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Evidence for the participation of two soluble noncatalytic proteins in hepatic microsomal cholesterol synthesis
Vol. 53, No. 1,1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EVIDENCEFORTHE PARTICIPATIONOF TWOSOLUBLENONCATALYTIC PROTEINSIN HEPATIC MICROSOMAL CHOLESTEROL SYNTHESIS1 R. C. Johnson and S. N. Shah2 Brain-Behavior Research Center SonomaState Hospital Eldridge, California 95431 Received
May 18,
1973 SOMMARY
The capacity of liver soluble fraction to stimulate hepatic microsomal conversion of squalene to cholesterol is lost on treatment with trypsin. Heat treatment of the soluble fraction results in a selective loss of its capacity to stimulate conversion of squalene to cholesterol; the ability to stimulate conversion of lanosterol and desmosterol to cholesterol is however retained. It is proposed that the liver soluble fraction contains at least two noncatalytic proteins, one heat-labile and the other heat-stable, which participate in microsomal cholesterol synthesis. The heat-labile protein mediates the conversion of squalene to lanosterol while the heat-stable protein is needed for the conversion of lanosterol and other sterol precursors to cholesterol. A requirement for a noncatalytic synthesis of cholesterol been clearly
for hepatic microsomal
from squalene and from other sterol precursors,
demonstrated (l-10).
which binds squalene, sterols synthesis has been purified (4-9).
soluble protein
The squalene and sterol
carrier
protein
and phospholipids and stimulates microsomal in the laboratories
has (SCP) sterol
of two groups of investigators
The major discrepancy in the results of these studies however, is that
the protein purified protein purified
by Ritter
by Scallen's
Bloch (10) have purified
and Dempsey(Q-6) is heat-stable group (7-9) is heat-labile.
a liver
supernatant protein
factor
while the
Recently,
Tai and
(SPF) which does
not bind squalene or oxidosqualene, but stimulates microsomal squalene epoxidase and is heat-labile.
To resolve these differences,
present study, the effects
we have reexamined, in the
of heat treatment and of trypsin
105,000 g supernatant fraction
from liver
on its
digestion of the
capacity to stimulate 1) the
1 Supported by Research Grants NW9301 and HBO5317from National Institutes of Health. 2 To whomcorrespondence should be addressed.
Copyright 0 19 73 by Academic Press, Inc. ,411 rights of reproduction in any form reserved.
105
Vol. 53, No. 1,1973
overall to
BIOCHEMICAL
AND BIOPHYSICAL
conversion of squalene to cholesterol, and 3) the reduction
cholesterol,
factors,
one heat-labile
is heat-labile,
soluble fraction
and the other heat-stable.
The results
contains two
The protein which
is required for squalene epoxidase system and the protein
which is heat-stable the lanosterol Materials
2) the conversion of lanosterol
of desmosterol to cholesterol.
obtained support the hypothesis that the liver protein
RESEARCH COMMUNICATIONS
participates
in microsomal cholesterol
synthesis beyond
step.
and Methods
C14CI Squalene and C1'Cl lanosterol
were prepared from DL-2 [14Cl
meva-
ionic acid DBEDsalt (New England Nuclear Corp., Boston, Ma.) by the method of Tchen (11).
26
[l”C]
was purchased from NewEngland Nuclear Corp.,
Desmosterol
Boston, Ma., NADPH,NAD , glucose-6-phosphate and soybean trypsin
inhibitor
were purchased from Calbiochem, San Diego, Ca. Glucose-6-phosphate dehydrogenase and trypsin, coated silica
were purchased from Sigma Chemical Co., St. Louis,
gel G plates and silver
nitrate
MO.
Pre-
were purchased from Eastman-Kodak,
Rochester, N. Y. Adult female Sprague-Dawley rats weighing approximately The livers
ficed by decapitation.
genized in 2.5 volumes of 0.1
M
were removed, perfused with saline and homo-
potassium phosphate buffer
0.5 mMDTT. The homogenate was centrifuged natant decanted and centrifuged was then centrifuged drawn off. collected
a buffer
(pH 7.4) containing
at 1,000 g for 10 min, and the supe-
at 18,000 g for 20 min.
