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Interaction of apoHDL with HDL and with other lipoproteins
Atherosclerosis Elsevier Publishing
Company,
INTERACTION
OF apoHDL
H. S. SODHI Department
R. GORDON
AND
439
- Printed in The Netherlands
WITH
HDL AND WITH
OTHER
LIPOPROTEINS
GOULD
Stanford Medical
of Medicine,
(Received
Amsterdam
School,
Stanford,
Calif.
(U.S.A.)
.4pril 24th, 1970)
SUMMARY
Ultracentrifugation which
at a density
one component
protein
was labeled
took place. Since no evidence
probable protein complex
that
these
subunits
results
(mol.wt.
(mol.wt.
approx.
has been previously of [iasI]apoHDL
approx.
The top fraction
results
small
and the large
HDL
obtained
by centrifuging
as
a mixture
of the labeled protein when
in addition
were obtained
of some complex
to the interchange on electrophoresis; to HDL
of HDL
to the bottom
in the
formation
between
of labeled protein
subunits.
[issI]HDL
after incubation
and to apoHDL
both
of which
1251.
Complex
formation
between
of a mixture.
d 1.063 but on recentrifugation
fraction
apoHDL
which sedimented
the occurrence
gave zones corresponding
ultracentrifugation
separated
of the relatively
which floated at d 1.21 in the presence
_ of [ lssI]HDL
indicating
and [i2sI]apoHDL
amounts
of labeled
it seems more
at the same density.
presence of apoHDL,
contained
was obtained
than that lipids move from HDL to apoHDL,
of [issI]apoHDL
was larger than the fraction
Similar
between
and HDL in
interchange
by interchange
15,000)
200,000)
of apoHDL
that
of intermediates
and HDL at d 1.21 lost less than 20%
The fraction
with apoHDL
iss1 showed
are explained
suggested.
it was recentrifuged
HDL
of 1.21 of mixtures
with
of [125IjapoHDL
[issI]apoHDL
60%
with serum of the
was demonstrated
of the labeled protein
half of the labeled protein
were mixed
by ultracentrifugation,
and LDL
A small fraction
label
and
sedimented. the
When trace
lipoprotein
was recovered
by
floated at fractions
in the
HDL
and only 1.5% in the LDL fraction.
This work was supported by U.S. Public Health Service Grant No. HE-08476 from the National Institutes of Health and, in part, by Grant No. MA-3431 from the Medical Research Council of Canada. Dr. H. S. SODHI was the recipient of a post-doctoral research fellowship from the N.I.H. His present address: Department of Medicine, University Hospital, University of Saskatchewan, Saskatoon, Canada. Atherosclerosis,
1970, 12: 439-450
440
H. S. SODHI, R. GORDON GOULD
Key words:
apoHDL
- Electrophoresis
- HDL
- 1251~labeled HDL
- Interaction
-
Interchange - LDL - Protein subunit - Ultracentrifugation
INTRODUCTION SCANU et al. have reported serum high density lipoprotein with lipoproteins
that
apoHDL,
(HDL)
in two ways2J.
by low temperature
When
part of it was found to have acquired authors interpreted a reconstituted
this to indicate
HDL2.
sufficient that
apoHDL
suspensions that
appeared
complex
apparent
conversion
determine
whether
with lipoproteins
after
It is unlikely
of <
of apoHDL
interacts with
properties
mixing
to HDL.
can actually
HDL,
of HDL;
with
the
to give
LDL
that apoHDL
was re(d 1.063-
could acquire was considered
and LDL. SCANU et al. recognized
with chylomicrons
as well as with
LDL.
may also occur with
HDL
was mixed
1.063 so this observation
to form complexes
formation
delipidizationl,
of lipids from HDL to apoHDL
of a complex of apoHDL
of triglycerides
from human
(d < 1.063), partly in the HDL fraction
1.21 fraction.
lipids to produce a density
to be due to the formation
obtained
apoHDL
the ultracentrifugal
[ 131IlapoHDL
covered partly in the LDL fraction 1.21) and partly in the d >
labeled
a transfer
However,
the protein
and even artificial
It appears
HDL
possible,
and account
therefore,
for some of the
We have carried out studies designed to
be reconstituted
and the results are reported
from apoHDL
by mixing
in this communication.
MATERIALS AND METHODS HDL, LDL, VLDL human collected density
serum
and chylomicrons
by ultracentrifugation
were isolated from normal postabsorptive
as previously
from the top 1 ml of the centrifuge of 1.006, VLDL
describedd.
tube after
were
x g at a
after 24 h at 1.006, LDL after 24 h at 1.063 and HDL after
24 h at 1.21. The HDL fraction
thus contained
both HDLz and HDLs.
ApoHDL
was obtained by delipidization
of HDL at -20X,
of the method
of SCANU et al.1 as previously
described4;
is free of all neutral
Chylomicrons
1 h at 104,000
lipids but contains
a few percent
using a modification
this gives a product of the original
which
amount
of
phospholipids. Preparation HDL inorganic
of [125I]HDL was labeled
and [l25I]apoHDL
with 1251 by the method
iodide was removed
G-100 column and dialysis. protein
and lipid staining
[125I]apoHDL
Electrophoresis material
1970, 12: 439-450
of HUNTER AND GREENWOODS;
of an ion exchange
resin or a Sephadex
of the product showed that 1251 activity,
all had the same mobility.
was prepared from [ 125I]HDL
of the total 1251 in [125I]HDL Atherosclerosis,
by means
as described
was found to be associated
above; about 20-30%
with the lipids.
INTERACTION
OF APOHDL
HDL
WITH
The lipids were extracted on thin-layer acetic
with chloroform-methanol
plates with a solvent
acid (75:25:1,
by vol.),
system
- ethyl ether - glacial
and the lipid zones were eluted
by the ferric chloride-sulfuric
by the hydroxamate
methods
and phospholipids
The protein in the ultracentrifugal by trichloroacetic
(2:1, v/v)s, fractionated
of light petroleum
lesterol was analyzed
precipitated
441
AND WITH OTHER LIPOPROTEINS
by the method
fractions
acid and thoroughly
acid to remove any traces of 1251 activitv
and analyzed.
acid color reaction’, containing
of
Cho-
triglycerides
BARTLETTg.
HDL and apoHDL
was
washed with dilute trichloroacetic
not associated
with the protein.
Paper electrophoresis Paper strip electrophoresis strength
was done in Verona1 buffer at pH 8.6 and at ionic
of 0.075 in a Spinco electrophoretic
were stained
with bromphenol
cell. The proteins
blue. The lipids were stained
the serum on paper strips with acetylated completion
Sudan Black
on the paper strips
either before applying
Bie or with Oil red 0 after
of the electrophoresisii.
In some experiments This was prepared determined
LDL containing
from human
LDL
in a liquid scintillation
determine
the distribution
counter
of radioactivity
cut into 0.125 or 0.25 inch wide sections in a well counter,
unesterified
[4-i%]cholesterol
and [4-iK]cholesterol
and 1251 in a NaI crystal
on paper electrophoretic and transferred
or into vials for counting
was used.
by exchange.
1% was
counter.
To
strips, they were
into test tubes for counting
in the liquid scintillation
counter.
RESULTS
Exchange of protein between HDL Ultracentr(fuge
short time at room temperature When [izsI]apoHDL
and aPoHDL
studies. Mixtures
and HDL
were incubated
at pH 8.6 and ultracentrifuged
was centrifuged
ed from the top 1 ml fraction
of apoHDL
at a density
alone at this density it was completely
(Table
l), but after incubation
mixture
at this density,
of [issI]HDL
some labeled
none was found in the bottom
and unlabeled
protein
was present
apoHDL
products
a mixture
most likely
due to sedimentation
and HDL
to the bottom
only [125I]apoHDL.
(Table
1). No lipids were delipidated
was centrifuged,
the
specific
was lower than that in the control
This decrease in the specific activity
of some unlabeled
protein
originally
was.
present
in
fraction.
These findings are consistent had acquired fraction
1 ml, but when a
to ultracentrifugation,
the absence of HDL or its partially
of [rssI]apoHDL
of the protein sedimenting
tube which contained the HDL
fraction
HDL,
When [isV]HDL
from this fraction.
