BIOCHEMICAL
Vol. 75, No. 3, 1977
ELEVATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF A THREONINE PHOSPHOLIPID
IN
POLYOMA VIRUS TRANSFORMED HAMSTER EMBRYO FIBROBLASTS
D. Mark-Malchoff,
University Dentistry, Microbiology
Received
February
J. D. Hare,
G. V. Marinetti,
A. Meisler
of Rochester School of Medicine and Departments of Biochemistry, and Medicine, Rochester, New York
16,1977
of the lipids of normal hamster embryo fibroblasts and SUMMARY: Analysis m virus transformed fibroblasts shows a decrease in phosphatidylcholine and phosphatidylethanolamine and a marked increase in a threonine phospholipid after transformation. Transformed cells also react differently with fluorodinitrobenzene and trinitrobenzenesulfonate. Phosphatidylethanolamine of transformed cells reacts to a greater extent with both probes. Phosphatidylserine and the threonine phospholipid of both cells do not react with trinitrobenzenesulfonate. The threonine phospholipid is provisionally identified as phosphatidylthreonine. INTRODUCTION: metabolism
is
the
cell
culture
of membrane
(16).
A review
recently
fibroblasts
to a reduction Abbreviations:
Copyright AI1 rights
fluidity
of the
but
changes
occur
modulation
of the surface
we report
of normal by Howard
a new type
upon transformation
and tumor
of lipid virus.
lipid
cholesterol
(l-4),
of these in part
to
of membrane
trans-
properties
of
recognition
and Howard
by polyoma
in phosphatidylethanolamine
with
to be related
and concomitant
lipids
to have altered
The significance
is believed
has been published paper
reported
(16-20).
or to modification
In this embryo
understood
are
These
or glycolipids
not well
properties
cells
composition.
(5-15)
alteration port
transformed
and lipid
phospholipids changes
Virus
cells
grown
in
(21).
alteration
in hamster
Transformation
and phosphatidylcholine
leads
and an
HEF = hamster embryo fib-oblasts; HFT = hamster fibroblasts transformed; FDNB = l-fluoro-2,4-dinitrobenzene; TNBS = 1,3,5trinitrobenzenesulfonate; DNP = dinitrophenyl; PE = phosphatidylethanolamine; PS = phosphatidylserine; PC = phosphatidylcholine; PT = threonine phospholipid (tentatively identified as phosphatidylthreonine); TNP = trinitrophenyl.
0 1977 by Academic of reproducfion in any
Press, Inc. form reserved.
589 ISSN
0006-29
IX
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.;
0 .i
0. I
:
0.c
5 g
0.2
5: : 0. I
MINUTES LEGENDS:
Figure 1. Hydrolysis
Analysis of Serine and Threonine Obtained of Phospholipids from HEF and HFT Cells.
by HCl
The phospholipids of HEF and HFT cells were extracted by the method of Bligh and Dyer (22) and hydrolyzed as explained in the text. The aqueous phase of the hydrolysates was evaporated to dryness at 23OC under nitrogen and dissolved in 1.0 ml of 0.07 M citrate buffer pH 3.5. Half ml aliquots were analyzed on the Spinco Amino Acid Analyzer. The tracings are shown in the figure. Profiles A and B represent HEF and HFT cells respectively. Very small amounts of aspartic and glutamic acid were found in both samples.