The 18,000 g supernatant
at 105,000 g for 90 min and the floating
The upper two-thirds and recentrifuged
microsomal material.
200 gmwere sacri-
portion of
the
layer of lipid
supernatant fraction
at 105,000 g for 60 min to
The sedimented microsomal pellet
remove (MS)
(Slo5) was
any residual was resuspended in
volume equal to that of the 18,000 g supernatant and recentrifuged
105,000 g for 60 min.
The supernatant fraction
at
and washed microsomal pellets
were stored at -86O if not used immediately. The protein sulfate
fraction
was obtained from S1o5 by precipitation
between 40070%saturation.
The precipitate
106
with ammonium
(AS)ws redissolved
in a
Vol.
53, No.
BIOCHEMICAL
I,1973
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE I CONVERSION OF SQUALENE AND STEROL PRECURSORSTO CHOLESTEROLBY RAT LIVER MICROSOMES
Additions
Substrate
Protein (mg)
l14C1 Squalene
C14C3 Lanosterol
tl’CI
lksmosterol
% [14C1 Recovepd in Cholesterol
Stimulation (-fold)
None
1.5
1.0
%;***
9.0 7.5
9.8 1.7
9.8
&G70%) AS(40-70%)**
5.0 5.3 3.6
1.1 9.8 0.9
1.1 9.8 0
None
1.5
1.63
p;*** s105** AS(40-70%) AS(40-70%)**
9.0 7.5 5.0 5.0 3.8
21.8 2.05 18.1 22.5 17.1
13.4 11.1 13.7 10.5
None s105*** SlO5**
1.5
7.5 9.0
4.7 3.6 52.5
11.2
&O-70%) AS(40-70%)**
5.3 5.0 3.6
53.8 50.3 51.7
11.9 10.7 11.0
Each reaction mixture contained 1.5 mg washed microsomal protein, 100 mM phosphate pH 7.4 and 1.2 mM NADPH. Squalene experiments buffer contained in addition, 0.6 mM NADt , 30 mM nicotinamide, 4.0 mM glucose-6-PO4 and 1 unit glucose-6-PO4 dehydrogenase. Labeled substrates (20,000 cpm) in 5 ul dioxane:propylene glycol (2:l) were routinely preincubated with S105 or AS preparations at 37O for 15 min under N2. Reactions were initiated by the addition of microsomes , and incubations were carried out aerobically (squalene and lanosterol substrates) for a period of 120 min or anaerobically (desmosterol substrate) for a period of 60 min. Protein was estimated Reaction products were isolated, separated by thin layer by biuret method (12). chromatography and assayed for radioactivity as previously described (13, 14). * ** ***
Values are the mean of closely Heated at 100° for 1 min Hicrosomes omitted
agreeing
107
duplicates
Vol. 53, No. 1, 1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II EFFECTOF TRYPSINHYDROLYSISONTHE CAPABILITY OF THE SOLUBLEPROTEINFRACTION PRECIPITATEDBY AMMONIUM SULFATETO STIMULATEMICROSOMAL CHOLESTEROL SYNTHESIS
Substrate
Additions
Protein (mg)
C1'C1 Squalene
None AS AS + Wyp AS+Tryp+Tryp Inhbtr.
[14Cl Lanosterol 3 As** *i As C14C1Desmosterol
+TT + izcLz . TwP
No#Fi As AS:: + Tryp As +~t~Ryp .
