- Email: [email protected]
Serum lipoproteins as cholesterol donors to mycoplasma membranes
Vol.68,No.
BIOCHEMICAL
2, 1976
SERUM LIPOPROTEINS
RESEARCH COMMUNICATIONS
AS CHOLESTEROL DONORS TO MYCOPLASMA MEMBRANES
G.M. Slutzky, Biomembrane
AND BIOPHYSICAL
S. Razin,
and I.
Research Laboratory, Department The Hebrew University-Hadassah Jerusalem, Israel
Kahane
of Clinical Microbiology Medical School
S. Eisenberg Lipid
Received
Research Laboratory, Department Hadassah University Hospital Jerusalem, Israel
of Medicine
B
November 19,1975
SUMMARY: MycopZasmahominis and AchoZepZasmaZaidZawii were grown in media in which a fraction of human serum lipoproteins provided the sole source of cholesterol. Increasing levels of very low density lipoproteins had an inhibitory effect on the growth of the organisms. Low and high density lipoproteins in all concentrations proved to be excellent sources of cholesterol. Both organisms were able to limit the amount of cholesterol taken up and to preferentially incorporate free cholesterol despite an excess of esterified cholesterol in the medium. When similar levels of free cholesterol were provided by low density or high density lipoproteins, the organisms incorporated from 20-45% more cholesterol from the former. This preference for cholesterol from low density lipoproteins partially supports the theory that the low density lipoproteins act as a donor while the high density lipoproteins are a scavenger of cholesterol.
Understanding the freely
circulating
significance, control
of cholesterol
The rationale
lipoproteins*
parasitic
role this
uptake has taken
of evidence
using
procaryotes
that
these are
capable
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
529
than
of incorporating
faulty
of
as a model
(less
*Abbreviations: VLDL, very low density lipoproteins, LDL, low density lipoproteins, d 1.006-1.063 g/ml; lipoproteins, d 1.063-1.21 g/ml.
Copyright All rights
that
as cholesterol
minute
from
on new
cause
mycoplasmas
of serum lipoproteins is
by cells
suggests
may be a fundamental
We propose
the behind
of cholesterol
amount
uptake
(1).
investigating
diameter)
plasma
as an increasing
atherosclerosis for
the mechanism
system donors.
0.5 urn in large
d cl.006 g/ml; HDL, high density
Vol.68,
No. 2, 1976
quantities
BIOCHEMICAL
of cholesterol
into
of them require
cholesterol
the mycoplasmas
have
hence
their
plasma
and can be readily addition, nor
these
their
membrane
Previous
isolated
by osmotic
Cholesterol
choTestero1 serum
cholesterol
exposed lysis
synthesize
esters
(3),
this
other
to the
of the
cell
growth
cells
they
most
procaryotes nor
or esterify
and since
walls,
medium
(2).
In
cholesterol are
can be observed
laboratory, closely
incapable
of
as a strictly
process,
or as part Since
independent
in these
mycoplasmas,
our
studies
cholesterol
source.
This
paper
and relates
the
to current
the
concepts
fraction
the
as well
by utilizing reports
as part
serum are
take
up cholesterol
of metabolic
studies
of a bovine
to extend
findings
the organisms
serum lipoproteins
to the parasitic
the sterol-requiring
sterol-nonrequiring
that
was provided
complex
using
related
has indicated
we decided
serum
directly
all
and in fact
membranes
cannot
adsorption
fraction).
Unlike
of cholesterol
and the
AchoZepZasmaspecies,
(4-6).
membrane,
intracytoplasmic is
from
species
a physical
growth.
RESEARCH COMMUNICATIONS
(2).
work
through
plasma
membrane
uptake
phenomenon
?$copZasma
neither
cholesteryl
pinocytosis,
their
for
organisms
hydrolyze
AND BIOPHYSICAL
of a Tween (Difco
natural
lipoproteins
of the
80-
PPLO
donors
as to tissue
results
activity
of
cells,
as the
of these
experiments
functioning
of
lipoproteins.