When activity
was subjected
in the bottom
found in the bottom fraction indicating
of 1.21.
sediment-
with unlabeled
labeled protein was found in the top 1 ml as well as in the bottom. was centrifuged
for a
the ultracentrifugal
of the HDL
had acquired
with the hypothesis characteristics
that a fraction
of HDL
the ultracentrifugal
of the apoHDL
and conversely
characteristics Atherosclerosis,
that
of apoHDL. 1970, 12: 439-450
a
442
H. S. SODHI, R. GORDON GOULD
TABLE
1
ULTRACENTRIFUGAL
BEHAVIOR
apoHDL
LABELED
OF
PROTEINS
IN
THE
MIXTURES
CONTAINING
LABELED
AND
UN-
HDL
AND
The protein mixtures were incubated at room temperature for 30 min in 0.05 M Tris buffer at pH 8.6 before being centrifuged at d 1.21 to separate apoHDL from HDL. Top and bottom 1 ml samples were removed for precipitation and washing. There were significant losses of proteins in these procedures for determination of its specific activity. composition
SpecQic activity of proteins (countslminlmg 10-3)
apoHD L (m&Y protein)
0f mixture Total 1251 content 0j top 1 ml proteins HDL counts/&n bottom 1 ml Img counts/min 10-3 protein) . 10-3
oh of Labeled protein recovered
to9
bottom
1.5a 1.5% 1.28 I.78 0 2.0 2.0
0 1.5 1.5 0 2.0% 2.0a 2.0a
0.3 10.1 10.7 0 36 31.3 49.0
16.3 8.3 8.2 47 0 19 8.3
0.5 18.7 15.7 1.0 178.3 154.9 242.4
30.1 15.4 12.1 198.3 0.5 94.4 41.2
top 1 ml
bottom 1 ml
123 76 75 248
55 46 318 155 269
131 40
a ‘251-labeled component.
The presence of [125I]apoHDL
in the top fraction
of the processes:
(i) formation
density
1.21 or (ii) by interchange
less than
of complexes
can be explained
of HDL of protein
by either or both
and [izsI]apoHDL subunits
having
between
HDL
a
and
apoHDL. As judged
from the changes
protein
carried
to the bottom
fraction
of labeled
apoHDL
in specific activity
by the presence
carried
values,
of apoHDL
the fraction was smaller
to the top by the presence
of HDL
of HDL than
the
(Table
1,
lines 7 and 6). PaPer electrojdaoresis. Mixtures amounts
on electrophoresis
of apoHDL
to only the HDL zone. When [125I]apoHDL electrophoresis, zone (Fig. mixture
more of the radioactivity
1). Conversely, with the HDL,
when [ rssI]HDL although
found in the zone containing hypothesis
mentioned
and HDL were incubated was incubated
apoHDL
in connection
(Fig.
between
small amounts
with the ultracentrifugal of apoHDL
on, among other factors,
formation
it is not clear whether
of an HDL-apoHDL
of apoHDL. Atherosclerosis,
1970, 12: 439-450
and the remained
results,
was
with the
that an inter-
and HDL and also complex
and HDL.
1). This decrease
However,
before
of the 1251 activity
(Fig. 1). Th ese findings are consistent
the electrophoretic
in the mixture.
with apoHDL,
fraction
In the presence of apoHDL depended
together
the major part of the activity
a significant
change of protein units may take place between apoHDL formation
equal
was present in the HDL than in the apoHDL
applied on paper for electrophoresis,
associated
and HDL in approximately
gave two protein zones with the lipid stain corresponding
mobility
of HDL was decreased
the amounts
of apoHDL
this effect is specifically
complex or is a non-specific
due to
effect due to the presence
WITH HDL
INTERACTION OF APOHDL
443
AND WITH OTHER LIPOPROTEINS
Fig. 1. Electrophoretic behavior of apoHDL, HDL and mixtures of the two. [1251]HDL, [1251]apoHDL, and mixtures of each with an equal amount of unlabeled apoHDL or HDL respectively were applied on paper for electrophoresis. The radioactivity is shown by bar diagrams and the protein by bromphenol blue staining. Arrows indicate the point of application. The double underline indicates the component in which the label was added in the mixture.
Relative a$inity of apoHDL Lipoproteins lipoproteins
for various lipoproteins
in serum. To determine the relative affinity of apoHDL
in serum, relatively
small amounts
(6 ,~g) of apoHDL
2 ml of serum and the mixtures were centrifuged 1.5% of the labeled apoHDL
at densities of 1.063 or 1.21. About
added to serum rose to the top fraction
1.063 and about 13% with the lipoproteins
for different
were mixed with with LDL at
floating at d 1.21, corresponding
to almost
11.5% being carried to the top with HDL (Table 2). In other experiments
of serum
of labeled apoHDL
with
VLDL,
LDL and HDL were separated by successive flotation
centrifuge.
6 ml aliquots
even smaller amounts
mixed
and after 30 min incubation,
As shown in Table 3, 60%
fraction and relatively
of the [125I]apoHDL
small amounts in the other fractions.
bulin was treated in the same manner as [ 125IlapoHDL; HDL fraction.
Most of it sedimented
fluence its distribution. chylomicron containing
fraction.
at d 1.21 indicating
in a preparative
ultra-
was found in the HDL As a control,
[issI]y-glo-
less than 1% was found in the that lipoproteins
A relatively large percentage of [125I]apoHDL This result was in part due to contamination
chylomicrons
(0.8 ,ug) were chylomicrons,
was foundin the since the fraction
was collected after only 1 h of centrifugation Atheroscleroisi,
did not in-
and the chylo1970, 12: 439-450
444
H. S. SODHI, R. GORDON GOULD
TABLE
2
DISTRIBUTIONOF [‘=I]apoHDL
AFTER INCUBATIONWITH SERUM
[ie5I]apoHDL (50,000 counts/min in 6 pup) was added to 2 ml aliquots of serum and the densities adjusted to either 1.063 or 1.21. The mixtures were centrifuged at 104,000 x g for 24 h and then the activity (counts/min) in the top and the bottom 1 ml fractions was determined. Density
Mean y0 of Mean 0/0 of
+c S.E.M. total f S.E.M. total
TABLE
Counts/min~
1.063 1.063 1.21 1.21
10-3
topfraction
bottomfraction
0.756 i 0.050 1.5 6.42 * 0.50 12.8
14.31 * 0.48 28.6 0.811 * 0.032 1.6
3
RELATIVE AM~IJNT~CJF~~OHDLAND AFTER
MIXING
WITH
Y-GLOBULIN
FOUND
IN
DIFFERENT
LIPOPROTEIN
FRACTIONS
SERUM
[iaaI]apoHDL (0.8 ,ug) and y-globulin (1 .O ,ug) were mixed with 6 ml serum and fractions containing chylomicrons, VLDL, LDL and HDL were successively collected by floating them at appropriate densities. Percent of the total iasI activity present in each fraction was determined.
oftotal
Percent
[iaaI]apoHDL [iesI]y-globulin
activity
Chylo
VLDL
LDL
HDL
d >
25a 188
0.7 0.07
1.5 0.1
60 0.6
14 82
1.21
a The values for the chylomicron fraction are too high by about 8% because the time of centrifugation was too short to sediment the labeled protein from the top fraction. The chylomicrons were not washed and may have contained other lipoproteins and proteins.
microns
were not washed free of other proteins or lipoproteins
The top 1 ml fraction
initially
excess over this amount ciation
present in the mixture.
8% of the [12sI]apoHDL
was probably
or y-globulin;
carried up in more or less non-specific
the asso-
with chylomicrons.
Individual
lipoproteins.
and with purified lipoproteins lipoprotein conditions lipoprotein,
apoHDL however,
than
was associated the
amounts
could not be correlated
cholesterol
esters, triglycerides,
The bottom Atherosclerosis,
those
1 ml fractions
1970, 12: 439-450
with washed
of [issI]apoHDL
at the appropriate
of [rssI]apoHDL
greater
was mixed
and the amounts
data for y-globulin
the amounts
were considerably proteins;
[ issI]apoHDL
after recentrifugation
Table 4. Corresponding
proteins
contained
density
present
in different
with chylomicrons
of apoHDL
that
with their content or with the amount
with the
for 24 h are shown in
as a control are included.
of [rasI]y-globulin
chylomicrons
associated
Under the same
lipoprotein
(Table
fractions
4). Per unit mass
more than with other lipo-
combined
with
of phospholipids,
different
lipo-
free cholesterol,
of proteins.
in the ultracentrifuge
tubes
contained
significant
INTERACTION TABLE
OF APOHDL
WITH
HDL AND WITH OTHER LIPOPROTEINS
445
4
AMOUNTS
OF
[issI]apoHDL
1 mg of [is5I]apoHDL room temperature and the lipoproteins. From top ml, the amount of
AND
[1251]y-GLOBULIiY
ASSOCIATED
WITH
PLASMA
LIPOPROTEINS
(or [ isaI]y-globulin) was incubated with various lipoproteins for 30 min at then centrifuged at 104,000 xg at the appropriate density for 24 h to float determination of the lipoprotein content and the counts/min of isa1 in the labeled protein per mg of lipoprotein was calculated.