% ["Cl Recoveffed in Cholesterol
Stimulation (-fold)
1.5 5.3 3.5
0.92 9.6 1.8
10.4 1.8
5.1
9.0
9.8
1.5 4.0 2.7
1.6 17.1 4.6
10.7 2.8
3.7
15.4
9.7
1.5 3.7 2.8
4.3 49.9 7.1
11.6 1.7
3.6
46.4
10.8
AS (40-70%, 8.0 mg) in 100 mMphosphate buffer, pH 8.5, were digested with 0.8 mg of trypsin for 30 min at 37O. Proteolysis was stopped by the addition of equivalent amounts of soybean trypsin inhibitor and incubating the digests an additional 15 min. Control flasks containing 40 mg AS (40-70%), to which 0.8 mg each of trypsin and soybean trypsin inhibitor were simultaneously added, were incubated for 45 min. After the incubations the pH was adjusted to 7.4. Aliquots of the trypsin treated and control samples were then tested for its ability to stimulate conversion of squalene, lanosterol and desmosterol to cholesterol by liver microsomes as described in Table I. * **
Values are the means of closely Heated at 100° for 1 min
volume of buffer
equivalent
agreeing duplicate
to that of the original
against 100 volumes of the samebuffer
test the heat stability
of AS, the fractions
were heated under nitrogen at 100° for one min. sedimented and the supernatant fluids squalene, lanosterol
Slo5 fraction,
dialyzed
for 12 hr, and assayed for its ability
to stimulate conversions of squalene, lanosterol To
determinations
and desmosterol to cholesterol. containing
5% (NH4)2SO4, w/v,
The coagulated protein
assayed for their
ability
or desmosterol conversion to cholesterol.
108
was
to stimulate
BIOCHEMICAL
Vol. 53, No. 1,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS The conversion of squalene, lanosterol rat liver
and desmosterol to cholesterol
microsomes is shown in Table I.
fraction
The presence of the supernatant
stimulated the conversion of squalene to cholesterol
to cholesterol
13-fold,
and desmosterol to cholesterol
ment of the supernatant fraction
stimulate the conversion of lanosterol
natant precipitated
cholesterol
precipitated
activator
fraction
by liver
from liver
at 40-708 saturation,
similar results.
super-
which has been of the
Treatment with trypsin
by ammoniumsulfate
results in
capacity for the conversion of squalene to
by microsomes (Table II).
heated ammoniumsulfate capacity
fraction
loss of its
fraction
as the source for the purification
SCP and SPF proteins gave essentially
a substantial
The heat treat-
and desmosterol to cholesterol
with ammoniumsulfate
of the unheated protein
11-fold.
lanosterol
however, no loss in the capacity to
The studies with the protein
used by other investigators,
lo-fold,
resulted in a loss of its capacity to stimulate
the conversion of squalene to cholesterol;
microsomeswas observed.
by
Similarly,
treatment with trypsin
of
results in an almost complete loss of its
to stimulate conversion of lanosterol
and desmosterol to cholesterol.
DISCUSSION The data presented here show that the capacity of the soluble fraction stimulate conversion of squalene to cholesterol
However, the capacity
and by heating at 100° for one minute. fraction
to stimulate the conversion of lanosterol
by heat treatment,
but is lost by trypsin
The results therefore participate
sors to cholesterol
treatment
of the soluble
and desmosterol is unaffected
treatment of the heated fraction.
support the hypothesis that at least two soluble proteins
in the overall
somes. They further
conversion of squalene to cholesterol
indicate
that the conversion of lanosterol
requires a heat-stable
version of squalene to cholesterol teins.
is lost by trypsin
to
It can be inferred
therefore
protein
factor
109
micro-
or sterol precur-
while the overall
requires both heat-labile that the heat-labile
by liver
and heat-stable
protein
conpro-
factor plays
Vol. 53, No. 1,1973
BIOCHEMICAL
AND BIOPHYSICAL
a role in the conversion of squalene to lanosterol.
RESEARCH COMMUNICATIONS
The results of Yamamoto,
Lin and Bloch (15), that microsomal 2, 3, oxidosqualene cyclase does not require
the supernatant factor,
heat-labile
protein
are in accord with the suggestion that the
is required solely
for the squalene epoxidase system.