MATERIALS
AND METHODS
MycopZasmahominis and AchoZepZasmaZaidZawii were grown in a modified Edward medium (7) in which the PPLO serum fraction was replaced by 0.5% fatty-acid-poor bovine serum albumin and elaidic acid (20 us/ml). Cholesterol was added as a component of human VLDL, LDL, or HDL prepared according to Have1 et aZ. (8). In some experiments LDL or HDL were labeled with 1251 as described by Bilheimer et ~2. (9), and more than 97% of the radioactivity was associated with the apoprotein moieties. A. ZaidZawii was harvested after 18 h and M. hominis after 36 h of incubation at 37 "C. Cell membranes were isolated by osmotic lysis (7) and extracted with chloroform:methanol (2:1, v/v). The lipid extracts from the membranes and from the lipoprotein fractions were analyzed frr total, free (unesterified), and esterified cholesterol (3, 10) and for lipid phosphorus (11).
530
Vol.68,
No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS
M. hominis and A. ZaidZLwii all
three
lipoprotein
required
the
free
inhibited
release
that
lipases
of both
(12)
on the
VLDL was found
membrane
(13).
growth
cholesterol
available
concentrations
20-45% more cholesterol
cholesterol free
in M.
cholesterol
content
cholesterol.
much lower
in HDL than
cells
grown
Thus,
the selectivitiy
on HDL were
higher in the
of cholesterol
in the ratio the ratio
was greater
in LDL only
was found
those
incorporation with
cholesterol than
at the
that
found
two highest
grown
of free
on
to esterified with
the large
increasing
excess
of
cholesterol
is
found
the membranes
of
from
in
cells
grown
over
to LDL.
by the
phosphorus.
in HDL, while
on LDL.
cholesterol
to HDL than
LDL concentrations.
531
donor
cholesterol
was reflected
to lipid
were
up by cells
of free
respect
M. hominis
to esterified
the ratios
than
serum,
of LDL and
to increase
despite
by the organisms
of free
At similar
of free
in LDL, but
was more pronounced
uptake
found
The ratio
horse
to be a better
The ratio
on HDL.
of the medium,
with
membranes.
was taken
membranes
hominis
esterified
esterified
grown
HDL.
Very
of the mycoplasmas,
quantities
LDL appeared
than
activity.
media.
of the mycoplasma
to the membranes
possibility
concentrations
when larger
of
of the erythrocyte
growth
of increasing
action
enzymatic
in mycoplasma
However,
to cells
by the Another
excellent
of cholesterol
LDL when compared
acids
the Mg++-ATPase
more cholesterol
in the medium.
fatty
10 ug/ml
may have been due to
on a vital
source
content
incorporated
This
in medium supplemented
I shows the effects
HDL on the cholesterol cells
effect
from
exceeding
VLDL triglycerides.
to stimulate
than
conventional Table
concentrations
LDL and HDL enabled
as good or better
cholesterol
VLDL in concentrations
of free
VLDL has a regulatory
free
organisms.
quantities
recently
the
However,
cholesterol
growth
of large
mycoplasma is
species.
to provide
medium
incorporated
increase
In all it
The
exceeded
cases that
I.
60.0
HDL
HDL
'
B
Only
free
free
to
(pg/mg
added
esterified
were
was found
to
cholesterol
reSpE!CtiVely.
of
of
cholesterol
ratio
72 (with
A. hidZa#ii
levels
Average
cholesterol.
0.25
24.0
HDL
free
0.25
6.0
LDL
lipoproteins
0.33
144.0
LDL
isolated
0.33
72.0
LDL
The
0.33
0.25
0.33
7.2
the
have
been
cholesterol
membrane
to
0.13
0.13
0.13
1.11
1.11
1.11
1.11
(molar
ratio)
(molar
(w/m!
1
lipid
cholesterol
and
2:l)
in
medium
62.1
55.1
49.4
111.1
107.4
78.4
66.2
(m/v)
protein
into
and
Free/
the
31 (with
in
0.88
0.81
0.59
1.33
1.16
0.92
0.83
provide
ratio)
Pi
membranes.