Lipoproteins
Chylo
VLDL
LDL
HDL
Amount of lipoprotein added (mg) ‘[issI]apoHDL (pg/mg lipoprotein) [issI]y-globulin (pg/mg lipoprotein)
2.280 13.0 4.0
17.971 3.0 0.6
45.476 1.5 0.4
13.326 6.0 0.7
amounts of [125I]apoHDL but no lipids indicating that apoHDL sedimenting at densities of 1.006 or 1.063 had not acquired any lipids from VLDL or LDL. Serum Z@ids. In the absence of plasma lipoproteins [issI]apoHDL sedimented .at d 1.21, but if plasma lipoproteins were present significant fractions of [rs5I]apoHDL rose to the top. It was of interest to determine the effect of serum lipids instead of plasma lipoproteins on the ultracentrifugal behavior of apoHDL. Lipids from 2 ml of serum samples dissolved in solutions of Tween 20 by the method of WHEREAT AND STAPLERS were mixed with 2 mg of [issI]apoHDL and the density adjusted to 1.063 g/ml. After centrifugation for 24 h at 104,000 x g, 1 ml of opalescent solution was removed from the top. The density of the remainder was adjusted to 1.21 and 1 ml ,of a clear solution was collected from the top after centrifugation for 24 h at 104,000 TABLE
5
RECENTRIFUGATION
OF
apoHDL-
LIPOPROTEIN
COMPLEXES
Each tube contained 0.85 mg [ issI]apoHDL; after addition of HDL or LDL, salt solution, Tris buffer and water the final volume was 9.0 ml and the pH 8.6. Tubes containing HDL were adjusted to d 1.21 and those containing LDL to d 1.063. After 30 min incubation at room temperature the tubes were centrifuged at 104,000 x g at 15°C for 40 h. The material in the top 1 ml fraction .of each tube was mixed with fresh salt solution of the appropriate density and centrifuged for 60 h and the top 1 ml fractions collected. is51 activity was measured and total cholesterol determined by analysis. The ratio of issI per mg cholesterol was calculated and also the percent of the 1251 per mg cholesterol present after one centrifugation which was recovered after the second centrifugation. Tube No.
Amount of 1ipopvotein (mg)
First centrifugation
Second centrifugation
countslmin 1zsr
ratio counts/ minlmg chol.
countslmin 1261
ratio counts/ minlmg chol.
Percent remaining
1 ‘2 3
HDL 11.2 22.4 44.8 LDL
13,300 11,450 13,800
3577 1694 974
5000 7000 7620
2971 1461 802
84 86 82
:: 3
22.4 11.2 44.8
2420 2150 3353
105 169 70
880 480 1000
55 68 39
52 40 56
Atherosclerosis,
1970, 12: 439-450
446
H. S. SODHI, R. GORDON GOULD
Fig. 2. Electrophoretic behavior of [iasI]apoHDL when mixed in relatively small amounts in serum or purified lipoproteins. Plbout 10 ~5 of [ i25IlapoHDL was incubated with 50 ~1 of serum, 800 p”g of LDL or 400 ~“g of HDL for 30 min at room temperature. After prestaining for lipids, with acetylated Sudan black B, 30 ,/_dof the mixtures were applied in duplicate for paper electrophoresis. The distribution of lipids and 1251 activity on the paper strips are shown.
x g. The amount initial amount; fraction
of 1251 activity
in the d 1.063-1.21
present in the d < or HDL fraction
was 1.5% of the 1.21
it was 30%.
Thin-layer
chromatography
showed that phospholipids
Recentvifugation After
of the lipids extracted
in about the same order of magnitude.
of [rssI]apoHDL containing
HDL
with HDL and
with salt solution of d 1.21 and recentrifuged. of loss of [issI]apoHDL
Cholesterol
and centrifugation
[issI]apoHDL
1970, 12: 439-450
(Table
diluted
The top 1 ml was collected to determine
from the HDL
were calculated.
on recentrifugation
at d 1.21,
was separated,
fraction.
analysis was used as an index of the amount
ratios of 1251 to cholesterol
Atherosclerosis,
fractions
but other lipids
stzcdies
incubation
the top 1 ml fraction the amount
from the three
were present mostly in the HDL fraction
were present in all fractions
change
1.063 fraction
it was 60% and in the d >
In the top fraction
5). In this experiment
of lipoprotein
and the
this ratio showed little
recentrifugation
of three
INTERACTION
OF
WITH HDL
APOHDL
AND
WITH
OTHER
447
LIPOPROTEINS
Fig. 3. Evidence that lipids are not transferred from LDL to apoHDL. 500 ,ug of LDL containing [14C]cholesterol was incubated with 100 ,ug of apoHDL and applied to paper electrophoresis (left hand figure). The same amount of LDL was used for control. The direction of electrophoresis was left to right. The top half of each strip was stained for lipids (oil red 0) and the bottom half for protein. Lipids are seen only in the LDL zone both by staining and by radioactivity. The apoHDL is seen in the “Test” diagram in zone 3. No protein or lipids are seen in zone 4 where HDL normally is found.
mixtures
of different
amounts
showed losses of only about In a second experiment Corresponding
Paper
experiments
staining.
of the [i2sI]apoHDL
of [125I]apoHDL
per mg of cholesterol.
changes were observed.
with LDL,
centrifuged
twice at d I .063, gave much the ratio of [issI]apoHDL
by about half.
Microgram
amounts
of [125I]apoHDL,
failed to give a well defined peak of radioactivity
in the absence
(HDL)
[125I]apoHDL
the [125I]apoHDL
nor 1251 activity with purified no detectable
than that in the p-lipoprotein
was mixed with purified HDL,
electrophoresis,
was observed
LDL,
corresponding
were found on paper strips (Fig. 2). The activity
zone was greater
(LDL)
in the a-lipozone. When
applied on paper for
was found only in the HDL zone. Neither
with the mobility
the [125I]apoHDL
1251 or protein
and the mixture
was found
was observed
of apoHDL associated
of
or to reveal protein by
When mixed with serum, however, two peaks of 1251 activity
to /3-and a-lipoproteins protein
amount
studies
electrophoresis.
lipoproteins,
with a standard
on the second centrifugation;
in LDL decreased
electrophoretic Paper
14-B%
no significant
greater losses of [i25I]apoHDL to cholesterol
of HDL
protein
(Fig. 3). When mixed only with LDL;
in the zone where
HDL
and
or apoHDL
would be expected. Atherosclerosis,
1970, 12: 439-450
448
H. S. SODHI, R. GORDON GOULD Control
plasma
experiments
lipoproteins
with
indicated
mixtures
of [ issI]y-globulin
that the mobility
with
of [125I]yglobulin
serum
or with
was not affected
by the presence of serum or purified lipoproteins. Electrophoresis gave little protein)
of a mixture
evidence
of either
(Fig. 3), indicating
of apoHDL
protein
and LDL
or isotope
that apoHDL
labeled
with [lT_i’cholesterol
with the mobility
was not converted
of HDL
(a-lipo-
into HDL by acquiring
lipids from LDL. In viva studies V’sI]apoHDL L the distribution
prepared
from dog serum was injected
of the label in the LDL,
in the intact dog at various time intervals.
into a normal
dog and
HDL and d > 1.21 fractions
was determined
37 min after the injection,
63% of the total
activity
in the plasma was present in the HDL fraction,
33% in the plasma proteins
of d >
1.21:
disappeared
fraction
in 4.h and from the d > 1.21 fraction
and 4%
in LDL
fraction.