The results of the previous investigators manner. The heat-labile lanosterol
can be reconciled
protein which is required for conversion of squalene to
is probably identical
with the supernatant protein
purified
by Tai and Bloch (10) and which participates
system.
The heat-stable
this protein
factor
(SPF)
in the squalene epoxidase
protein which stimulates the conversion of lanosterol
and desmosterol to cholesterol that reported by Nitter
in the following
by liver
microsomes is perhaps identical
While the binding of squalene to
and Dempsey(4-6).
has been reported (6) its ability
between squalene and lanosterol
with
to stimulate
the enzymatic steps
has not been adequately documented.
REFERFNCES 1.
Olson, J. A. Jr.,
Lindberg M. and Bloch, K., J. Biol.
2.
. Avigan, J.,Goodman, D. S. and Steinberg,
3.
Dempsey, M. E., J. Biol.
4.
Bitter,
5.
Pitter, M. C. and Dempsey, M. E., Biochem. Biophys. Res. Commun.38, 921 (1970).
6.
Bitter,
7.
Scallen, (1971).
T. J.,
Schuster, M. W. and Dhar, A. K., J. Biol.
Chem. 246, 224
8.
Scallen, (1968).
T. J.,
Dean, W. J. and Schuster, M. W., J. Biol.
Chem. 243, 5202
9.
Scallen, T. J., Srikantaiah, Letters 25, 227 (1972).
Chem.E,
D., J. Biol.
Chem. 226, 941 (1957). Chem.238, 1283 (1963).
4176 (1965).
M. C. and Dempsey, M. E., Proc. Nat. Acad. Sci. 2,
M. C. and Dempsey, M. E., J. of Biol.
M. V.,
J. Biol.
Skrdlant,
Chem.z,
265 (1973).
Chem.-'246 1536 (1971).
H. B. and Hansbury, E.,FEBS
3767 (1972).
10.
Tai, H. and Bloch, K.,
11.
T. T. Tchen Methods in Enzymology Vol. VI, eds. S. P. Col&rich and N. 0. Kaplan (Academic Press, New York and London) P. 509 (1963).
110
Vol. 53, No. 1, 1973
BIOCHEMICAL
A., Bardawill,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
12.
Gornall,
C. and David, M., J. Biol.
13.
Shah, S. N., FEBSLetters 20, 75 (1972).
14.
Hinse, C. H. and Shah, S. N., J. Neurochem. 18, 1989 (1971).
15.
Yamamoto, s., Lin, K. and Bloch, K., F?oc. Nat. Acad. Sci. 63, 110 (1969).
111
Chem. 177, 751 (1949).
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EVIDENCEFORTHE PARTICIPATIONOF TWOSOLUBLENONCATALYTIC PROTEINSIN HEPATIC MICROSOMAL CHOLESTEROL SYNTHESIS1 R. C. Johnson and S. N. Shah2 Brain-Behavior Research Center SonomaState Hospital Eldridge, California 95431 Received
May 18,
1973 SOMMARY
The capacity of liver soluble fraction to stimulate hepatic microsomal conversion of squalene to cholesterol is lost on treatment with trypsin. Heat treatment of the soluble fraction results in a selective loss of its capacity to stimulate conversion of squalene to cholesterol; the ability to stimulate conversion of lanosterol and desmosterol to cholesterol is however retained. It is proposed that the liver soluble fraction contains at least two noncatalytic proteins, one heat-labile and the other heat-stable, which participate in microsomal cholesterol synthesis. The heat-labile protein mediates the conversion of squalene to lanosterol while the heat-stable protein is needed for the conversion of lanosterol and other sterol precursors to cholesterol. A requirement for a noncatalytic synthesis of cholesterol been clearly
for hepatic microsomal
from squalene and from other sterol precursors,
demonstrated (l-10).
which binds squalene, sterols synthesis has been purified (4-9).