increase
in a threonine
phospholipid
which
has the properties
of phosphatidyl-
threonine. METHODS AND REAGENTS: Hamster embryo fibroblasts (HEF) and transformed fibroblasts (HFT) (transformed by polyoma virus) were grown in cell culture in Eagle's minimal essential medium modified with Earle's salts (Auto-Pow Flow Labs) containing 5% bovine fetal serum. Cells (between 90-115 x lo6
590
Vol. 75, No.3,
1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells) were extracted three times with chloroform-methanol 1:l and the extracts were rectified by the method of Bligh and Dyer (22) to remove nonlipid contaminants. Aliquots of lipids were analyzed directly by thin layer chromatography on silica gel plates (Merck & Darmstadt SG 5763) using a solvent mixture of chloroform:methanol:acetic acid:water (65:25:2:4 vol/vol). Other aliquots of the lipid extracts were hydrolyzed in 3N HCl at 1OOoC for 2 hours. The aqueous HCl phase was removed and evaporated to dryness under nitrogen at 23OC. The residues were analyzed for serine and threonine on the Spinco amino Other aliquots of the HCl hydrolysate were brought up to 2 ml acid analyzer. of 120mM NaHCO -4DmM NaCl buffer pH 8.5 and treated with 2mM fluorodinitrobenzene for 2 Elours at 37OC. The reaction medium was acidified with HCl extracted with ethyl acetate to obtain the dinitrophenyl derivatives of the amino acids (23). These derivatives were analyzed by thin layer chromatography on silica gel plates using chloroform:methanol:acetic acid:water (65:25:2:4 vol/vol) as solvent and compared to authentic standard DNP derivatives of serine, threonine and ethanolamine. The labeling of washed trypsinized HEF* and HFT* cells with 2mM fluorodinitrobenzene (FDNB)* or 2mM trinitrobenzenesulfonate (TNBS)* was carried out in 120mM NaCl -4OmM NaHCO buffer pH 8.5 at 230C for 1 hour. For these experiments suspensions of 3 O-40 x 106 cells were used per experiment. The cells were washed with buffer and the lipids extracted by the Bligh and Dyer technique (22). The intact lipids and the HCl hydrolysis products of the lipids were separated by thin layer chromatography as described above. The DNP and TNP derivatives of the amino-phospholipids and the DNP derivatives of serine and threonine were eluted with methanol and measured spectrophotometrically. The unreacted amino-phospholipids were eluted with 1N HCl in methanol and measured by analysis of their phosphate content after digestion with 70% perchloric acid. The percent of toal PE, PS or PT which reacted with FDNB or TNBS was then calculated. FDNB and TNBS were obtained from Pierce Chem. Co., Rockford, Ill. RESULTS:
Evidence
provisionally embryo major
for
identify
fibroblasts amino
analyzed
is
acid
shown
of the
cells
nearly
equal
cells,
serine
transformed phospholipid
amounts.
cells,
threonine
Analyzer.
used for
to authentic
the
serine
(profile
profile
for
and serine
are
given
were
rectified
are
in Table
is barely
we
is the
cells
to the same amount
studies
amino
to the dinitrophenyl
threonine
close
these
characterization
of normal
and threonine
occurs
which hamster
can be seen that
However,
data
component
to remove free
and comparison
It
hydrolysate
The quantitative
phospholipid
in transformed
1.
B) shows both
the major
The further by conversion
Amino Acid
is
(22)
in Figure
phospholipid
(profile
extract
Dyer method
of a threonine
as phosphatidylthreonine
on the Spinco
transformed
tography
the occurrence
A) the
present
1.
in
In normal
detectable.
In
as serine.
The
by the
Bligh
and
acids. of threonine
derivatives, samples
591
and serine
analysis
by thin
of dinitrophenylserine
was accomplished layer
chrcma-
(ONPSer)
BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Serine
and Threonine
Lipids
from
1
Content
Normal
of the
HCl Hydrolysate
and Transformed
Fibroblastsa
Normal
Cells
Transformed
nmoles Experiment
of
Cells
nmoles
1 Serine
94
59
3
46
526
163
13
151
Threonine
Experiment
2 Serine Threonine
a Lipid samples were hydrolyzed with 3 N HCl for 2 hours at 1OO'C. The hydrolysis products were analyzed on the Spinco Amino Acid Analyzer. Different amounts of lipids were used in experiments 1 and 2 and each experiment used a different batch of cells.
and dinitrophenylthreonine by the
layer
chromatography
and DNPSer are water
DNPThr).
0.40
65:25:2:4
It
is
phospholipid
have essentially
acid:water.
Since
expected
to have similar
2.
HEF cells
nearly
equal
amounts
formed
cells
is
cell
types
for
plates.
respectively noteworthy the
that
The Rf values
separated of DNPThr
the
intact
acid-
PS and the
threonine-
in chlorofonn:methanol:acetic
by only
group,
as compared
a methylene
they
would
be
mobilities.
very
of PS and PT.
decreased
completely
same mobility
in PE and PC content PS but
are
in chloroform-methanol-acetic
chromatographic
contain
1 hour
compounds
gel
PS and PT differ
The decrease Table
on silica
and 0.31
v/v.
These
of transformed
little
The total
PT whereas phospholipid
to normal
at 37OC in 120 mM NaHC03-40
592
cells.
cells
is shown
HFT cells
contain
content
of trans-
Incubation
mM NaCl buffer
in
of both pH 8.5
shows
BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Phospholipid
Content Hamster
2
of Normal
and Transformed
Embryo Fibroblasts
Normal
Cells
Transformed
nmoles Total
chloroform-methanol
Total
Bligh-Dyer
soluble
lipid
Pb
PC
Phosphatidylethanolamine Phosphatidylserine threonine
b
This
extract
includes
'
This
extract
represents
d
Represents
mainly
e
Represents
about
61.3
+ 1.1
(14)
55
+ 0.8
(11)
50
+ 1.6
(14)
14.4
+ 0.3
(5)
11.8 t 0.5
(7)
9.5
+ 0.8
(5)d
8.6 + 1.5
(7)e
+ 1
(3)
experiments
done
in duplicate
some non-lipid total
lipid
P free
phosphatidylserine equal
amounts
based
in Figure
little
degradation
of the
phospholipids
in the
transformed
treated
with
extent
with
reacts
to a greater with
threonine
TNBS and FDNB.