cholesterol)
supplemented
to
(molar
lipid
the
for
serum
M. hominis
horse
levels
0.44
0.53
0.44
0.55
0.65
0.55
0.26
of
Pi ratic)
were and
(molar
lipid
CholesterolC/
A. laidluwii
indicated
with
31.3
39.8
35.4
52.3
4.5.9
50.3
25.7
(Wms)
protein
CholesterolC/
membranesB
Ifpop;-o.Leins
choies;terol/
Free
on medium no esterified
grcrwn
suPficient
ratio)
A. ZaidZatrii
3.50
3.06
1.68
3.58
2.50
1.49
1.09
(molar
cholesterol
esterified
quantities
organisms
in
Cholesterol/
serum
Cholesterol
from
M. hominis
AchoZepZasma ZaidZawii
incorporated
of
protein)
growth
ratio)
Pi
cholesterol/
Free/
Free
mediumA
esterified
in
Myycoplama 3wminis
cholesterol
28.2
A
by
Cholesterol
uptake
Free
Cholesterol
LDL
added
Lipoproteins
TABLE
Vol.68,No.
2, 1976
TABLE II.
BIOCHEMICAL
Comparison
of binding
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the protein
LDL and HDL by mycoplasma 125
I-labeled
and cholesterol
moieties
of
membranes
M. hominis
A. ZaidZawii
lipoporteinA Type
Radioactivity
Percent
in medium
lipoprotein 125 I protein bound
(total
cpm)
of
Percent
of
Percent
lipoprotein
of
Percent lipoprotein
free
lipoprotein 125 I protein
cholesterol
bound
cholesterol
of
free
bound
bound
LDL
3.72
x lo6
0.78
10.5
0.70
5.8
LDL
7.40
x lo6
0.99
8.3
0.52
3.4
HDL
1.04
x lo7
0.07
20.0
0.02
8.8
HDL
2.02
x lo7
0.05
11.0
0.02
4.6
A LDL (s ecific activity 1.60 x lo6 cpm/mg protein) or HDL (specific activity 1.96 x 10 Ii cpm/mg protein) was added to the growth medium to provide cholesterol concentrations of 28 and 55 ug cholesterol/ml medium from LDL and 13 and 25 ug cholesterol/ml medium from HDL. More than 97% of the added label was associated with the apoprotein moieties of the lipoproteins. Radioactivity of isolated cell membranes was measured with a Packard Autogamma Scintillation spectrometer. The amount of radioactivity found in the membrane lipids extracted with chloroformmethanol was negligible.
of A. ZaidZawii was about
The growth
of LDL or HDL as well independence
from
concentration
cholesterol.
of free
a maximum of only at the
lowest
free
cholesterol
incorporated
into
to phospholipid found
about
from
in LDL but
total
absence,
Table
cholesterol
in the medium,
levels, LDL than
it from
was Present the membranes
in the membranes exceeded
that
HDL.
that
in the medium, o? A. ZaidZawii.
found
in HDL.
533
cases
known
the high
the cells
Although
was in all
its
despite
membrane
was found
concentrations
reflecting
I shows that
50 ug cholesterol/mg
lipoprotein
more cholesterol than
as in their
the same in all
incorporated
protein.
Except
the cells
incorporated
much more esterified only
the latter
The ratio lower
than
was
of cholesterol that
VoL68,No.
BIOCHEMICAL
2, 1976
The binding to mycoplasma
II.
Table that
of the protein membranes
The table
of 1251-HDL
shows that
is
protein
was taken
that
the binding
that
of cholesterol.
moieties
compared
shows that
particles,
percentage
cholesterol;
labeled
125
of
particles
of cholesterol
I-LDL
protein
the table
derived
protein
the
labeled
from
moiety,
material
not
represent large
cholesterol
the
indicating related
bound
intact
in
exceeded
was not directly
contain
and no esterified
LDL and HDL
In addition,
to the
protein
Moreover,
the A. ZaidZatii
uptake
of cholesterol
up as compared
of the
as these
to the
binding
of A. ZaidZawii could
membranes
of 1251-labeled
by 10 to 30 fold.
a much higher
lipoprotein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to
to the
lipoprotein
amounts
of esterified
was found
associated
with
membranes.