soluble protein
The squalene and sterol
carrier
protein
and phospholipids and stimulates microsomal in the laboratories
has (SCP) sterol
of two groups of investigators
The major discrepancy in the results of these studies however, is that
the protein purified protein purified
by Ritter
by Scallen's
Bloch (10) have purified
and Dempsey(Q-6) is heat-stable group (7-9) is heat-labile.
a liver
supernatant protein
factor
while the
Recently,
Tai and
(SPF) which does
not bind squalene or oxidosqualene, but stimulates microsomal squalene epoxidase and is heat-labile.
To resolve these differences,
present study, the effects
we have reexamined, in the
of heat treatment and of trypsin
105,000 g supernatant fraction
from liver
on its
digestion of the
capacity to stimulate 1) the
1 Supported by Research Grants NW9301 and HBO5317from National Institutes of Health. 2 To whomcorrespondence should be addressed.
Copyright 0 19 73 by Academic Press, Inc. ,411 rights of reproduction in any form reserved.
105
Vol. 53, No. 1,1973
overall to
BIOCHEMICAL
AND BIOPHYSICAL
conversion of squalene to cholesterol, and 3) the reduction
cholesterol,
factors,
one heat-labile
is heat-labile,
soluble fraction
and the other heat-stable.
The results
contains two
The protein which
is required for squalene epoxidase system and the protein
which is heat-stable the lanosterol Materials
2) the conversion of lanosterol
of desmosterol to cholesterol.
obtained support the hypothesis that the liver protein
RESEARCH COMMUNICATIONS
participates
in microsomal cholesterol
synthesis beyond
step.
and Methods
C14CI Squalene and C1'Cl lanosterol
were prepared from DL-2 [14Cl
meva-
ionic acid DBEDsalt (New England Nuclear Corp., Boston, Ma.) by the method of Tchen (11).
26
[l”C]
was purchased from NewEngland Nuclear Corp.,
Desmosterol
Boston, Ma., NADPH,NAD , glucose-6-phosphate and soybean trypsin
inhibitor
were purchased from Calbiochem, San Diego, Ca. Glucose-6-phosphate dehydrogenase and trypsin, coated silica
were purchased from Sigma Chemical Co., St. Louis,
gel G plates and silver
nitrate
MO.
Pre-
were purchased from Eastman-Kodak,
Rochester, N. Y. Adult female Sprague-Dawley rats weighing approximately The livers
ficed by decapitation.
genized in 2.5 volumes of 0.1
M
were removed, perfused with saline and homo-
potassium phosphate buffer
0.5 mMDTT. The homogenate was centrifuged natant decanted and centrifuged was then centrifuged drawn off. collected
a buffer
(pH 7.4) containing
at 1,000 g for 10 min, and the supe-
at 18,000 g for 20 min.
The 18,000 g supernatant
at 105,000 g for 90 min and the floating
The upper two-thirds and recentrifuged
microsomal material.
200 gmwere sacri-
portion of
the
layer of lipid
supernatant fraction
at 105,000 g for 60 min to
The sedimented microsomal pellet
remove (MS)
(Slo5) was
any residual was resuspended in
volume equal to that of the 18,000 g supernatant and recentrifuged
105,000 g for 60 min.
The supernatant fraction
at
and washed microsomal pellets
were stored at -86O if not used immediately. The protein sulfate
fraction
was obtained from S1o5 by precipitation
between 40070%saturation.
The precipitate
106
with ammonium
(AS)ws redissolved
in a
Vol.
53, No.