FDNB and TNBS.
FDNB.
with
In normal
cells
phospholipid that
even with
of both excess
(2)
from most on the
non-lipid
data
P.
in Figure
1.
and threonine
phospholipid,
1.
by lipases cells
cells
reacts'to
does PS.
10% of PS reacts cells
do not react
FDNB, the
these
in Table
phospholipid
FDNB than
under
is seen
PE of transformed
The threonine
extent
+ 1.3
or triplicate.
of phosphatidylserine
on the data
change
13.2
phosphate.
based
Another
worthy
(11)
21.6
Mean + SD of 2-11
acts
+ 1.1
phospholipid
a
are
cellsa
65.9
+
Phosphatidylcholine
very
P/lo6
Cells
conditions. 3 when cells a greater
of transformed Only 8% of this
cells lipid
re-
with
FDNB.
PS and the
with
TNBS.
It
PE does not react
is also
to completion
noteshowing
Vol.75,No.3,
1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Extent
of Reaction
of Phosphatidylethanolamine,
Phosphatidylserine, of Normal
(PS)
and Threonine
and Transformed Percent Which
3
Reacts
Phospholipid
Fibroblasts
of Total
(PE)
with
(PT)
FDNB and TNBSa
PE
Percent
With
Which
FDNB or TNBS FDNB Normal
Cells
64:
Transformed
Cells
of Total Reacts
FDNB or
TNBS
With TNBSb
FDNB
1
11.8
+
0.8
88 + 14
18.8
+ 12
PS or PT
TNBS
8.2 + 6 10.5
0
+ 3
a
Cells were reacted with FDNB or TNBS as explained represent the mean f SD of three experiments done
b
With normal cells PS was the major component which reacts with FDNB. However, with transformed cells, PT was the major component which reacts with FDNB. This was determined by thin layer chromatographic analysis of the HCl hydrolysis products of the phospholipids as explained in the
that
36% of this
lipid
is
transformed
cells.
phospholipid
is
DISCUSSION:
The present
addition
cholesterol
found
by others
stood
but
the
The inaccessible probably
to phospholipid
lipid,
cell
is
not accessible
tightly
work
presumed
content
The significance
that
contain
Assuming
that
our
to 12% in
in phospholipid
cells.
Changes
of transformed of these
cells
changes
changes
is
in the
type
in
in phosphohave been not well
under-
properties
of
lipids.
identification
is correct,
compared
text.
protein.
a change
to critical
these
Values
of PE, PS and the threonine
of transformed
to be related
cells
to membrane
demonstrates
content
membrane
phosphatidylthreonine
fraction bound
and glycolipid (1-21).
in normal
in the text. in triplicate.
0
of the
the following
594
threonine reactions
phospholipid
as
show the possible
BIOCHEMICAL
Vol. 75, No. 3, 1977
ways in which
phosphatidylthreonine PS + threonine
=
PT + serine
(2)
PE + threonine
2
PT + ethanolamine
(3)
PC + threonine
=
PT + choline
(4)
CDP-diacylglycerol
(5)
CDP-threonine
demonstrated
l-3
for
of reaction
Reaction (32,
(34).
to our
knowledge
Any of the
reaction
the most
base exchange threonine
Our results
with
of transformed
chemical
of those
tain
both
with
FDNB suggesting
lipids.
phospholipid
compared
or is masked
phospholipid
of intact
this
These
probe.
to PS.
appears
in some other cells
results
reacts suggest
(30,31).
of PT.
Since
been shown to occur we
in the
way. with that
595
differences
of labeling
find
synthesis so that
cells
indicating
in transformed
latter
of this
PS is more tightly
Furthermore TNBS yet 12-19%
cells
the
arrangement that
neither 12-19%
of the
in the
of the aminophospho-
primarily
topological It
cells
5 is hypothetical,
demonstrate
phospholipid,
a different
yet
serine
to have been altered
in transformed We also
PS and the threonine
but not
as serine.