Radioactivity
in 30 h. From 30-50
from the LDL
h the 1251 activity
was present only in the HDL fraction. DISCUSSIOK ApoHDL
is known to have a marked
:‘SCANU~~and by us4 that aqueous dispersions Aqueous
solutions
of apoHDL
affinity
also combine
and free fatty acids in light petroleum
for lipids; it has been shown by
of phospholipids
with phospholipids,
solution, indicating
with fresh light petroleum [ iaiI]apoHDL
When
centrifuged
at a density
[iaiI]apoHDL
acquired
and that [iarI]apoHDL any evidence
apoHDL
was mixed
containing
lipoproteins.
lipids from plasma between
with LDL.
apoHDL
results are generally
apoHDL
and centrifuged
protein present initially lipids in the bottom HDL-apoHDL
complexes
Sedimentation
of the labeled
protein
with a decrease in the specific activity Conversely,
when labeled apoHDL
was centrifuged
sedimented
initially
Atherosclerosis,
12:439-450
of the labeled
Since there were no
delipidated
[issI]HDL
cannot
be entertained.
of apoHDL
in HDL
was mixed with unlabeled
This was associated
1970,
to the bottom.
present
was mixed with
fraction
or
was also associated
of the protein floating in the top 1 ml at d 1.21.
at d 1.21, a significant
the HDL fraction.
that
[iarI]HDL
with those reported
[issI]HDL
of partially
a high ratio
was carried
These authors did not find
in good agreement
the possibility
with
apoHDL
and HDL.
at d 1.21, a significant
in [ rs5I]HDL
fractions,
with plasma
and the mixture
to reconstitute
by SCANU AND HUGHES~ and SCANU AND PAGE a. When unlabeled
or LDL
SCANU et al. suggested
lipoproteins
also formed complexes
of intermediates
Our experimental
also interacted
with HDL
of 1.21 or 1.063, some of the labeled
to the top in the fractions
has sufficient
to water-d. On subsequent
the lipids were not removed.
It was shown by SCANU et al .2,3 that lipoproteins.
free cholesterol
that apoHDL
affinity for lipids to transfer these lipids from light petroleum .extraction
combine with apoHDL.
fraction
HDL and the mixture
of the labeled
apoHDL
floated
with a decrease in the specific activity
in
of the
INTERACTION OF APOHDL protein
sedimenting
fraction,
HDL AND WITH OTHER LIPOPROTEINS
WITH
to the bottom.
the exchange
of protein
In view of the absence moieties
between
449
of lipids in the bottom
apoHDL
and HDL
appears
to
be the most likely explanation. The paper When
electrophoretic
a mixture
of labeled
electrophoresis, suggesting
apoHDL
Conversely,
either
with apoHDL.
fractions
or both.
[issI]apoHDL
behavior
The hypothesis
only
mixture
a complex
be-
If, however,
in the HDL fraction
in
into HDL
Since the molecular of LDL
is about
or with a subunit
or
weight
2.5 * 106,
of apoHDL
would
into HDL was further
on recentrifugation and HDL,
of the material floating
its ultracentrifugation
fraction
the [ 125IlapoHDL
of the top fraction
reversal
of [is5I]HDL
was actually
incorporated
would be expected in the amount
in of the
to sediment to have
into little
of labeled protein per
it may be inferred that almost all of the [i2sI]apoHDL was incorporated.
in the
at d 1.21 had
to result in a partial
an appreciable
effect. Since only a 15% decrease was observed unit weight of HDL,
is subjected
of the [issI]apoHDL
becomes incorporated
of [ issI]apoHDL
on recentrifugation.
The absence of intermediates
present
in the conversion
to HDL noted by SCANU AND HUGHES~ has been confirmed in the present
studies.
This observation
present
in HDL
also supports
may exchange weights
a conglomerate
of many
very largely
can be separated
as low as 13 or 14,00015J6. protein
subunits
by hydrophobic
under
certain
HDL
must be thought
as well as many
bonds.
The protein
of exchanging conditions.
en masse to apoHDL,
and is not supported
subunits
into several subunits
other
The preliminary
small protein
that apoHDL
are all capable
HDL can transfer
that
It has recently
and phospholipids lipoproteins
the hypothesis
with similar units in apoHDL.
by several laboratories
with molecular
unlikely
and that
of d 1.21 might be expected
recentrifugation
HDL
of [rssI]apoHDL
at d 1.21. If the material
thus releasing
together
exchange
more like that of LDL than apoHDL.
formation
reported
was seen associated
of protein
and unlabeled
into LDL.
of 30,000
that [125I]apoHDL
and HDL
fresh salt solution
of apoHDL
of radioactivity
was applied on
when mixed with LDL and centrifuged
with apoHDL
by the results of the experiment
[125I]HDL,
or was complexed
apoHDL
the presence
is reconstituted
of a lipoprotein
have ultracentrifugal
contained
to paper for
carried to the top and this can hardly be explained
is in the neighborhood
[125I]apoHDL
into HDL
the concept
of [rssI]apoHDL
of 1.063 is partially
complex
complex
fraction supports
or paper electrophoresis,
by assuming that the apoHDL
supported
was applied
of HDL4.
was found in the HDL zone
and unlabeled
could be due to either incorporation
to complex formation
a l-l
HDL
a significant
when a mixture
to ultracentrifugation
of apoHDL
HDL
of radioactivity
that
and HDL.
However,
at a density
is less than
had been converted
This last observation
tween apoHDL
of apoHDL
and unlabeled
amount
when labeled
paper for electrophoresis,
HDL
apoHDL
a considerable
that
with HDL.
mobility
lipid molecules
subunits,
as was originally
that
considered
of as bound
free cholesterol
with corresponding
The possibility
been
molecules
in
all the lipids in to occur, seems
by the evidence.
experiments
in dogs support
the hypothesis Atherosclerosis,
that
the small
1970, 12: 439-450
450
H. S. SODHI, R. GORDON GOULD
amount of 1251 recovered from the LDL fraction was [iV]apoHDL
loosely com-
plexed to LDL since it disappeared within a few hours. The large amount of [issI]apoHDL present in the d > 1.21 fraction at 37 min disappeared from this fraction in 30 h, but it cannot be determined from the available data whether it was converted into HDL by passage through the liver or was broken down. REFERENCES SCANU, A., L. A. LEWIS AND F. M. BUMPUS, Separation and characterization of the protein moiety of human ai-lipoprotein, Arch. B&hem. Biophys., 1958, 74: 390. 2 SCANU, A. AND W. L. HUGHES, Recombining capacity towards lipids of the protein moiety of human serum a-lipoprotein, J. Biol. Chem., 1960, 235: 2876. 3 SCANU, A. AND I. H. PAGE, Recombination with lipids of the lipid-free protein from canine serum (d. 1.063-1.21, al) lipoprotein, J. Lipid Res., 1961, 2: 161. 4 SODHI, H. S. AND R. G. GOULD, Combination of delipidized high density lipoprotein with lipids, J. Biol. Chem.. 1967, 242: 1205. 5 HUNTER, W. M. AND F. C. GREENWOOD, Preparation of iodine-131 labelled human growth hormone of high specific activity, Nature, 1962, 194: 495. 6 FOLCH, J., M. LEES AND G. H. SLOANE-STANLEY, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 1957, 226: 487. 7 ZAK, B., R. C. DICKENMAN, E. G. WHITE, H. BURNETT AND P. J. CHERNEY, Rapid estimation of free and total cholesterol, Am. J. CZin. Pathol., 1954, 24: 1307. 8 STERN, I. AND B. SHAPIRO, Rapid and simple method for determination of esterified fatty acids and for total fatty acids in blood, J. C&z. Pathol., 1953, 6: 158. 9 BARTLETT, G. R., Phosphorus assay in column chromatography, J. Biol. Chem., 1959,234: 466. Ill MCDONALD, H. J., A new approach to the staining of lipoproteins. In: G. E. W. WOLSTENHOLME AND E. C. P. MILLAR (Eds.), Ciba Foundation Symfiosium on P@er Electrophoresis, Little, Brown, Boston, 1956, p. 183. 11 JENCKS, W. P., M. R. HYATT, M. R. JETTON, T. W. MATTINGLY AND E. L. DURRUM, Study of serum lipoproteins in normal and atherosclerotic patients by paper electrophoretic techniques, J. Clin. Invest., 1956, 35: 980. 12 WHEREAT, A. F. AND E. STAPLE, The preparation of serum lipoproteins labeled with radioactive cholesterol, Arch. Biochem. Biophys., 1960, 90: 224. 13 SCANU, A., Binding of human serum high density lipoprotein apoprotein with aqueous dispersions of phospholipids, J. Biol. Chem., 1967, 242: 711. 14 FRAZER, A. C., Blood plasma lipoproteins, with special reference to fat transport and metabolism, Discussions Faraday Sot., 1949, 6: 81. 15 SHORE, B. AND V. SHORE, Heterogeneity in protein subunits of human serum high density lipoproteins, Biochemistry, 1968, 7: 2773. 16 SCANU, A., J. TOTH, C. EDELSTEIN, S. KOGA AND E. STILLER, Fractionation of human serum high density lipoprotein in urea solutions. Evidence for polypeptide heterogeneity, Biochemistry, 1969, 8: 3309. 1
Atherosclerosis,
1970, 12: 439-450
Company,
INTERACTION
OF apoHDL
H. S. SODHI Department
R. GORDON
AND
439
- Printed in The Netherlands
WITH
HDL AND WITH
OTHER
LIPOPROTEINS
GOULD
Stanford Medical
of Medicine,
(Received
Amsterdam
School,
Stanford,
Calif.