BIOCHEMICAL
I,1973
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE I CONVERSION OF SQUALENE AND STEROL PRECURSORSTO CHOLESTEROLBY RAT LIVER MICROSOMES
Additions
Substrate
Protein (mg)
l14C1 Squalene
C14C3 Lanosterol
tl’CI
lksmosterol
% [14C1 Recovepd in Cholesterol
Stimulation (-fold)
None
1.5
1.0
%;***
9.0 7.5
9.8 1.7
9.8
&G70%) AS(40-70%)**
5.0 5.3 3.6
1.1 9.8 0.9
1.1 9.8 0
None
1.5
1.63
p;*** s105** AS(40-70%) AS(40-70%)**
9.0 7.5 5.0 5.0 3.8
21.8 2.05 18.1 22.5 17.1
13.4 11.1 13.7 10.5
None s105*** SlO5**
1.5
7.5 9.0
4.7 3.6 52.5
11.2
&O-70%) AS(40-70%)**
5.3 5.0 3.6
53.8 50.3 51.7
11.9 10.7 11.0
Each reaction mixture contained 1.5 mg washed microsomal protein, 100 mM phosphate pH 7.4 and 1.2 mM NADPH. Squalene experiments buffer contained in addition, 0.6 mM NADt , 30 mM nicotinamide, 4.0 mM glucose-6-PO4 and 1 unit glucose-6-PO4 dehydrogenase. Labeled substrates (20,000 cpm) in 5 ul dioxane:propylene glycol (2:l) were routinely preincubated with S105 or AS preparations at 37O for 15 min under N2. Reactions were initiated by the addition of microsomes , and incubations were carried out aerobically (squalene and lanosterol substrates) for a period of 120 min or anaerobically (desmosterol substrate) for a period of 60 min. Protein was estimated Reaction products were isolated, separated by thin layer by biuret method (12). chromatography and assayed for radioactivity as previously described (13, 14). * ** ***
Values are the mean of closely Heated at 100° for 1 min Hicrosomes omitted
agreeing
107
duplicates
Vol. 53, No. 1, 1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II EFFECTOF TRYPSINHYDROLYSISONTHE CAPABILITY OF THE SOLUBLEPROTEINFRACTION PRECIPITATEDBY AMMONIUM SULFATETO STIMULATEMICROSOMAL CHOLESTEROL SYNTHESIS
Substrate
Additions
Protein (mg)
C1'C1 Squalene
None AS AS + Wyp AS+Tryp+Tryp Inhbtr.
[14Cl Lanosterol 3 As** *i As C14C1Desmosterol
+TT + izcLz . TwP
No#Fi As AS:: + Tryp As +~t~Ryp .
% ["Cl Recoveffed in Cholesterol
Stimulation (-fold)
1.5 5.3 3.5
0.92 9.6 1.8
10.4 1.8
5.1
9.0
9.8
1.5 4.0 2.7
1.6 17.1 4.6
10.7 2.8
3.7
15.4
9.7
1.5 3.7 2.8
4.3 49.9 7.1
11.6 1.7
3.6
46.4
10.8
AS (40-70%, 8.0 mg) in 100 mMphosphate buffer, pH 8.5, were digested with 0.8 mg of trypsin for 30 min at 37O. Proteolysis was stopped by the addition of equivalent amounts of soybean trypsin inhibitor and incubating the digests an additional 15 min. Control flasks containing 40 mg AS (40-70%), to which 0.8 mg each of trypsin and soybean trypsin inhibitor were simultaneously added, were incubated for 45 min. After the incubations the pH was adjusted to 7.4. Aliquots of the trypsin treated and control samples were then tested for its ability to stimulate conversion of squalene, lanosterol and desmosterol to cholesterol by liver microsomes as described in Table I. * **
Values are the means of closely Heated at 100° for 1 min
volume of buffer
equivalent
agreeing duplicate
to that of the original
against 100 volumes of the samebuffer
test the heat stability
of AS, the fractions
were heated under nitrogen at 100° for one min. sedimented and the supernatant fluids squalene, lanosterol
Slo5 fraction,
dialyzed
for 12 hr, and assayed for its ability
to stimulate conversions of squalene, lanosterol To
determinations
and desmosterol to cholesterol. containing
5% (NH4)2SO4, w/v,
The coagulated protein
assayed for their
ability
or desmosterol conversion to cholesterol.