The extent
cells.
cells
between
ones involved
appears
probes
by FDNB and TNBS is greater
accessability
likely
This
and CDP-
synthesis
4 has not
reaction
enzymes as well
CDP-choline
to the
been
threonine.
in prokaryote
in mammalian
reaction
have
(24-30).
for
Base exchange
recently
2 and 3 as being
cells
has been reported for
(35,36).
and since
can now recognize
membranes
cells
cells
One of the
of PT.
primarily
above can lead
in eukaryote
reactions
serine
in PC and PE, since
which
been demonstrated
in eukaryote
listed
cells:
PT + CMP
transformations
5 has been demonstrated
5 reactions
unequivocally
protein
has not
CDP-serine
transformed
and eukaryote
3) has been postulated
we see a decrease
lipids
prokaryote
for
in the
2 PT f CMP
base exchange
in both
but not for
and PC (Reaction
it
represent
Reaction
=
+ diacyglycerol
serine
A similar
ethanolamine
favor
+ threonine
4 has been demonstrated
33).
proven
may be formed
(1)
Reactions
type
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a greater that
con-
reacts unique bound
to
PS or the threonine
of the
PE reacts
PE and essentially
with none
Vol. 75, No.3,
BIOCHEMICAL
1977
of the
PS or the threonine
plasma
membrane
in these
Threonine egg (37) istence
and tuna
reported
(38).
type
of
than
PS or PT.
lipid
and conversion
phatidylserylthreonine.
yield
lower primarily
surface
of the
mobility
that type
of lipid
in our
serylthreonine
ACKNOWLEDGEMENTS: Supported in part Cancer Institute and Grant HL 02063,
in our of
suggest
threonine
chromatographic on HCl hydrolysis
is
unlikely
its
isolation We
may be phos-
since
for
since
hydrolysis.
phospholipid
system
ex-
system
require
alkaline
is unlikely
the
The threonine
chromatography
PT will
by mild the
as ours
but this
mobility
characterization
of hen
of phospholipid.
of phosphatidylglycerol
possibility This
as well
as a new type
have a lower
The complete
the
outer
in phospholipids
studies
to glycerophosphorylthreonine
have considered
a considerably
ester
would
on the
to occur
These
of phosphatidylthreonine
this
occurs
RESEARCH COMMUNICATIONS
cells.
muscle
as an 0-acyl
would
phospholipid
has been
may exist
AND BIOPHYSICAL
it
would
have
and furthermore 2 hours.
by Contract No. l-CP-45611, Heart and Lung Institute.
National
REFERENCES: :: 3. 4. Z: 7. f : I: 12. 13. 14. ::: :;: 19.
Bailey, J. M., Proc. Sac. Exp. Biol. Med. 107, 30 (1961). Rothblatt, G. H., Hartzell, R. W., Mailhe, H., Kritchevsky, D., Biochim. Biophys. Acta 116, 133 (1966). Rothblatt, G. H., Buckho, M. K., Kritchevsky, D., Biochim. Biophys. Acta, 164, 327 (1968). H. P. and McIntrye, J., J. Lipid Res. 17, 313 (1971) Harry, 0. S., Morris, Peterson, J. A., and Rubin, H., Exp. Cell Res. 58, 365 (1969). Cunningham, D. D., J. Biol. Chem. 247, 2464 (1970). Pasternak, G. A. and Friedricks, B., Biochem. J., 119, 473 (1970). Lucas, D. D., Shohet, S. F. and Merler, E., J. Immunol. 106, 768 (1971). Quigley, J. P., Rifkin, D. P. and Reich, E., Virology 46, 106 (1971). Rytter, D. J. and Cornatzer, W. E., Lipids 7, 142 (1972). Wallach, D. F. H., Phospholipids in Membrane Molecular Biology of NeoPlastic Cells, Elsevier Scientific Publishing Co., Amsterdam, 1975 p. 141. Cunningham, D., J. Biol. Chem. 247, 2464 (1972). Peterson, J. A. and Rubins, H., Exp. Cell Res. 60, 383 (1970). Bergelson, L. S., Dyatlovitskaya, E. V., Torkhovskaya, T. I., Sorokina, I. B. and Gorkova, N. P., Biochim. Biophys. Acta 210, 287 (1970). Howard, B. V. and Kritchevsky, D., rnt. 3. Cancer 4, 393 (1969). Fishman, P. H. and Brady, R. O., Science 194, 906 (1976). Hirschberg, C. B., and Robbins, P. W., Virolo 61, 602 (1974). Biophys. Acta 417, 55 9"1975). Hakomori, S., Biochim. Itaya, K., Hakomori, S., and Klein, G., Proc. Nat. Acad. Sci., U.S.A. 73, 1568 (1976).
