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The heterogeneous composition of the smooth microsomal membranes in rat liver
Life Sciences Vol . 7, Part I, pp . 905-911, 1968 . Printed in Great Britain.
Pergamon Press
THE HETEROGENE(7US COD~OSITION OF THE 31~OTH MICROSOMAL LII~RANES IN EAT LIVER H . Gla»_m~~n , A . von der Decken and G . Dallner Department of Pathology at 3abbatsberg Hospital, Karolinaka Institutet ; Wenner-Gren Inat . and Ins t . of Biochemistry, IIniv . of Stockholm, Sweden
(Received 14 March 1968 ; in final form 10 June 1968) THE microsomel fraction of liver consists of rough- and smooth-surfaced vesicles, differing in ultrastruatnre, partly in en~rmic oompoeition, and in their participation in enzyme and membrane synthesis (1,2) .
After
rough and smooth microsomes have been separated on a sucrose gradient containing a monovalent cation, the smooth miarosomes can be further subfractionated in the presence of a divalent cation (3) "
As the enzyme
pattern of one fraction of smooth miarosomes differ greatly from the other, the question arises se to whether both really belong to the endoplasmio reticulum (ER) .
Our findings indicate a close relationship between the
two smooth subfraotions, but the differences in lipid oompoeition and turnover, and behavior during the phenobarbital-induced process suggest specialized functions for these two types of membranes . üethods microsomes, mitochondria, outer mitoehondrial membranes (4) and plasma membranes (5) were prepared as described previously .
Subfraation-
ation of microsomes, eztraction, cüromatographic separation and meaeure mente of PLP (phoepholipida) and NL (neutral lipids) were performed as earlier (2,3,6) .
In incorporation experiments, glycerol-2 3H (20 ~0~100
g) or sodium acetate-3 H (1 ma~100 g) were injected intraperitoneally (ip~~ and the radioactivity of the extracted and washed PLP or Ch (cholesterol) was measured in a Beckman liquid scintillation counter .
905
DL-leucine- 14C
906
Vol. 7, No. 17
SMOOTH MICROSOMAL MEMBRANES
(10 Wo~100 g) was injected into the femoral vein . miprosomes were eonioated and centrifuged (7) .
The aubfraotionated
The soluble fractions
were ]yophilised and subjected to immunoeleptrophoresie (8) .
The pre-
oipitates were used for protein measurement and counting (9) .
En~rme
aptivitiee were determined as deapribed earlier (4,10,11) . eeu to The Ch content of the various miprosomal membranes is shown in Table 1 .
On a PLP basis, the Ch pontent of rough, smooth I and smooth TABLE 1 PLP and Cholesterol Content of Microsomal Subfraotione from Control and Phenobarbital-Treated Rate C 0 N T 8 0 L
Fraotion
PLP mg per g liver
P H E N 0 B d R B I T A L
Ch
Ch
mg per g liver
per PLP
PLP
Ch
mg per g liver
mg per g liver
Ch per PLP
Total
6 .7
0 .60
0 .09
11 .9
0 .95
o .os
Rough
3 .5
0 " 24
0 .07
6 .2
0 .37
0 .06
Smooth I
2 .1
0 .23
0 .11
4 .0
0 .44
0 .11
Smooth II
0 .4
0 .07
0 .17
0 .4
0 .07
0 .16
Phenobarbital was injected ip daily for 5 days (8 mg~100 g) " PLP . phoepholipids, Ch . cholesterol . Median of 8 experiments . II miproaomes is 7, 11 and 17 ~, reapeptively .
In spite of the fact that
the PLP pontent of the ,rough and smooth I fractions inpreasee after phenobarbital-treatment, while the PLP of smooth II membranes remains unchanged] there is no alteration of the Ch~PLP ratio in a~lyr fraction .
When the ad-
sorbed and luminal protein was removed by washing propedurea, the membranes of all eubfraotions exhibited the same PLP~protein ratio, ti 0 .40 . Paper ohromatographip ana]yeie of the phoephatides in the various micro-
Vol . 7, No . 17
SMOOTH MICROSOMAL MEMBRANES
907
aomal membranes did not reveal arm qualitative differences (not shown is Table) .