(U.S.A.)
.4pril 24th, 1970)
SUMMARY
Ultracentrifugation which
at a density
one component
protein
was labeled
took place. Since no evidence
probable protein complex
that
these
subunits
results
(mol.wt.
(mol.wt.
approx.
has been previously of [iasI]apoHDL
approx.
The top fraction
results
small
and the large
HDL
obtained
by centrifuging
as
a mixture
of the labeled protein when
in addition
were obtained
of some complex
to the interchange on electrophoresis; to HDL
of HDL
to the bottom
in the
formation
between
of labeled protein
subunits.
[issI]HDL
after incubation
and to apoHDL
both
of which
1251.
Complex
formation
between
of a mixture.
d 1.063 but on recentrifugation
fraction
apoHDL
which sedimented
the occurrence
gave zones corresponding
ultracentrifugation
separated
of the relatively
which floated at d 1.21 in the presence
_ of [ lssI]HDL
indicating
and [i2sI]apoHDL
amounts
of labeled
it seems more
at the same density.
presence of apoHDL,
contained
was obtained
than that lipids move from HDL to apoHDL,
of [issI]apoHDL
was larger than the fraction
Similar
between
and HDL in
interchange
by interchange
15,000)
200,000)
of apoHDL
that
of intermediates
and HDL at d 1.21 lost less than 20%
The fraction
with apoHDL
iss1 showed
are explained
suggested.
it was recentrifuged
HDL
of 1.21 of mixtures
with
of [125IjapoHDL
[issI]apoHDL
60%
with serum of the
was demonstrated
of the labeled protein
half of the labeled protein
were mixed
by ultracentrifugation,
and LDL
A small fraction
label
and
sedimented. the
When trace
lipoprotein
was recovered
by
floated at fractions
in the
HDL
and only 1.5% in the LDL fraction.
This work was supported by U.S. Public Health Service Grant No. HE-08476 from the National Institutes of Health and, in part, by Grant No. MA-3431 from the Medical Research Council of Canada. Dr. H. S. SODHI was the recipient of a post-doctoral research fellowship from the N.I.H. His present address: Department of Medicine, University Hospital, University of Saskatchewan, Saskatoon, Canada. Atherosclerosis,
1970, 12: 439-450
440
H. S. SODHI, R. GORDON GOULD
Key words:
apoHDL
- Electrophoresis
- HDL
- 1251~labeled HDL
- Interaction
-
Interchange - LDL - Protein subunit - Ultracentrifugation
INTRODUCTION SCANU et al. have reported serum high density lipoprotein with lipoproteins
that
apoHDL,
(HDL)
in two ways2J.
by low temperature
When
part of it was found to have acquired authors interpreted a reconstituted
this to indicate
HDL2.
sufficient that
apoHDL
suspensions that
appeared
complex
apparent
conversion
determine
whether
with lipoproteins
after
It is unlikely
of <
of apoHDL
interacts with
properties
mixing
to HDL.
can actually
HDL,
of HDL;
with
the
to give
LDL
that apoHDL
was re(d 1.063-
could acquire was considered
and LDL. SCANU et al. recognized
with chylomicrons
as well as with
LDL.
may also occur with
HDL
was mixed
1.063 so this observation
to form complexes
formation
delipidizationl,
of lipids from HDL to apoHDL
of a complex of apoHDL
of triglycerides
from human
(d < 1.063), partly in the HDL fraction
1.21 fraction.
lipids to produce a density
to be due to the formation
obtained
apoHDL
the ultracentrifugal
[ 131IlapoHDL
covered partly in the LDL fraction 1.21) and partly in the d >
labeled
a transfer
However,
the protein
and even artificial
It appears
HDL
possible,
and account
therefore,
for some of the
We have carried out studies designed to
be reconstituted
and the results are reported
from apoHDL
by mixing
in this communication.
MATERIALS AND METHODS HDL, LDL, VLDL human collected density
serum
and chylomicrons
by ultracentrifugation
were isolated from normal postabsorptive
as previously
from the top 1 ml of the centrifuge of 1.006, VLDL
describedd.
tube after
were
x g at a
after 24 h at 1.006, LDL after 24 h at 1.063 and HDL after
24 h at 1.21. The HDL fraction
thus contained
both HDLz and HDLs.
ApoHDL
was obtained by delipidization
of HDL at -20X,
of the method
of SCANU et al.1 as previously
described4;
is free of all neutral
Chylomicrons
1 h at 104,000
lipids but contains
a few percent
using a modification
this gives a product of the original
which
amount
of
phospholipids. Preparation HDL inorganic
of [125I]HDL was labeled
and [l25I]apoHDL
with 1251 by the method
iodide was removed
G-100 column and dialysis. protein
and lipid staining
[125I]apoHDL
Electrophoresis material
1970, 12: 439-450
of HUNTER AND GREENWOODS;
of an ion exchange
resin or a Sephadex
of the product showed that 1251 activity,
all had the same mobility.
was prepared from [ 125I]HDL
of the total 1251 in [125I]HDL Atherosclerosis,
by means
as described
was found to be associated
above; about 20-30%
with the lipids.
INTERACTION
OF APOHDL
HDL
WITH
The lipids were extracted on thin-layer acetic
with chloroform-methanol
plates with a solvent
acid (75:25:1,
by vol.),
system
- ethyl ether - glacial
and the lipid zones were eluted
by the ferric chloride-sulfuric
by the hydroxamate
methods
and phospholipids
The protein in the ultracentrifugal by trichloroacetic
(2:1, v/v)s, fractionated
of light petroleum
lesterol was analyzed
precipitated
441
AND WITH OTHER LIPOPROTEINS
by the method
fractions
acid and thoroughly
acid to remove any traces of 1251 activitv
and analyzed.
acid color reaction’, containing
of
Cho-
triglycerides
BARTLETTg.
HDL and apoHDL
was
washed with dilute trichloroacetic
not associated
with the protein.
Paper electrophoresis Paper strip electrophoresis strength
was done in Verona1 buffer at pH 8.6 and at ionic
of 0.075 in a Spinco electrophoretic
were stained
with bromphenol
cell. The proteins
blue. The lipids were stained
the serum on paper strips with acetylated completion
Sudan Black
on the paper strips
either before applying
Bie or with Oil red 0 after
of the electrophoresisii.
In some experiments This was prepared determined
LDL containing
from human
LDL
in a liquid scintillation
determine
the distribution
counter
of radioactivity
cut into 0.125 or 0.25 inch wide sections in a well counter,
unesterified
[4-i%]cholesterol
and [4-iK]cholesterol
and 1251 in a NaI crystal
on paper electrophoretic and transferred
or into vials for counting
was used.
by exchange.
1% was
counter.
To
strips, they were
into test tubes for counting
in the liquid scintillation
counter.
RESULTS
Exchange of protein between HDL Ultracentr(fuge
short time at room temperature When [izsI]apoHDL
and aPoHDL
studies. Mixtures
and HDL
were incubated
at pH 8.6 and ultracentrifuged
was centrifuged
ed from the top 1 ml fraction
of apoHDL
at a density
alone at this density it was completely
(Table
l), but after incubation
mixture
at this density,
of [issI]HDL
some labeled
none was found in the bottom
and unlabeled
protein
was present
apoHDL
products
a mixture
most likely
due to sedimentation
and HDL
to the bottom
only [125I]apoHDL.
(Table
1). No lipids were delipidated
was centrifuged,
the
specific
was lower than that in the control
This decrease in the specific activity
of some unlabeled
protein
originally
was.
present
in
fraction.
These findings are consistent had acquired fraction
1 ml, but when a
to ultracentrifugation,
the absence of HDL or its partially
of [rssI]apoHDL
of the protein sedimenting
tube which contained the HDL
fraction
HDL,
When [isV]HDL
from this fraction.