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20. 21. 22. 23.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hakomori, S. and Murakami, W. T., Proc. Nat. Acad. Sci., U.S.A. 59, 254 (1968). Pharmacol 10, 135 (1975). Howard, B. V., Howard, W. J., J. Prog. Biochem. Bligh, E. G. and Dyer, W. J., Can. J. Biochem. Physiol. 37, 911 (1959). Gordesky, S. E., Marinetti, G. V. and Love, R., J. Memb. Biol. 20, 111
Tai, !!'$!i*Matsumoto, M., Jap. J. Exp. Med. 43, 219 (1973). Borkenhagen, L. F., Kennedy, E. P., and Fielding, L., J. Biol. Chem. 236, 236 (1961). Dils, R. R., and Hubscher, G., Biochim. Biophys. Acta 46, 505 (1961). 2267: Crone, H. D., Riochem. J., 104, 695 (1967). 28. Procellati, G., Arienti, G., Pirotta, M. and Girogini, G , J. Neurochem. 18, 1395 (1971). 29. Kanfer, J. N., J. Lipid Res. 13, 468 (1972). 30. Marggraf, W., and Anderer, F. A., Hoppe-Seyler's Z. Phys 01. Chem. 355, 1299 (1974). 31. Diringer, H., Hoppe-Seyler's Z. Physiol. Chem. 354, 577 1973). 32. Kanfer, J. and Kennedy, E. P., J. Biol. Chem. 239, 1720 1964). Metabolism, ed. by Salih J. Wak 1, Academic 33. Lennarz, W. J., in Lipid Press, New York 1970, pp. 155-277. 34. Hoffman, R., Ristow, H. J., Pachowsky, H. and Frank, W., Eur. J. Biochem. 49, 317 (1974). 35. 2nd Ed., Worth Publishers, Inc. pp. 671Lehninger, A. L., Biochemistry, 675 (1976). 36. McMurray, W. C. and Magee, W. L., Ann. Rev. Biochem. 41, 129 (1972). Rhodes, D. N. and Lea, C. H., Biochem. 65, 526 (1957). 37. 38. Igarashi, H., Zama, K., and Kalada, M., Nature 181, 1282 (1958).
24. 25.
597
Vol. 75, No. 3, 1977
ELEVATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF A THREONINE PHOSPHOLIPID
IN
POLYOMA VIRUS TRANSFORMED HAMSTER EMBRYO FIBROBLASTS
D. Mark-Malchoff,
University Dentistry, Microbiology
Received
February
J. D. Hare,
G. V. Marinetti,
A. Meisler
of Rochester School of Medicine and Departments of Biochemistry, and Medicine, Rochester, New York
16,1977
of the lipids of normal hamster embryo fibroblasts and SUMMARY: Analysis m virus transformed fibroblasts shows a decrease in phosphatidylcholine and phosphatidylethanolamine and a marked increase in a threonine phospholipid after transformation. Transformed cells also react differently with fluorodinitrobenzene and trinitrobenzenesulfonate. Phosphatidylethanolamine of transformed cells reacts to a greater extent with both probes. Phosphatidylserine and the threonine phospholipid of both cells do not react with trinitrobenzenesulfonate. The threonine phospholipid is provisionally identified as phosphatidylthreonine. INTRODUCTION: metabolism
is
the
cell
culture
of membrane
(16).
A review
recently
fibroblasts
to a reduction Abbreviations:
Copyright AI1 rights
fluidity
of the
but
changes
occur
modulation
of the surface
we report
of normal by Howard
a new type
upon transformation
and tumor
of lipid virus.
lipid
cholesterol
(l-4),
of these in part
to
of membrane
trans-
properties
of
recognition
and Howard
by polyoma
in phosphatidylethanolamine
with
to be related
and concomitant
lipids
to have altered
The significance
is believed
has been published paper
reported
(16-20).
or to modification
In this embryo
understood
are
These
or glycolipids
not well
properties
cells
composition.
(5-15)
alteration port
transformed
and lipid
phospholipids changes
Virus
cells
grown
in
(21).
alteration
in hamster
Transformation
and phosphatidylcholine
leads
and an
HEF = hamster embryo fib-oblasts; HFT = hamster fibroblasts transformed; FDNB = l-fluoro-2,4-dinitrobenzene; TNBS = 1,3,5trinitrobenzenesulfonate; DNP = dinitrophenyl; PE = phosphatidylethanolamine; PS = phosphatidylserine; PC = phosphatidylcholine; PT = threonine phospholipid (tentatively identified as phosphatidylthreonine); TNP = trinitrophenyl.