Rough 20-
o
Smooth 1
a,
~
Smooth I1
96
120
oi
r
v~ K 24
48
72 Hours FIG. 1
Distribution of protein in miorosomal aubfractione after phenobarbital treatment . The rats were in acted intraperitoneal],q at 0 and every subsequent 24 hours (8 m$/1oo g~ " The great increase in the amount of miaroeomal membranes after
5
injections of phenobarbital ie restricted to the rough sad smooth I aubfractions (Fig . 1) .
a
160
Incorporation of (a) g]ycerol-3H iato PLP and (b) acetate- 3H into the cholesterol of miorosomal subfraotione . The values are taken from 3 oonaeoutive ezperimente .
908
SMOOTH MICROSOMAL MEMBRANES
Vol . 7, No. 17
The in vivo incorporation rates of glycerol- 3H into PLP and of acetate- 3 H into Ch suggest that PLP and Ch biosynthesis occurs in all three subfractions .
However, the incorporation rate into the PLP of the
smooth II subfraotion is significantly lower than that in the two other aubfractions, while in the case of Ch the smooth I fraction is the less active one (Fig . 2) . The incorporation of leucine-14C into albumin isolated from rough microsomes is much higher after smooth counterparts (Fig. 3) .
5
and 10 minutes than that of its two
The radioactivity of the albumin of both
c .d.
0 a vi E E a
U
10 20 Minutes after leucine-Cu injection FIG .
30
3
Incorporation of leucine- 14C into the albumin of miorosomal subfraotione . The radioactivity refers to oounte~min%mg protein present in antigenantibody precipitate (albumin pluo y-globulin) . smooth I and II miarosomes increases slowly and reaches the level of the rough microsomes at 20 min, which is in agreement with the concept of albumin transport from the rough to the smooth part of the ER (12) .
A
simultaneous occurrence of radioactivity in the smooth microaomal subfractions strongly indicates that both these fractions participate in
Vol . 7, No . 17
SMOOTH MICROSOMAL MEMBRANES
909
the transport prooese . TABLE 2 Distribution of Soae Ensy~ee Aotivities in Yariaue Cellfraotions Fraotion
AIIPase i
Md0ase2
Cyt .a oxidas " 3
Aoid P~hosphatea " 4
51 .0
Homogenate
0 .5
Plasma membranes
7 .2
1 .0
1[i.toohondria
0 .1
14"0
Outer mitoohondrial membranes
94 " 5
108 .0
Rough miorosomes
0.4
3.1
0.5
3.0
smooth I miorosomee
0 .6
3.0
0.3
2 .0
Smooth II miorosaaes
1 .0
2 .2
0 .2
0 .9
AMPase . adenosine moaophoephatase ; MAOase . monoamiao ozidass ; ayt . . oytoohrome . ' i ~+molee Pi ~20 min~ag protein ; 3 E+molee O~mix~g liver ;
2mWmolee ben$3rlemine~mi~/mg protein ;
4 ~moles Pi~20 mintg liver .
The distribution of oerte3n marker ensyme activities for various oell organelles does not show a~yr oonoentratioa in either the smooth I or smooth II fractions (Table 2) . Discussion The experiments on PLP content and oompoeition~ albumin transport and marker ens~mee Strongly indicate that both smooth and rough Subfraotions of liver microsomes either derive from the ER or from closely related membranee . turnover data
On the other handy the NL and en~me oomposition~ the
the absence of phenobarbital effect on the smooth II mioro-
somee, as well as the differentiated sensitivity to divalent canons (13) demonstrate heterogeneity and specialisation within the membranes of the miorosomsl fraction.
The aimultaneoue transport of albumin into both
SMOOTH MICROSOMAL MEMBRANES
91 0
Vol. 7, No . 17
smooth microsomal fraotiona does not neoesaarily mean that the albumina in the two fractions are identical (of . 14) .
To prove the membranous
nature of cholesterol, we used various washing procedures as well as diluted detergents but were not able to decreeae its content in the membranes .
Phenobarbital has been reported to enhance the amount of
microsomal membranes with e concomitant inareaee of PLP (15) and cholesterol synthesis (16) .