DISCUSSION Animal studying
cells
in culture
cholesterol
interpretation
uptake
synthesis
cholesterol
by the cells
pinocytosed
by tissue
plasma
hamper the
membrane
the analysis
lipoprotein from
show that
both
mechanisms
for
up from
18).
the
(14,
with
of sterols,
(16).
of the
tissue
tissue
fact
Moreover, cells
of the direct
system However,
cells
is complicated
that
interaction
the
the
of free
lipoproteins
difficulties
from
for
15).
the esterification
and by the
cells
experiments
are
in separating
intracytoplasmic
of the plasma
surface
Esterified
utilizing
mycoplasma
M. hominis and A. ZaidZawii appear restricting
the amount
the growth
of the
postulate particle
cholesterol
of free
Models
medium.
human LDL and HDL usually near
lipoproteins
obtained
(15),
as a model
membranes membrane
with
particle.
Results
taken
from
of the results
by the endogenous
the
have been employed
where is
usually
534
presence it
to possess
or esterified
suggested
the
as a made1 system
cholesterol
for
the structures
of free
cholesterol
may be readily thought
effective
to occur
exchanged in a more
of
(17,
BIOCHEMICAL
Vol. 68, No. 2, 1976
secluded
region,
perhaps
of the esterified could
while
from
Our results
(19). while
showing
from
HDL and the
lend
support
to this
It
to incorporate
uptake
affinity
cell
as free
large that
amounts
of the cells
or
cholesterol, of it
LDL may be a donor
of cholesterol
of cholesterol
which
membrane,
available
has been suggested
the greater
The location
the amount
to the
may be as readily
HDL may be a scavenger
greater
restrict
particle
of the membranes
may be restricted
of the particle.
may in itself
an intact
cholesterol
the ability
of cholesterol
the center
cholesterol
be transfered
the esterified
near
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(14,
15, 20).
from LDL than
to 125 I-protein
from
LDL
suggestion.
ACKNOWLEDGEMENT This research was supported by a grant from the United StatesIsrael Binational Science Foundation (BSF), Jerusalem, Israel. Our thanks are due to Miss P. Edelstein and Miss G. Halevi for expert technical assistance.
REFERENCES
:: 3. 4. 5.
;: 8. 9.
Papahadjopoulos, D. (1974) J. Theor. Biol. 43, 329-337. Razin, S. (1974) Progress in Surface and Mei&-ane Science (Danielli, J.F., Rosenberg, M.D., and Cadenhead, D.A., eds.), 2, pp. 257-312, Academic Press, New York. Argaman, M., and Razin, S. (1965) J. Gen. Microbial. 38, 153-168. Gershfeld, N.L., Wormser, M., and Razin, S. (1974) Bizhim. Biophys. Acta 352, 371-384. Razin, S., Wormser, M., and Gershfeld, N.L. (1974) Biochim. Biophys. Acta 352, 385-396. Razin, S. (1974) FEBS Lett. 47, 81-85. Razin, S., and Rottem, S. (1974) Methods in Enzymology (Fleischer, S ., and Packer, L., eds.), 32-, pp. 459-468, Academic Press, New York. Havel, R.J., Eder, H.A., and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353. Bilhelmer, D.W., Eisenberg, S., and Levy, R.I. (1972) Biochim, Biophys. Acta 260, 212-221. and Morris, M.D. (1973) J. Lipid Res. 14, 364-366. Rudel, L.L., Ames, B.N. (1966) Methods in Enzymology (Neufeld, ET., and S, pp. 115-118, Academic Press, New York. Ginsburg, V., eds.), S. (1964) J. Gen. Microbial. 37, 123-134. Rottem, S., and Razin, Shore, V., and Shore, B. (1975) Biochem. Biophys. Res. Commun. 65, 1250-1256.
1’7: 12. 13.
535
Vol. 68, No. 2, 1976
14. 15. 16. 17. 18. 19. 20.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bates, S.R., and Rothblat, G.H. (1974) Biochim. Biophys. Acta 2, 38-55. Goldstein, J.L., Dana, S.E., and Brown, M.S. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4288-4292. Stein, O., Stein, Y., and Eisenberg, S. (1973) Z. Zellforsch. 138, 223-237. Jackson, R.L., Morrisett, J.D., Gotto, A.M., and Segrest, J.P. (1975) Molec. and Cell Biochem. 6, 43-50. Eisenberg, S., and Levy, R.I. (1975rAdvances in Lipid Research (in press) Small, D.H. (1970) Surface Chemistry of Biological Systems (Blank, M., ed.), pp. 55-83, Plenum Press, New York. Stein, Y., Glangeaud, M.C., Fainam, M., and Stein, 0. (1975) Biochim. Biophys. Acta 380, 106-118.