0 1977 by Academic of reproducfion in any
Press, Inc. form reserved.
589 ISSN
0006-29
IX
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.;
0 .i
0. I
:
0.c
5 g
0.2
5: : 0. I
MINUTES LEGENDS:
Figure 1. Hydrolysis
Analysis of Serine and Threonine Obtained of Phospholipids from HEF and HFT Cells.
by HCl
The phospholipids of HEF and HFT cells were extracted by the method of Bligh and Dyer (22) and hydrolyzed as explained in the text. The aqueous phase of the hydrolysates was evaporated to dryness at 23OC under nitrogen and dissolved in 1.0 ml of 0.07 M citrate buffer pH 3.5. Half ml aliquots were analyzed on the Spinco Amino Acid Analyzer. The tracings are shown in the figure. Profiles A and B represent HEF and HFT cells respectively. Very small amounts of aspartic and glutamic acid were found in both samples.
increase
in a threonine
phospholipid
which
has the properties
of phosphatidyl-
threonine. METHODS AND REAGENTS: Hamster embryo fibroblasts (HEF) and transformed fibroblasts (HFT) (transformed by polyoma virus) were grown in cell culture in Eagle's minimal essential medium modified with Earle's salts (Auto-Pow Flow Labs) containing 5% bovine fetal serum. Cells (between 90-115 x lo6
590
Vol. 75, No.3,
1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells) were extracted three times with chloroform-methanol 1:l and the extracts were rectified by the method of Bligh and Dyer (22) to remove nonlipid contaminants. Aliquots of lipids were analyzed directly by thin layer chromatography on silica gel plates (Merck & Darmstadt SG 5763) using a solvent mixture of chloroform:methanol:acetic acid:water (65:25:2:4 vol/vol). Other aliquots of the lipid extracts were hydrolyzed in 3N HCl at 1OOoC for 2 hours. The aqueous HCl phase was removed and evaporated to dryness under nitrogen at 23OC. The residues were analyzed for serine and threonine on the Spinco amino Other aliquots of the HCl hydrolysate were brought up to 2 ml acid analyzer. of 120mM NaHCO -4DmM NaCl buffer pH 8.5 and treated with 2mM fluorodinitrobenzene for 2 Elours at 37OC. The reaction medium was acidified with HCl extracted with ethyl acetate to obtain the dinitrophenyl derivatives of the amino acids (23). These derivatives were analyzed by thin layer chromatography on silica gel plates using chloroform:methanol:acetic acid:water (65:25:2:4 vol/vol) as solvent and compared to authentic standard DNP derivatives of serine, threonine and ethanolamine. The labeling of washed trypsinized HEF* and HFT* cells with 2mM fluorodinitrobenzene (FDNB)* or 2mM trinitrobenzenesulfonate (TNBS)* was carried out in 120mM NaCl -4OmM NaHCO buffer pH 8.5 at 230C for 1 hour. For these experiments suspensions of 3 O-40 x 106 cells were used per experiment. The cells were washed with buffer and the lipids extracted by the Bligh and Dyer technique (22). The intact lipids and the HCl hydrolysis products of the lipids were separated by thin layer chromatography as described above. The DNP and TNP derivatives of the amino-phospholipids and the DNP derivatives of serine and threonine were eluted with methanol and measured spectrophotometrically. The unreacted amino-phospholipids were eluted with 1N HCl in methanol and measured by analysis of their phosphate content after digestion with 70% perchloric acid. The percent of toal PE, PS or PT which reacted with FDNB or TNBS was then calculated. FDNB and TNBS were obtained from Pierce Chem. Co., Rockford, Ill. RESULTS:
Evidence
provisionally embryo major
for
identify
fibroblasts amino
analyzed
is
acid
shown
of the
cells
nearly
equal
cells,
serine
transformed phospholipid
amounts.
cells,
threonine
Analyzer.
used for
to authentic
the
serine
(profile
profile
for
and serine
are
given
were
rectified
are
in Table
is barely
we
is the
cells
to the same amount
studies
amino
to the dinitrophenyl
threonine
close
these
characterization
of normal
and threonine
occurs
which hamster
can be seen that
However,
data
component
to remove free
and comparison
It
hydrolysate
The quantitative
phospholipid
in transformed
1.
B) shows both
the major
The further by conversion
Amino Acid
is
(22)
in Figure
phospholipid
(profile
extract
Dyer method
of a threonine
as phosphatidylthreonine
on the Spinco
transformed
tography
the occurrence
A) the
present
1.
in
In normal
detectable.
In
as serine.