In spite of these changes, the oholeaterol~PLP
ratio still remains the same .
Also, the long half-life of membrane
cholesterol (17) supports the hypothesis of its being a true constituent . Liver from fed animals exhibits a higher content of oholeeterol and also an increased rate of incorporation of various precursors when compared with that from starved animals (18) .
Probably this inareaee reflects a
stimulation of bile said synthesis, while in the case of starvation, as in our experiments, most of the cholesterol could belong to a compartment of relatively low metabolic nativity . Esoh of the two types of smooth microsomea might still represent heterogeneous fraotiona, but this could be decided only by elaboration of further subfraotioaation procedures .
Such heterogeneity seems to be
present among rough microsomal vesicles (19) . Acknowledgment This work has been supported by grants from the Swedish Cancer Society (72-K68-02%) and from the Swedish Natural Research Council (2520-7) .
We thank Dr . Bo Kuylenatierna for his help in the measurement
of 1dA0ase activity . References G .E . PALADE and P . SiEKEVITZ, J . BlOU ve . Bioahem . Cyt. é, 171 (1956) . G . DALLNER, P . SIEKEVITZ and G .E . PALADE, J . Cell Biol . ~, 73, 97
(1966) " 3" 4"
G . DALLNER, Aata Path . e t ~ücrobiol . Scand ., Suppl . 166 (1963) . G .L . SOTTCCASA, B . KUYLENSTIERNA, L . ERNSTER and A . BERGSTR6ND,
Vol. 7, No . 17
SMOOTH MICROSOMAL MEMBRANES
911
5"
P . E6II~LOT, C .J . BOS, E .L . BENEDETTI and Ph . R~1~E, Biochem. Biophys . Aota .20, 126 (1964) .
6.
G .V . MARINETTI, J . Lipid . Rea . 6, 315 (1965) "
7.
A . YON DER DECKEN and P .H . CAMPBELL, Biochem . J . ~, 195 (1964) "
e.
P . GRABAR and C .A . WILLIAMS, Bioohim. Biophys . Acta ~, 67 (1955) "
9.
A . VON DER DECKEN, Anal . Biochem . ]8, 444 (1967) "
10 . C .S . SONG and 0 . BODAxSRY, J . Biol . Chem . 2e~2, 694 (1967) " 11 . C . SCHNAITMAN, Y .G . ERWIN and J .W . GREENAWALT, J . Cell Biol . ~, 719 (1967) " 12 . T . PETERS, J . Biol . Chern . ~, 1186 (1962) . 13 " G-. DALLNER and R . NILSSON, J . Cell Biol . ~, 181 (1966) . 14 . P .W . JIINGBLIIT, Biochem . ZeitsahriYt ~, 267, 285 and 297 (1963) " 15 . S . ORRENIIIS, J .L .E . ERICSSON and L . ERNSTER, J . Cell Biol . ~, 627 (1965) " 16 . A .L . JONES and D .T . ARMSTRONG, Proo . Soo . Ezp . Biol . Yed. 1 1~, (1965) "
, 1136
17 " A .A . KHAN and J . FOLCH-PI, J . Nenrochem . 1~e, 1099 (1967) . 18 . G .R . JANSEN, M .E . ZANETTI and C .F . HIITCHINSOH, Biochem . J . ~ , 811 (1966) . 19 . G . DALLNER, A . BERGSTRAND and R . NIL330N, Abstracts . Oslo, p . 132 (1967) "
FEHS 4th YeetinR ,
Pergamon Press
THE HETEROGENE(7US COD~OSITION OF THE 31~OTH MICROSOMAL LII~RANES IN EAT LIVER H . Gla»_m~~n , A . von der Decken and G . Dallner Department of Pathology at 3abbatsberg Hospital, Karolinaka Institutet ; Wenner-Gren Inat . and Ins t . of Biochemistry, IIniv . of Stockholm, Sweden
(Received 14 March 1968 ; in final form 10 June 1968) THE microsomel fraction of liver consists of rough- and smooth-surfaced vesicles, differing in ultrastruatnre, partly in en~rmic oompoeition, and in their participation in enzyme and membrane synthesis (1,2) .