536
BIOCHEMICAL
2, 1976
SERUM LIPOPROTEINS
RESEARCH COMMUNICATIONS
AS CHOLESTEROL DONORS TO MYCOPLASMA MEMBRANES
G.M. Slutzky, Biomembrane
AND BIOPHYSICAL
S. Razin,
and I.
Research Laboratory, Department The Hebrew University-Hadassah Jerusalem, Israel
Kahane
of Clinical Microbiology Medical School
S. Eisenberg Lipid
Received
Research Laboratory, Department Hadassah University Hospital Jerusalem, Israel
of Medicine
B
November 19,1975
SUMMARY: MycopZasmahominis and AchoZepZasmaZaidZawii were grown in media in which a fraction of human serum lipoproteins provided the sole source of cholesterol. Increasing levels of very low density lipoproteins had an inhibitory effect on the growth of the organisms. Low and high density lipoproteins in all concentrations proved to be excellent sources of cholesterol. Both organisms were able to limit the amount of cholesterol taken up and to preferentially incorporate free cholesterol despite an excess of esterified cholesterol in the medium. When similar levels of free cholesterol were provided by low density or high density lipoproteins, the organisms incorporated from 20-45% more cholesterol from the former. This preference for cholesterol from low density lipoproteins partially supports the theory that the low density lipoproteins act as a donor while the high density lipoproteins are a scavenger of cholesterol.
Understanding the freely
circulating
significance, control
of cholesterol
The rationale
lipoproteins*
parasitic
role this
uptake has taken
of evidence
using
procaryotes
that
these are
capable
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
529
than
of incorporating
faulty
of
as a model
(less
*Abbreviations: VLDL, very low density lipoproteins, LDL, low density lipoproteins, d 1.006-1.063 g/ml; lipoproteins, d 1.063-1.21 g/ml.
Copyright All rights
that
as cholesterol
minute
from
on new
cause
mycoplasmas
of serum lipoproteins is
by cells
suggests
may be a fundamental
We propose
the behind
of cholesterol
amount
uptake
(1).
investigating
diameter)
plasma
as an increasing
atherosclerosis for
the mechanism
system donors.
0.5 urn in large
d cl.006 g/ml; HDL, high density
Vol.68,
No. 2, 1976
quantities
BIOCHEMICAL
of cholesterol
into
of them require
cholesterol
the mycoplasmas
have
hence
their
plasma
and can be readily addition, nor
these
their
membrane
Previous
isolated
by osmotic
Cholesterol
choTestero1 serum
cholesterol
exposed lysis
synthesize
esters
(3),
this
other
to the
of the
cell
growth
cells
they
most
procaryotes nor
or esterify
and since
walls,
medium
(2).
In
cholesterol are
can be observed
laboratory, closely
incapable
of
as a strictly
process,
or as part Since
independent
in these
mycoplasmas,
our
studies
cholesterol
source.
This
paper
and relates
the
to current
the
concepts
fraction
the
as well
by utilizing reports
as part
serum are
take
up cholesterol
of metabolic
studies
of a bovine
to extend
findings
the organisms
serum lipoproteins
to the parasitic
the sterol-requiring
sterol-nonrequiring
that
was provided
complex
using
related
has indicated
we decided
serum
directly
all
and in fact
membranes
cannot
adsorption
fraction).
Unlike
of cholesterol
and the
AchoZepZasmaspecies,
(4-6).
membrane,
intracytoplasmic is
from
species
a physical
growth.
RESEARCH COMMUNICATIONS
(2).
work
through
plasma
membrane
uptake
phenomenon
?$copZasma
neither
cholesteryl
pinocytosis,
their
for
organisms
hydrolyze
AND BIOPHYSICAL
of a Tween (Difco
natural
lipoproteins
of the
80-
PPLO
donors
as to tissue
results
activity
of
cells,
as the
of these
experiments
functioning
of
lipoproteins.