When activity
was subjected
in the bottom
found in the bottom fraction indicating
of 1.21.
sediment-
with unlabeled
labeled protein was found in the top 1 ml as well as in the bottom. was centrifuged
for a
the ultracentrifugal
of the HDL
had acquired
with the hypothesis characteristics
that a fraction
of HDL
the ultracentrifugal
of the apoHDL
and conversely
characteristics Atherosclerosis,
that
of apoHDL. 1970, 12: 439-450
a
442
H. S. SODHI, R. GORDON GOULD
TABLE
1
ULTRACENTRIFUGAL
BEHAVIOR
apoHDL
LABELED
OF
PROTEINS
IN
THE
MIXTURES
CONTAINING
LABELED
AND
UN-
HDL
AND
The protein mixtures were incubated at room temperature for 30 min in 0.05 M Tris buffer at pH 8.6 before being centrifuged at d 1.21 to separate apoHDL from HDL. Top and bottom 1 ml samples were removed for precipitation and washing. There were significant losses of proteins in these procedures for determination of its specific activity. composition
SpecQic activity of proteins (countslminlmg 10-3)
apoHD L (m&Y protein)
0f mixture Total 1251 content 0j top 1 ml proteins HDL counts/&n bottom 1 ml Img counts/min 10-3 protein) . 10-3
oh of Labeled protein recovered
to9
bottom
1.5a 1.5% 1.28 I.78 0 2.0 2.0
0 1.5 1.5 0 2.0% 2.0a 2.0a
0.3 10.1 10.7 0 36 31.3 49.0
16.3 8.3 8.2 47 0 19 8.3
0.5 18.7 15.7 1.0 178.3 154.9 242.4
30.1 15.4 12.1 198.3 0.5 94.4 41.2
top 1 ml
bottom 1 ml
123 76 75 248
55 46 318 155 269
131 40
a ‘251-labeled component.
The presence of [125I]apoHDL
in the top fraction
of the processes:
(i) formation
density
1.21 or (ii) by interchange
less than
of complexes
can be explained
of HDL of protein
by either or both
and [izsI]apoHDL subunits
having
between
HDL
a
and
apoHDL. As judged
from the changes
protein
carried
to the bottom
fraction
of labeled
apoHDL
in specific activity
by the presence
carried
values,
of apoHDL
the fraction was smaller
to the top by the presence
of HDL
of HDL than
the
(Table
1,
lines 7 and 6). PaPer electrojdaoresis. Mixtures amounts
on electrophoresis
of apoHDL
to only the HDL zone. When [125I]apoHDL electrophoresis, zone (Fig. mixture
more of the radioactivity
1). Conversely, with the HDL,
when [ rssI]HDL although
found in the zone containing hypothesis
mentioned
and HDL were incubated was incubated
apoHDL
in connection
(Fig.
between
small amounts
with the ultracentrifugal of apoHDL
on, among other factors,
formation
it is not clear whether
of an HDL-apoHDL
of apoHDL. Atherosclerosis,
1970, 12: 439-450
and the remained
results,
was
with the
that an inter-
and HDL and also complex
and HDL.
1). This decrease
However,
before
of the 1251 activity
(Fig. 1). Th ese findings are consistent
the electrophoretic
in the mixture.
with apoHDL,
fraction
In the presence of apoHDL depended
together
the major part of the activity
a significant
change of protein units may take place between apoHDL formation
equal
was present in the HDL than in the apoHDL
applied on paper for electrophoresis,
associated
and HDL in approximately
gave two protein zones with the lipid stain corresponding
mobility
of HDL was decreased
the amounts
of apoHDL
this effect is specifically
complex or is a non-specific
due to
effect due to the presence
WITH HDL
INTERACTION OF APOHDL
443
AND WITH OTHER LIPOPROTEINS
Fig. 1. Electrophoretic behavior of apoHDL, HDL and mixtures of the two. [1251]HDL, [1251]apoHDL, and mixtures of each with an equal amount of unlabeled apoHDL or HDL respectively were applied on paper for electrophoresis. The radioactivity is shown by bar diagrams and the protein by bromphenol blue staining. Arrows indicate the point of application. The double underline indicates the component in which the label was added in the mixture.
Relative a$inity of apoHDL Lipoproteins lipoproteins
for various lipoproteins
in serum. To determine the relative affinity of apoHDL
in serum, relatively
small amounts
(6 ,~g) of apoHDL
2 ml of serum and the mixtures were centrifuged 1.5% of the labeled apoHDL
at densities of 1.063 or 1.21. About
added to serum rose to the top fraction
1.063 and about 13% with the lipoproteins
for different
were mixed with with LDL at
floating at d 1.21, corresponding
to almost
11.5% being carried to the top with HDL (Table 2). In other experiments
of serum
of labeled apoHDL
with
VLDL,
LDL and HDL were separated by successive flotation
centrifuge.
6 ml aliquots
even smaller amounts
mixed
and after 30 min incubation,
As shown in Table 3, 60%
fraction and relatively
of the [125I]apoHDL
small amounts in the other fractions.
bulin was treated in the same manner as [ 125IlapoHDL; HDL fraction.
Most of it sedimented
fluence its distribution. chylomicron containing
fraction.
at d 1.21 indicating
in a preparative
ultra-
was found in the HDL As a control,
[issI]y-glo-
less than 1% was found in the that lipoproteins
A relatively large percentage of [125I]apoHDL This result was in part due to contamination
chylomicrons
(0.8 ,ug) were chylomicrons,
was foundin the since the fraction
was collected after only 1 h of centrifugation Atheroscleroisi,
did not in-
and the chylo1970, 12: 439-450
444
H. S. SODHI, R. GORDON GOULD
TABLE
2
DISTRIBUTIONOF [‘=I]apoHDL
AFTER INCUBATIONWITH SERUM
[ie5I]apoHDL (50,000 counts/min in 6 pup) was added to 2 ml aliquots of serum and the densities adjusted to either 1.063 or 1.21. The mixtures were centrifuged at 104,000 x g for 24 h and then the activity (counts/min) in the top and the bottom 1 ml fractions was determined. Density
Mean y0 of Mean 0/0 of
+c S.E.M. total f S.E.M. total
TABLE
Counts/min~
1.063 1.063 1.21 1.21
10-3
topfraction
bottomfraction
0.756 i 0.050 1.5 6.42 * 0.50 12.8
14.31 * 0.48 28.6 0.811 * 0.032 1.6
3
RELATIVE AM~IJNT~CJF~~OHDLAND AFTER
MIXING
WITH
Y-GLOBULIN
FOUND
IN
DIFFERENT
LIPOPROTEIN
FRACTIONS
SERUM
[iaaI]apoHDL (0.8 ,ug) and y-globulin (1 .O ,ug) were mixed with 6 ml serum and fractions containing chylomicrons, VLDL, LDL and HDL were successively collected by floating them at appropriate densities. Percent of the total iasI activity present in each fraction was determined.
oftotal
Percent
[iaaI]apoHDL [iesI]y-globulin
activity
Chylo
VLDL
LDL
HDL
d >
25a 188
0.7 0.07
1.5 0.1
60 0.6
14 82
1.21
a The values for the chylomicron fraction are too high by about 8% because the time of centrifugation was too short to sediment the labeled protein from the top fraction. The chylomicrons were not washed and may have contained other lipoproteins and proteins.
microns
were not washed free of other proteins or lipoproteins
The top 1 ml fraction
initially
excess over this amount ciation
present in the mixture.
8% of the [12sI]apoHDL
was probably
or y-globulin;
carried up in more or less non-specific
the asso-
with chylomicrons.
Individual
lipoproteins.
and with purified lipoproteins lipoprotein conditions lipoprotein,
apoHDL however,
than
was associated the
amounts
could not be correlated
cholesterol
esters, triglycerides,
The bottom Atherosclerosis,
those
1 ml fractions
1970, 12: 439-450
with washed
of [issI]apoHDL
at the appropriate
of [rssI]apoHDL
greater
was mixed
and the amounts
data for y-globulin
the amounts
were considerably proteins;
[ issI]apoHDL
after recentrifugation
Table 4. Corresponding
proteins
contained
density
present
in different
with chylomicrons
of apoHDL
that
with their content or with the amount
with the
for 24 h are shown in
as a control are included.
of [rasI]y-globulin
chylomicrons
associated
Under the same
lipoprotein
(Table
fractions
4). Per unit mass
more than with other lipo-
combined
with
of phospholipids,
different
lipo-
free cholesterol,
of proteins.
in the ultracentrifuge
tubes
contained
significant
INTERACTION TABLE
OF APOHDL
WITH
HDL AND WITH OTHER LIPOPROTEINS
445
4
AMOUNTS
OF
[issI]apoHDL
1 mg of [is5I]apoHDL room temperature and the lipoproteins. From top ml, the amount of
AND
[1251]y-GLOBULIiY
ASSOCIATED
WITH
PLASMA
LIPOPROTEINS
(or [ isaI]y-globulin) was incubated with various lipoproteins for 30 min at then centrifuged at 104,000 xg at the appropriate density for 24 h to float determination of the lipoprotein content and the counts/min of isa1 in the labeled protein per mg of lipoprotein was calculated.