The
by the
Bligh
and
acids. of threonine
derivatives, samples
591
and serine
analysis
by thin
of dinitrophenylserine
was accomplished layer
chrcma-
(ONPSer)
BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Serine
and Threonine
Lipids
from
1
Content
Normal
of the
HCl Hydrolysate
and Transformed
Fibroblastsa
Normal
Cells
Transformed
nmoles Experiment
of
Cells
nmoles
1 Serine
94
59
3
46
526
163
13
151
Threonine
Experiment
2 Serine Threonine
a Lipid samples were hydrolyzed with 3 N HCl for 2 hours at 1OO'C. The hydrolysis products were analyzed on the Spinco Amino Acid Analyzer. Different amounts of lipids were used in experiments 1 and 2 and each experiment used a different batch of cells.
and dinitrophenylthreonine by the
layer
chromatography
and DNPSer are water
DNPThr).
0.40
65:25:2:4
It
is
phospholipid
have essentially
acid:water.
Since
expected
to have similar
2.
HEF cells
nearly
equal
amounts
formed
cells
is
cell
types
for
plates.
respectively noteworthy the
that
The Rf values
separated of DNPThr
the
intact
acid-
PS and the
threonine-
in chlorofonn:methanol:acetic
by only
group,
as compared
a methylene
they
would
be
mobilities.
very
of PS and PT.
decreased
completely
same mobility
in PE and PC content PS but
are
in chloroform-methanol-acetic
chromatographic
contain
1 hour
compounds
gel
PS and PT differ
The decrease Table
on silica
and 0.31
v/v.
These
of transformed
little
The total
PT whereas phospholipid
to normal
at 37OC in 120 mM NaHC03-40
592
cells.
cells
is shown
HFT cells
contain
content
of trans-
Incubation
mM NaCl buffer
in
of both pH 8.5
shows
BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Phospholipid
Content Hamster
2
of Normal
and Transformed
Embryo Fibroblasts
Normal
Cells
Transformed
nmoles Total
chloroform-methanol
Total
Bligh-Dyer
soluble
lipid
Pb
PC
Phosphatidylethanolamine Phosphatidylserine threonine
b
This
extract
includes
'
This
extract
represents
d
Represents
mainly
e
Represents
about
61.3
+ 1.1
(14)
55
+ 0.8
(11)
50
+ 1.6
(14)
14.4
+ 0.3
(5)
11.8 t 0.5
(7)
9.5
+ 0.8
(5)d
8.6 + 1.5
(7)e
+ 1
(3)
experiments
done
in duplicate
some non-lipid total
lipid
P free
phosphatidylserine equal
amounts
based
in Figure
little
degradation
of the
phospholipids
in the
transformed
treated
with
extent
with
reacts
to a greater with
threonine
TNBS and FDNB.
FDNB and TNBS.
FDNB.
with
In normal
cells
phospholipid that
even with
of both excess
(2)
from most on the
non-lipid
data
P.
in Figure
1.
and threonine
phospholipid,
1.
by lipases cells
cells
reacts'to
does PS.
10% of PS reacts cells
do not react
FDNB, the
these
in Table
phospholipid
FDNB than
under
is seen
PE of transformed
The threonine
extent
+ 1.3
or triplicate.
of phosphatidylserine
on the data
change
13.2
phosphate.
based
Another
worthy
(11)
21.6
Mean + SD of 2-11
acts
+ 1.1
phospholipid
a
are
cellsa
65.9
+
Phosphatidylcholine
very
P/lo6
Cells
conditions. 3 when cells a greater
of transformed Only 8% of this
cells lipid
re-
with
FDNB.
PS and the
with
TNBS.
It
PE does not react
is also
to completion
noteshowing
Vol.75,No.3,
1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Extent
of Reaction
of Phosphatidylethanolamine,
Phosphatidylserine, of Normal
(PS)
and Threonine
and Transformed Percent Which
3
Reacts
Phospholipid
Fibroblasts
of Total
(PE)
with
(PT)
FDNB and TNBSa
PE
Percent
With
Which
FDNB or TNBS FDNB Normal
Cells
64:
Transformed
Cells
of Total Reacts
FDNB or
TNBS
With TNBSb
FDNB
1
11.8
+
0.8
88 + 14
18.8
+ 12
PS or PT
TNBS
8.2 + 6 10.5
0
+ 3
a
Cells were reacted with FDNB or TNBS as explained represent the mean f SD of three experiments done
b
With normal cells PS was the major component which reacts with FDNB. However, with transformed cells, PT was the major component which reacts with FDNB. This was determined by thin layer chromatographic analysis of the HCl hydrolysis products of the phospholipids as explained in the
that
36% of this
lipid
is
transformed
cells.
phospholipid
is
DISCUSSION:
The present
addition
cholesterol
found
by others
stood
but
the
The inaccessible probably
to phospholipid
lipid,
cell
is
not accessible
tightly
work
presumed
content
The significance
that
contain
Assuming
that
our
to 12% in
in phospholipid
cells.