MATERIALS
AND METHODS
MycopZasmahominis and AchoZepZasmaZaidZawii were grown in a modified Edward medium (7) in which the PPLO serum fraction was replaced by 0.5% fatty-acid-poor bovine serum albumin and elaidic acid (20 us/ml). Cholesterol was added as a component of human VLDL, LDL, or HDL prepared according to Have1 et aZ. (8). In some experiments LDL or HDL were labeled with 1251 as described by Bilheimer et ~2. (9), and more than 97% of the radioactivity was associated with the apoprotein moieties. A. ZaidZawii was harvested after 18 h and M. hominis after 36 h of incubation at 37 "C. Cell membranes were isolated by osmotic lysis (7) and extracted with chloroform:methanol (2:1, v/v). The lipid extracts from the membranes and from the lipoprotein fractions were analyzed frr total, free (unesterified), and esterified cholesterol (3, 10) and for lipid phosphorus (11).
530
Vol.68,
No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS
M. hominis and A. ZaidZLwii all
three
lipoprotein
required
the
free
inhibited
release
that
lipases
of both
(12)
on the
VLDL was found
membrane
(13).
growth
cholesterol
available
concentrations
20-45% more cholesterol
cholesterol free
in M.
cholesterol
content
cholesterol.
much lower
in HDL than
cells
grown
Thus,
the selectivitiy
on HDL were
higher in the
of cholesterol
in the ratio the ratio
was greater
in LDL only
was found
those
incorporation with
cholesterol than
at the
that
found
two highest
grown
of free
on
to esterified with
the large
increasing
excess
of
cholesterol
is
found
the membranes
of
from
in
cells
grown
over
to LDL.
by the
phosphorus.
in HDL, while
on LDL.
cholesterol
to HDL than
LDL concentrations.
531
donor
cholesterol
was reflected
to lipid
were
up by cells
of free
respect
M. hominis
to esterified
the ratios
than
serum,
of LDL and
to increase
despite
by the organisms
of free
At similar
of free
in LDL, but
was more pronounced
uptake
found
The ratio
horse
to be a better
The ratio
on HDL.
of the medium,
with
membranes.
was taken
membranes
hominis
esterified
esterified
grown
HDL.
Very
of the mycoplasmas,
quantities
LDL appeared
than
activity.
media.
of the mycoplasma
to the membranes
possibility
concentrations
when larger
of
of the erythrocyte
growth
of increasing
action
enzymatic
in mycoplasma
However,
to cells
by the Another
excellent
of cholesterol
LDL when compared
acids
the Mg++-ATPase
more cholesterol
in the medium.
fatty
10 ug/ml
may have been due to
on a vital
source
content
incorporated
This
in medium supplemented
I shows the effects
HDL on the cholesterol cells
effect
from
exceeding
VLDL triglycerides.
to stimulate
than
conventional Table
concentrations
LDL and HDL enabled
as good or better
cholesterol
VLDL in concentrations
of free
VLDL has a regulatory
free
organisms.
quantities
recently
the
However,
cholesterol
growth
of large
mycoplasma is
species.
to provide
medium
incorporated
increase
In all it
The
exceeded
cases that
I.
60.0
HDL
HDL
'
B
Only
free
free
to
(pg/mg
added
esterified
were
was found
to
cholesterol
reSpE!CtiVely.
of
of
cholesterol
ratio
72 (with
A. hidZa#ii
levels
Average
cholesterol.
0.25
24.0
HDL
free
0.25
6.0
LDL
lipoproteins
0.33
144.0
LDL
isolated
0.33
72.0
LDL
The
0.33
0.25
0.33
7.2
the
have
been
cholesterol
membrane
to
0.13
0.13
0.13
1.11
1.11
1.11
1.11
(molar
ratio)
(molar
(w/m!
1
lipid
cholesterol
and
2:l)
in
medium
62.1
55.1
49.4
111.1
107.4
78.4
66.2
(m/v)
protein
into
and
Free/
the
31 (with
in
0.88
0.81
0.59
1.33
1.16
0.92
0.83
provide
ratio)
Pi
membranes.
cholesterol)
supplemented
to
(molar
lipid
the
for
serum
M. hominis
horse
levels
0.44
0.53
0.44
0.55
0.65
0.55
0.26
of
Pi ratic)
were and
(molar
lipid
CholesterolC/
A. laidluwii
indicated
with
31.3
39.8
35.4
52.3
4.5.9
50.3
25.7
(Wms)
protein
CholesterolC/
membranesB
Ifpop;-o.Leins
choies;terol/
Free
on medium no esterified
grcrwn
suPficient
ratio)
A. ZaidZatrii
3.50
3.06
1.68
3.58
2.50
1.49
1.09
(molar
cholesterol
esterified
quantities
organisms
in
Cholesterol/
serum
Cholesterol
from
M. hominis
AchoZepZasma ZaidZawii
incorporated
of
protein)
growth
ratio)
Pi
cholesterol/
Free/
Free
mediumA
esterified
in
Myycoplama 3wminis
cholesterol
28.2
A
by
Cholesterol
uptake
Free
Cholesterol
LDL
added
Lipoproteins
TABLE
Vol.68,No.