Lipoproteins
Chylo
VLDL
LDL
HDL
Amount of lipoprotein added (mg) ‘[issI]apoHDL (pg/mg lipoprotein) [issI]y-globulin (pg/mg lipoprotein)
2.280 13.0 4.0
17.971 3.0 0.6
45.476 1.5 0.4
13.326 6.0 0.7
amounts of [125I]apoHDL but no lipids indicating that apoHDL sedimenting at densities of 1.006 or 1.063 had not acquired any lipids from VLDL or LDL. Serum Z@ids. In the absence of plasma lipoproteins [issI]apoHDL sedimented .at d 1.21, but if plasma lipoproteins were present significant fractions of [rs5I]apoHDL rose to the top. It was of interest to determine the effect of serum lipids instead of plasma lipoproteins on the ultracentrifugal behavior of apoHDL. Lipids from 2 ml of serum samples dissolved in solutions of Tween 20 by the method of WHEREAT AND STAPLERS were mixed with 2 mg of [issI]apoHDL and the density adjusted to 1.063 g/ml. After centrifugation for 24 h at 104,000 x g, 1 ml of opalescent solution was removed from the top. The density of the remainder was adjusted to 1.21 and 1 ml ,of a clear solution was collected from the top after centrifugation for 24 h at 104,000 TABLE
5
RECENTRIFUGATION
OF
apoHDL-
LIPOPROTEIN
COMPLEXES
Each tube contained 0.85 mg [ issI]apoHDL; after addition of HDL or LDL, salt solution, Tris buffer and water the final volume was 9.0 ml and the pH 8.6. Tubes containing HDL were adjusted to d 1.21 and those containing LDL to d 1.063. After 30 min incubation at room temperature the tubes were centrifuged at 104,000 x g at 15°C for 40 h. The material in the top 1 ml fraction .of each tube was mixed with fresh salt solution of the appropriate density and centrifuged for 60 h and the top 1 ml fractions collected. is51 activity was measured and total cholesterol determined by analysis. The ratio of issI per mg cholesterol was calculated and also the percent of the 1251 per mg cholesterol present after one centrifugation which was recovered after the second centrifugation. Tube No.
Amount of 1ipopvotein (mg)
First centrifugation
Second centrifugation
countslmin 1zsr
ratio counts/ minlmg chol.
countslmin 1261
ratio counts/ minlmg chol.
Percent remaining
1 ‘2 3
HDL 11.2 22.4 44.8 LDL
13,300 11,450 13,800
3577 1694 974
5000 7000 7620
2971 1461 802
84 86 82
:: 3
22.4 11.2 44.8
2420 2150 3353
105 169 70
880 480 1000
55 68 39
52 40 56
Atherosclerosis,
1970, 12: 439-450
446
H. S. SODHI, R. GORDON GOULD
Fig. 2. Electrophoretic behavior of [iasI]apoHDL when mixed in relatively small amounts in serum or purified lipoproteins. Plbout 10 ~5 of [ i25IlapoHDL was incubated with 50 ~1 of serum, 800 p”g of LDL or 400 ~“g of HDL for 30 min at room temperature. After prestaining for lipids, with acetylated Sudan black B, 30 ,/_dof the mixtures were applied in duplicate for paper electrophoresis. The distribution of lipids and 1251 activity on the paper strips are shown.
x g. The amount initial amount; fraction
of 1251 activity
in the d 1.063-1.21
present in the d < or HDL fraction
was 1.5% of the 1.21
it was 30%.
Thin-layer
chromatography
showed that phospholipids
Recentvifugation After
of the lipids extracted
in about the same order of magnitude.
of [rssI]apoHDL containing
HDL
with HDL and
with salt solution of d 1.21 and recentrifuged. of loss of [issI]apoHDL
Cholesterol
and centrifugation
[issI]apoHDL
1970, 12: 439-450
(Table
diluted
The top 1 ml was collected to determine
from the HDL
were calculated.
on recentrifugation
at d 1.21,
was separated,
fraction.
analysis was used as an index of the amount
ratios of 1251 to cholesterol
Atherosclerosis,
fractions
but other lipids
stzcdies
incubation
the top 1 ml fraction the amount
from the three
were present mostly in the HDL fraction
were present in all fractions
change
1.063 fraction
it was 60% and in the d >
In the top fraction
5). In this experiment
of lipoprotein
and the
this ratio showed little
recentrifugation
of three
INTERACTION
OF
WITH HDL
APOHDL
AND
WITH
OTHER
447
LIPOPROTEINS
Fig. 3. Evidence that lipids are not transferred from LDL to apoHDL. 500 ,ug of LDL containing [14C]cholesterol was incubated with 100 ,ug of apoHDL and applied to paper electrophoresis (left hand figure). The same amount of LDL was used for control. The direction of electrophoresis was left to right. The top half of each strip was stained for lipids (oil red 0) and the bottom half for protein. Lipids are seen only in the LDL zone both by staining and by radioactivity. The apoHDL is seen in the “Test” diagram in zone 3. No protein or lipids are seen in zone 4 where HDL normally is found.
mixtures
of different
amounts
showed losses of only about In a second experiment Corresponding
Paper
experiments
staining.
of the [i2sI]apoHDL
of [125I]apoHDL
per mg of cholesterol.
changes were observed.
with LDL,
centrifuged
twice at d I .063, gave much the ratio of [issI]apoHDL
by about half.
Microgram
amounts
of [125I]apoHDL,
failed to give a well defined peak of radioactivity
in the absence
(HDL)
[125I]apoHDL
the [125I]apoHDL
nor 1251 activity with purified no detectable
than that in the p-lipoprotein
was mixed with purified HDL,
electrophoresis,
was observed
LDL,
corresponding
were found on paper strips (Fig. 2). The activity
zone was greater
(LDL)
in the a-lipozone. When
applied on paper for
was found only in the HDL zone. Neither
with the mobility
the [125I]apoHDL
1251 or protein
and the mixture
was found
was observed
of apoHDL associated
of
or to reveal protein by
When mixed with serum, however, two peaks of 1251 activity
to /3-and a-lipoproteins protein
amount
studies
electrophoresis.
lipoproteins,
with a standard
on the second centrifugation;
in LDL decreased
electrophoretic Paper
14-B%
no significant
greater losses of [i25I]apoHDL to cholesterol
of HDL
protein
(Fig. 3). When mixed only with LDL;
in the zone where
HDL
and
or apoHDL
would be expected. Atherosclerosis,
1970, 12: 439-450
448
H. S. SODHI, R. GORDON GOULD Control
plasma
experiments
lipoproteins
with
indicated
mixtures
of [ issI]y-globulin
that the mobility
with
of [125I]yglobulin
serum
or with
was not affected
by the presence of serum or purified lipoproteins. Electrophoresis gave little protein)
of a mixture
evidence
of either
(Fig. 3), indicating
of apoHDL
protein
and LDL
or isotope
that apoHDL
labeled
with [lT_i’cholesterol
with the mobility
was not converted
of HDL
(a-lipo-
into HDL by acquiring
lipids from LDL. In viva studies V’sI]apoHDL L the distribution
prepared
from dog serum was injected
of the label in the LDL,
in the intact dog at various time intervals.
into a normal
dog and
HDL and d > 1.21 fractions
was determined
37 min after the injection,
63% of the total
activity
in the plasma was present in the HDL fraction,
33% in the plasma proteins
of d >
1.21:
disappeared
fraction
in 4.h and from the d > 1.21 fraction
and 4%
in LDL
fraction.