108
was
to stimulate
BIOCHEMICAL
Vol. 53, No. 1,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS The conversion of squalene, lanosterol rat liver
and desmosterol to cholesterol
microsomes is shown in Table I.
fraction
The presence of the supernatant
stimulated the conversion of squalene to cholesterol
to cholesterol
13-fold,
and desmosterol to cholesterol
ment of the supernatant fraction
stimulate the conversion of lanosterol
natant precipitated
cholesterol
precipitated
activator
fraction
by liver
from liver
at 40-708 saturation,
similar results.
super-
which has been of the
Treatment with trypsin
by ammoniumsulfate
results in
capacity for the conversion of squalene to
by microsomes (Table II).
heated ammoniumsulfate capacity
fraction
loss of its
fraction
as the source for the purification
SCP and SPF proteins gave essentially
a substantial
The heat treat-
and desmosterol to cholesterol
with ammoniumsulfate
of the unheated protein
11-fold.
lanosterol
however, no loss in the capacity to
The studies with the protein
used by other investigators,
lo-fold,
resulted in a loss of its capacity to stimulate
the conversion of squalene to cholesterol;
microsomeswas observed.
by
Similarly,
treatment with trypsin
of
results in an almost complete loss of its
to stimulate conversion of lanosterol
and desmosterol to cholesterol.
DISCUSSION The data presented here show that the capacity of the soluble fraction stimulate conversion of squalene to cholesterol
However, the capacity
and by heating at 100° for one minute. fraction
to stimulate the conversion of lanosterol
by heat treatment,
but is lost by trypsin
The results therefore participate
sors to cholesterol
treatment
of the soluble
and desmosterol is unaffected
treatment of the heated fraction.
support the hypothesis that at least two soluble proteins
in the overall
somes. They further
conversion of squalene to cholesterol
indicate
that the conversion of lanosterol
requires a heat-stable
version of squalene to cholesterol teins.
is lost by trypsin
to
It can be inferred
therefore
protein
factor
109
micro-
or sterol precur-
while the overall
requires both heat-labile that the heat-labile
by liver
and heat-stable
protein
conpro-
factor plays
Vol. 53, No. 1,1973
BIOCHEMICAL
AND BIOPHYSICAL
a role in the conversion of squalene to lanosterol.
RESEARCH COMMUNICATIONS
The results of Yamamoto,
Lin and Bloch (15), that microsomal 2, 3, oxidosqualene cyclase does not require
the supernatant factor,
heat-labile
protein
are in accord with the suggestion that the
is required solely
for the squalene epoxidase system.
The results of the previous investigators manner. The heat-labile lanosterol
can be reconciled
protein which is required for conversion of squalene to
is probably identical
with the supernatant protein
purified
by Tai and Bloch (10) and which participates
system.
The heat-stable
this protein
factor
(SPF)
in the squalene epoxidase
protein which stimulates the conversion of lanosterol
and desmosterol to cholesterol that reported by Nitter
in the following
by liver
microsomes is perhaps identical
While the binding of squalene to
and Dempsey(4-6).
has been reported (6) its ability
between squalene and lanosterol
with
to stimulate
the enzymatic steps
has not been adequately documented.
REFERFNCES 1.
Olson, J. A. Jr.,
Lindberg M. and Bloch, K., J. Biol.
2.
. Avigan, J.,Goodman, D. S. and Steinberg,
3.
Dempsey, M. E., J. Biol.
4.
Bitter,
5.
Pitter, M. C. and Dempsey, M. E., Biochem. Biophys. Res. Commun.38, 921 (1970).
6.
Bitter,
7.
Scallen, (1971).
T. J.,
Schuster, M. W. and Dhar, A. K., J. Biol.
Chem. 246, 224
8.
Scallen, (1968).
T. J.,
Dean, W. J. and Schuster, M. W., J. Biol.
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