2, 1976
TABLE II.
BIOCHEMICAL
Comparison
of binding
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the protein
LDL and HDL by mycoplasma 125
I-labeled
and cholesterol
moieties
of
membranes
M. hominis
A. ZaidZawii
lipoporteinA Type
Radioactivity
Percent
in medium
lipoprotein 125 I protein bound
(total
cpm)
of
Percent
of
Percent
lipoprotein
of
Percent lipoprotein
free
lipoprotein 125 I protein
cholesterol
bound
cholesterol
of
free
bound
bound
LDL
3.72
x lo6
0.78
10.5
0.70
5.8
LDL
7.40
x lo6
0.99
8.3
0.52
3.4
HDL
1.04
x lo7
0.07
20.0
0.02
8.8
HDL
2.02
x lo7
0.05
11.0
0.02
4.6
A LDL (s ecific activity 1.60 x lo6 cpm/mg protein) or HDL (specific activity 1.96 x 10 Ii cpm/mg protein) was added to the growth medium to provide cholesterol concentrations of 28 and 55 ug cholesterol/ml medium from LDL and 13 and 25 ug cholesterol/ml medium from HDL. More than 97% of the added label was associated with the apoprotein moieties of the lipoproteins. Radioactivity of isolated cell membranes was measured with a Packard Autogamma Scintillation spectrometer. The amount of radioactivity found in the membrane lipids extracted with chloroformmethanol was negligible.
of A. ZaidZawii was about
The growth
of LDL or HDL as well independence
from
concentration
cholesterol.
of free
a maximum of only at the
lowest
free
cholesterol
incorporated
into
to phospholipid found
about
from
in LDL but
total
absence,
Table
cholesterol
in the medium,
levels, LDL than
it from
was Present the membranes
in the membranes exceeded
that
HDL.
that
in the medium, o? A. ZaidZawii.
found
in HDL.
533
cases
known
the high
the cells
Although
was in all
its
despite
membrane
was found
concentrations
reflecting
I shows that
50 ug cholesterol/mg
lipoprotein
more cholesterol than
as in their
the same in all
incorporated
protein.
Except
the cells
incorporated
much more esterified only
the latter
The ratio lower
than
was
of cholesterol that
VoL68,No.
BIOCHEMICAL
2, 1976
The binding to mycoplasma
II.
Table that
of the protein membranes
The table
of 1251-HDL
shows that
is
protein
was taken
that
the binding
that
of cholesterol.
moieties
compared
shows that
particles,
percentage
cholesterol;
labeled
125
of
particles
of cholesterol
I-LDL
protein
the table
derived
protein
the
labeled
from
moiety,
material
not
represent large
cholesterol
the
indicating related
bound
intact
in
exceeded
was not directly
contain
and no esterified
LDL and HDL
In addition,
to the
protein
Moreover,
the A. ZaidZatii
uptake
of cholesterol
up as compared
of the
as these
to the
binding
of A. ZaidZawii could
membranes
of 1251-labeled
by 10 to 30 fold.
a much higher
lipoprotein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to
to the
lipoprotein
amounts
of esterified
was found
associated
with
membranes.
DISCUSSION Animal studying
cells
in culture
cholesterol
interpretation
uptake
synthesis
cholesterol
by the cells
pinocytosed
by tissue
plasma
hamper the
membrane
the analysis
lipoprotein from
show that
both
mechanisms
for
up from
18).
the
(14,
with
of sterols,
(16).
of the
tissue
tissue
fact
Moreover, cells
of the direct
system However,
cells
is complicated
that
interaction
the
the
of free
lipoproteins
difficulties
from
for
15).
the esterification
and by the
cells
experiments
are
in separating
intracytoplasmic
of the plasma
surface
Esterified
utilizing
mycoplasma
M. hominis and A. ZaidZawii appear restricting
the amount
the growth
of the
postulate particle
cholesterol
of free
Models
medium.
human LDL and HDL usually near
lipoproteins
obtained
(15),
as a model
membranes membrane
with
particle.