Changes
of transformed of these
cells
changes
changes
is
in the
type
in
in phosphohave been not well
under-
properties
of
lipids.
identification
is correct,
compared
text.
protein.
a change
to critical
these
Values
of PE, PS and the threonine
of transformed
to be related
cells
to membrane
demonstrates
content
membrane
phosphatidylthreonine
fraction bound
and glycolipid (1-21).
in normal
in the text. in triplicate.
0
of the
the following
594
threonine reactions
phospholipid
as
show the possible
BIOCHEMICAL
Vol. 75, No. 3, 1977
ways in which
phosphatidylthreonine PS + threonine
=
PT + serine
(2)
PE + threonine
2
PT + ethanolamine
(3)
PC + threonine
=
PT + choline
(4)
CDP-diacylglycerol
(5)
CDP-threonine
demonstrated
l-3
for
of reaction
Reaction (32,
(34).
to our
knowledge
Any of the
reaction
the most
base exchange threonine
Our results
with
of transformed
chemical
of those
tain
both
with
FDNB suggesting
lipids.
phospholipid
compared
or is masked
phospholipid
of intact
this
These
probe.
to PS.
appears
in some other cells
results
reacts suggest
(30,31).
of PT.
Since
been shown to occur we
in the
way. with that
595
differences
of labeling
find
synthesis so that
cells
indicating
in transformed
latter
of this
PS is more tightly
Furthermore TNBS yet 12-19%
cells
the
arrangement that
neither 12-19%
of the
in the
of the aminophospho-
primarily
topological It
cells
5 is hypothetical,
demonstrate
phospholipid,
a different
yet
serine
to have been altered
in transformed We also
PS and the threonine
but not
as serine.
The extent
cells.
cells
between
ones involved
appears
probes
by FDNB and TNBS is greater
accessability
likely
This
and CDP-
synthesis
4 has not
reaction
enzymes as well
CDP-choline
to the
been
threonine.
in prokaryote
in mammalian
reaction
have
(24-30).
for
Base exchange
recently
2 and 3 as being
cells
has been reported for
(35,36).
and since
can now recognize
membranes
cells
cells
One of the
of PT.
primarily
above can lead
in eukaryote
reactions
serine
in PC and PE, since
which
been demonstrated
in eukaryote
listed
cells:
PT + CMP
transformations
5 has been demonstrated
5 reactions
unequivocally
protein
has not
CDP-serine
transformed
and eukaryote
3) has been postulated
we see a decrease
lipids
prokaryote
for
in the
2 PT f CMP
base exchange
in both
but not for
and PC (Reaction
it
represent
Reaction
=
+ diacyglycerol
serine
A similar
ethanolamine
favor
+ threonine
4 has been demonstrated
33).
proven
may be formed
(1)
Reactions
type
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a greater that
con-
reacts unique bound
to
PS or the threonine
of the
PE reacts
PE and essentially
with none
Vol. 75, No.3,
BIOCHEMICAL
1977
of the
PS or the threonine
plasma
membrane
in these
Threonine egg (37) istence
and tuna
reported
(38).
type
of
than
PS or PT.
lipid
and conversion
phatidylserylthreonine.
yield
lower primarily
surface
of the
mobility
that type
of lipid
in our
serylthreonine
ACKNOWLEDGEMENTS: Supported in part Cancer Institute and Grant HL 02063,
in our of
suggest
threonine
chromatographic on HCl hydrolysis
is
unlikely
its
isolation We
may be phos-
since
for
since
hydrolysis.
phospholipid
system
ex-
system
require
alkaline
is unlikely
the
The threonine
chromatography
PT will
by mild the
as ours
but this
mobility
characterization
of hen
of phospholipid.
of phosphatidylglycerol
possibility This
as well
as a new type
have a lower
The complete
the
outer
in phospholipids
studies
to glycerophosphorylthreonine
have considered
a considerably
ester
would
on the
to occur
These
of phosphatidylthreonine
this
occurs
RESEARCH COMMUNICATIONS
cells.
muscle
as an 0-acyl
would
phospholipid
has been
may exist
AND BIOPHYSICAL
it
would
have
and furthermore 2 hours.
by Contract No. l-CP-45611, Heart and Lung Institute.
National
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597