After
rough and smooth microsomes have been separated on a sucrose gradient containing a monovalent cation, the smooth miarosomes can be further subfractionated in the presence of a divalent cation (3) "
As the enzyme
pattern of one fraction of smooth miarosomes differ greatly from the other, the question arises se to whether both really belong to the endoplasmio reticulum (ER) .
Our findings indicate a close relationship between the
two smooth subfraotions, but the differences in lipid oompoeition and turnover, and behavior during the phenobarbital-induced process suggest specialized functions for these two types of membranes . üethods microsomes, mitochondria, outer mitoehondrial membranes (4) and plasma membranes (5) were prepared as described previously .
Subfraation-
ation of microsomes, eztraction, cüromatographic separation and meaeure mente of PLP (phoepholipida) and NL (neutral lipids) were performed as earlier (2,3,6) .
In incorporation experiments, glycerol-2 3H (20 ~0~100
g) or sodium acetate-3 H (1 ma~100 g) were injected intraperitoneally (ip~~ and the radioactivity of the extracted and washed PLP or Ch (cholesterol) was measured in a Beckman liquid scintillation counter .
905
DL-leucine- 14C
906
Vol. 7, No. 17
SMOOTH MICROSOMAL MEMBRANES
(10 Wo~100 g) was injected into the femoral vein . miprosomes were eonioated and centrifuged (7) .
The aubfraotionated
The soluble fractions
were ]yophilised and subjected to immunoeleptrophoresie (8) .
The pre-
oipitates were used for protein measurement and counting (9) .
En~rme
aptivitiee were determined as deapribed earlier (4,10,11) . eeu to The Ch content of the various miprosomal membranes is shown in Table 1 .
On a PLP basis, the Ch pontent of rough, smooth I and smooth TABLE 1 PLP and Cholesterol Content of Microsomal Subfraotione from Control and Phenobarbital-Treated Rate C 0 N T 8 0 L
Fraotion
PLP mg per g liver
P H E N 0 B d R B I T A L
Ch
Ch
mg per g liver
per PLP
PLP
Ch
mg per g liver
mg per g liver
Ch per PLP
Total
6 .7
0 .60
0 .09
11 .9
0 .95
o .os
Rough
3 .5
0 " 24
0 .07
6 .2
0 .37
0 .06
Smooth I
2 .1
0 .23
0 .11
4 .0
0 .44
0 .11
Smooth II
0 .4
0 .07
0 .17
0 .4
0 .07
0 .16
Phenobarbital was injected ip daily for 5 days (8 mg~100 g) " PLP . phoepholipids, Ch . cholesterol . Median of 8 experiments . II miproaomes is 7, 11 and 17 ~, reapeptively .
In spite of the fact that
the PLP pontent of the ,rough and smooth I fractions inpreasee after phenobarbital-treatment, while the PLP of smooth II membranes remains unchanged] there is no alteration of the Ch~PLP ratio in a~lyr fraction .
When the ad-
sorbed and luminal protein was removed by washing propedurea, the membranes of all eubfraotions exhibited the same PLP~protein ratio, ti 0 .40 . Paper ohromatographip ana]yeie of the phoephatides in the various micro-
Vol . 7, No . 17
SMOOTH MICROSOMAL MEMBRANES
907
aomal membranes did not reveal arm qualitative differences (not shown is Table) .
Rough 20-
o
Smooth 1
a,
~
Smooth I1
96
120
oi
r
v~ K 24
48
72 Hours FIG. 1
Distribution of protein in miorosomal aubfractione after phenobarbital treatment . The rats were in acted intraperitoneal],q at 0 and every subsequent 24 hours (8 m$/1oo g~ " The great increase in the amount of miaroeomal membranes after
5
injections of phenobarbital ie restricted to the rough sad smooth I aubfractions (Fig . 1) .
a
160
Incorporation of (a) g]ycerol-3H iato PLP and (b) acetate- 3H into the cholesterol of miorosomal subfraotione . The values are taken from 3 oonaeoutive ezperimente .
908
SMOOTH MICROSOMAL MEMBRANES
Vol . 7, No. 17
The in vivo incorporation rates of glycerol- 3H into PLP and of acetate- 3 H into Ch suggest that PLP and Ch biosynthesis occurs in all three subfractions .