Radioactivity
in 30 h. From 30-50
from the LDL
h the 1251 activity
was present only in the HDL fraction. DISCUSSIOK ApoHDL
is known to have a marked
:‘SCANU~~and by us4 that aqueous dispersions Aqueous
solutions
of apoHDL
affinity
also combine
and free fatty acids in light petroleum
for lipids; it has been shown by
of phospholipids
with phospholipids,
solution, indicating
with fresh light petroleum [ iaiI]apoHDL
When
centrifuged
at a density
[iaiI]apoHDL
acquired
and that [iarI]apoHDL any evidence
apoHDL
was mixed
containing
lipoproteins.
lipids from plasma between
with LDL.
apoHDL
results are generally
apoHDL
and centrifuged
protein present initially lipids in the bottom HDL-apoHDL
complexes
Sedimentation
of the labeled
protein
with a decrease in the specific activity Conversely,
when labeled apoHDL
was centrifuged
sedimented
initially
Atherosclerosis,
12:439-450
of the labeled
Since there were no
delipidated
[issI]HDL
cannot
be entertained.
of apoHDL
in HDL
was mixed with unlabeled
This was associated
1970,
to the bottom.
present
was mixed with
fraction
or
was also associated
of the protein floating in the top 1 ml at d 1.21.
at d 1.21, a significant
the HDL fraction.
that
[iarI]HDL
with those reported
[issI]HDL
of partially
a high ratio
was carried
These authors did not find
in good agreement
the possibility
with
apoHDL
and HDL.
at d 1.21, a significant
in [ rs5I]HDL
fractions,
with plasma
and the mixture
to reconstitute
by SCANU AND HUGHES~ and SCANU AND PAGE a. When unlabeled
or LDL
SCANU et al. suggested
lipoproteins
also formed complexes
of intermediates
Our experimental
also interacted
with HDL
of 1.21 or 1.063, some of the labeled
to the top in the fractions
has sufficient
to water-d. On subsequent
the lipids were not removed.
It was shown by SCANU et al .2,3 that lipoproteins.
free cholesterol
that apoHDL
affinity for lipids to transfer these lipids from light petroleum .extraction
combine with apoHDL.
fraction
HDL and the mixture
of the labeled
apoHDL
floated
with a decrease in the specific activity
in
of the
INTERACTION OF APOHDL protein
sedimenting
fraction,
HDL AND WITH OTHER LIPOPROTEINS
WITH
to the bottom.
the exchange
of protein
In view of the absence moieties
between
449
of lipids in the bottom
apoHDL
and HDL
appears
to
be the most likely explanation. The paper When
electrophoretic
a mixture
of labeled
electrophoresis, suggesting
apoHDL
Conversely,
either
with apoHDL.
fractions
or both.
[issI]apoHDL
behavior
The hypothesis
only
mixture
a complex
be-
If, however,
in the HDL fraction
in
into HDL
Since the molecular of LDL
is about
or with a subunit
or
weight
2.5 * 106,
of apoHDL
would
into HDL was further
on recentrifugation and HDL,
of the material floating
its ultracentrifugation
fraction
the [ 125IlapoHDL
of the top fraction
reversal
of [is5I]HDL
was actually
incorporated
would be expected in the amount
in of the
to sediment to have
into little
of labeled protein per
it may be inferred that almost all of the [i2sI]apoHDL was incorporated.
in the
at d 1.21 had
to result in a partial
an appreciable
effect. Since only a 15% decrease was observed unit weight of HDL,
is subjected
of the [issI]apoHDL
becomes incorporated
of [ issI]apoHDL
on recentrifugation.
The absence of intermediates
present
in the conversion
to HDL noted by SCANU AND HUGHES~ has been confirmed in the present
studies.
This observation
present
in HDL
also supports
may exchange weights
a conglomerate
of many
very largely
can be separated
as low as 13 or 14,00015J6. protein
subunits
by hydrophobic
under
certain
HDL
must be thought
as well as many
bonds.
The protein
of exchanging conditions.
en masse to apoHDL,
and is not supported
subunits
into several subunits
other
The preliminary
small protein
that apoHDL
are all capable
HDL can transfer
that
It has recently
and phospholipids lipoproteins
the hypothesis
with similar units in apoHDL.
by several laboratories
with molecular
unlikely
and that
of d 1.21 might be expected
recentrifugation
HDL
of [rssI]apoHDL
at d 1.21. If the material
thus releasing
together
exchange
more like that of LDL than apoHDL.
formation
reported
was seen associated
of protein
and unlabeled
into LDL.
of 30,000
that [125I]apoHDL
and HDL
fresh salt solution
of apoHDL
of radioactivity
was applied on
when mixed with LDL and centrifuged
with apoHDL
by the results of the experiment
[125I]HDL,
or was complexed
apoHDL
the presence
is reconstituted
of a lipoprotein
have ultracentrifugal
contained
to paper for
carried to the top and this can hardly be explained
is in the neighborhood
[125I]apoHDL
into HDL
the concept
of [rssI]apoHDL
of 1.063 is partially
complex
complex
fraction supports
or paper electrophoresis,
by assuming that the apoHDL
supported
was applied
of HDL4.
was found in the HDL zone
and unlabeled
could be due to either incorporation
to complex formation
a l-l
HDL
a significant
when a mixture
to ultracentrifugation
of apoHDL
HDL
of radioactivity
that
and HDL.
However,
at a density
is less than
had been converted
This last observation
tween apoHDL
of apoHDL
and unlabeled
amount
when labeled
paper for electrophoresis,
HDL
apoHDL
a considerable
that
with HDL.
mobility
lipid molecules
subunits,
as was originally
that
considered
of as bound
free cholesterol
with corresponding
The possibility
been
molecules
in
all the lipids in to occur, seems
by the evidence.
experiments
in dogs support
the hypothesis Atherosclerosis,
that
the small
1970, 12: 439-450
450
H. S. SODHI, R. GORDON GOULD
amount of 1251 recovered from the LDL fraction was [iV]apoHDL
loosely com-
plexed to LDL since it disappeared within a few hours. The large amount of [issI]apoHDL present in the d > 1.21 fraction at 37 min disappeared from this fraction in 30 h, but it cannot be determined from the available data whether it was converted into HDL by passage through the liver or was broken down. REFERENCES SCANU, A., L. A. LEWIS AND F. M. BUMPUS, Separation and characterization of the protein moiety of human ai-lipoprotein, Arch. B&hem. Biophys., 1958, 74: 390. 2 SCANU, A. AND W. L. HUGHES, Recombining capacity towards lipids of the protein moiety of human serum a-lipoprotein, J. Biol. Chem., 1960, 235: 2876. 3 SCANU, A. AND I. H. PAGE, Recombination with lipids of the lipid-free protein from canine serum (d. 1.063-1.21, al) lipoprotein, J. Lipid Res., 1961, 2: 161. 4 SODHI, H. S. AND R. G. GOULD, Combination of delipidized high density lipoprotein with lipids, J. Biol. Chem.. 1967, 242: 1205. 5 HUNTER, W. M. AND F. C. GREENWOOD, Preparation of iodine-131 labelled human growth hormone of high specific activity, Nature, 1962, 194: 495. 6 FOLCH, J., M. LEES AND G. H. SLOANE-STANLEY, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 1957, 226: 487. 7 ZAK, B., R. C. DICKENMAN, E. G. WHITE, H. BURNETT AND P. J. CHERNEY, Rapid estimation of free and total cholesterol, Am. J. CZin. Pathol., 1954, 24: 1307. 8 STERN, I. AND B. SHAPIRO, Rapid and simple method for determination of esterified fatty acids and for total fatty acids in blood, J. C&z. Pathol., 1953, 6: 158. 9 BARTLETT, G. R., Phosphorus assay in column chromatography, J. Biol. Chem., 1959,234: 466. Ill MCDONALD, H. J., A new approach to the staining of lipoproteins. In: G. E. W. WOLSTENHOLME AND E. C. P. MILLAR (Eds.), Ciba Foundation Symfiosium on P@er Electrophoresis, Little, Brown, Boston, 1956, p. 183. 11 JENCKS, W. P., M. R. HYATT, M. R. JETTON, T. W. MATTINGLY AND E. L. DURRUM, Study of serum lipoproteins in normal and atherosclerotic patients by paper electrophoretic techniques, J. Clin. Invest., 1956, 35: 980. 12 WHEREAT, A. F. AND E. STAPLE, The preparation of serum lipoproteins labeled with radioactive cholesterol, Arch. Biochem. Biophys., 1960, 90: 224. 13 SCANU, A., Binding of human serum high density lipoprotein apoprotein with aqueous dispersions of phospholipids, J. Biol. Chem., 1967, 242: 711. 14 FRAZER, A. C., Blood plasma lipoproteins, with special reference to fat transport and metabolism, Discussions Faraday Sot., 1949, 6: 81. 15 SHORE, B. AND V. SHORE, Heterogeneity in protein subunits of human serum high density lipoproteins, Biochemistry, 1968, 7: 2773. 16 SCANU, A., J. TOTH, C. EDELSTEIN, S. KOGA AND E. STILLER, Fractionation of human serum high density lipoprotein in urea solutions. Evidence for polypeptide heterogeneity, Biochemistry, 1969, 8: 3309. 1
Atherosclerosis,
1970, 12: 439-450