Results
taken
from
of the results
by the endogenous
the
have been employed
where is
usually
534
presence it
to possess
or esterified
suggested
the
as a made1 system
cholesterol
for
the structures
of free
cholesterol
may be readily thought
effective
to occur
exchanged in a more
of
(17,
BIOCHEMICAL
Vol. 68, No. 2, 1976
secluded
region,
perhaps
of the esterified could
while
from
Our results
(19). while
showing
from
HDL and the
lend
support
to this
It
to incorporate
uptake
affinity
cell
as free
large that
amounts
of the cells
or
cholesterol, of it
LDL may be a donor
of cholesterol
of cholesterol
which
membrane,
available
has been suggested
the greater
The location
the amount
to the
may be as readily
HDL may be a scavenger
greater
restrict
particle
of the membranes
may be restricted
of the particle.
may in itself
an intact
cholesterol
the ability
of cholesterol
the center
cholesterol
be transfered
the esterified
near
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(14,
15, 20).
from LDL than
to 125 I-protein
from
LDL
suggestion.
ACKNOWLEDGEMENT This research was supported by a grant from the United StatesIsrael Binational Science Foundation (BSF), Jerusalem, Israel. Our thanks are due to Miss P. Edelstein and Miss G. Halevi for expert technical assistance.
REFERENCES
:: 3. 4. 5.
;: 8. 9.
Papahadjopoulos, D. (1974) J. Theor. Biol. 43, 329-337. Razin, S. (1974) Progress in Surface and Mei&-ane Science (Danielli, J.F., Rosenberg, M.D., and Cadenhead, D.A., eds.), 2, pp. 257-312, Academic Press, New York. Argaman, M., and Razin, S. (1965) J. Gen. Microbial. 38, 153-168. Gershfeld, N.L., Wormser, M., and Razin, S. (1974) Bizhim. Biophys. Acta 352, 371-384. Razin, S., Wormser, M., and Gershfeld, N.L. (1974) Biochim. Biophys. Acta 352, 385-396. Razin, S. (1974) FEBS Lett. 47, 81-85. Razin, S., and Rottem, S. (1974) Methods in Enzymology (Fleischer, S ., and Packer, L., eds.), 32-, pp. 459-468, Academic Press, New York. Havel, R.J., Eder, H.A., and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353. Bilhelmer, D.W., Eisenberg, S., and Levy, R.I. (1972) Biochim, Biophys. Acta 260, 212-221. and Morris, M.D. (1973) J. Lipid Res. 14, 364-366. Rudel, L.L., Ames, B.N. (1966) Methods in Enzymology (Neufeld, ET., and S, pp. 115-118, Academic Press, New York. Ginsburg, V., eds.), S. (1964) J. Gen. Microbial. 37, 123-134. Rottem, S., and Razin, Shore, V., and Shore, B. (1975) Biochem. Biophys. Res. Commun. 65, 1250-1256.
1’7: 12. 13.
535
Vol. 68, No. 2, 1976
14. 15. 16. 17. 18. 19. 20.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bates, S.R., and Rothblat, G.H. (1974) Biochim. Biophys. Acta 2, 38-55. Goldstein, J.L., Dana, S.E., and Brown, M.S. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4288-4292. Stein, O., Stein, Y., and Eisenberg, S. (1973) Z. Zellforsch. 138, 223-237. Jackson, R.L., Morrisett, J.D., Gotto, A.M., and Segrest, J.P. (1975) Molec. and Cell Biochem. 6, 43-50. Eisenberg, S., and Levy, R.I. (1975rAdvances in Lipid Research (in press) Small, D.H. (1970) Surface Chemistry of Biological Systems (Blank, M., ed.), pp. 55-83, Plenum Press, New York. Stein, Y., Glangeaud, M.C., Fainam, M., and Stein, 0. (1975) Biochim. Biophys. Acta 380, 106-118.
536