order article via Infotrieve]
-
Igarashi, H.,
Zama, K.,
and Katada, M.
(1958)
Nature
181,
1282-1283[Medline]
[Order article via Infotrieve]
-
Mark-Malchoff, D.,
Marinetti, G. B.,
Hare, G. D.,
and Meisler, A.
(1977)
Biochem. Biophys. Res. Commun.
75,
589-597[Medline]
[Order article via Infotrieve]
-
Mark-Malchoff, D.,
Marinetti, G. B.,
Hare, G. D.,
and Meisler, A.
(1978)
Biochemistry
17,
2684-2688[Medline]
[Order article via Infotrieve]
-
Lehninger, A. L.
(1982)
Principles of Biochemistry, pp. 620-621, Worth Publishers, Inc., New York
-
Shimada, N.,
Graf, R.,
Rosner, G.,
and Heiss, W. D.
(1993)
J. Neurochem.
60,
66-71[Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.