However, the incorporation rate into the PLP of the
smooth II subfraotion is significantly lower than that in the two other aubfractions, while in the case of Ch the smooth I fraction is the less active one (Fig . 2) . The incorporation of leucine-14C into albumin isolated from rough microsomes is much higher after smooth counterparts (Fig. 3) .
5
and 10 minutes than that of its two
The radioactivity of the albumin of both
c .d.
0 a vi E E a
U
10 20 Minutes after leucine-Cu injection FIG .
30
3
Incorporation of leucine- 14C into the albumin of miorosomal subfraotione . The radioactivity refers to oounte~min%mg protein present in antigenantibody precipitate (albumin pluo y-globulin) . smooth I and II miarosomes increases slowly and reaches the level of the rough microsomes at 20 min, which is in agreement with the concept of albumin transport from the rough to the smooth part of the ER (12) .
A
simultaneous occurrence of radioactivity in the smooth microaomal subfractions strongly indicates that both these fractions participate in
Vol . 7, No . 17
SMOOTH MICROSOMAL MEMBRANES
909
the transport prooese . TABLE 2 Distribution of Soae Ensy~ee Aotivities in Yariaue Cellfraotions Fraotion
AIIPase i
Md0ase2
Cyt .a oxidas " 3
Aoid P~hosphatea " 4
51 .0
Homogenate
0 .5
Plasma membranes
7 .2
1 .0
1[i.toohondria
0 .1
14"0
Outer mitoohondrial membranes
94 " 5
108 .0
Rough miorosomes
0.4
3.1
0.5
3.0
smooth I miorosomee
0 .6
3.0
0.3
2 .0
Smooth II miorosaaes
1 .0
2 .2
0 .2
0 .9
AMPase . adenosine moaophoephatase ; MAOase . monoamiao ozidass ; ayt . . oytoohrome . ' i ~+molee Pi ~20 min~ag protein ; 3 E+molee O~mix~g liver ;
2mWmolee ben$3rlemine~mi~/mg protein ;
4 ~moles Pi~20 mintg liver .
The distribution of oerte3n marker ensyme activities for various oell organelles does not show a~yr oonoentratioa in either the smooth I or smooth II fractions (Table 2) . Discussion The experiments on PLP content and oompoeition~ albumin transport and marker ens~mee Strongly indicate that both smooth and rough Subfraotions of liver microsomes either derive from the ER or from closely related membranee . turnover data
On the other handy the NL and en~me oomposition~ the
the absence of phenobarbital effect on the smooth II mioro-
somee, as well as the differentiated sensitivity to divalent canons (13) demonstrate heterogeneity and specialisation within the membranes of the miorosomsl fraction.
The aimultaneoue transport of albumin into both
SMOOTH MICROSOMAL MEMBRANES
91 0
Vol. 7, No . 17
smooth microsomal fraotiona does not neoesaarily mean that the albumina in the two fractions are identical (of . 14) .
To prove the membranous
nature of cholesterol, we used various washing procedures as well as diluted detergents but were not able to decreeae its content in the membranes .
Phenobarbital has been reported to enhance the amount of
microsomal membranes with e concomitant inareaee of PLP (15) and cholesterol synthesis (16) .
In spite of these changes, the oholeaterol~PLP
ratio still remains the same .
Also, the long half-life of membrane
cholesterol (17) supports the hypothesis of its being a true constituent . Liver from fed animals exhibits a higher content of oholeeterol and also an increased rate of incorporation of various precursors when compared with that from starved animals (18) .
Probably this inareaee reflects a
stimulation of bile said synthesis, while in the case of starvation, as in our experiments, most of the cholesterol could belong to a compartment of relatively low metabolic nativity . Esoh of the two types of smooth microsomea might still represent heterogeneous fraotiona, but this could be decided only by elaboration of further subfraotioaation procedures .
Such heterogeneity seems to be
present among rough microsomal vesicles (19) . Acknowledgment This work has been supported by grants from the Swedish Cancer Society (72-K68-02%) and from the Swedish Natural Research Council (2520-7) .
We thank Dr . Bo Kuylenatierna for his help in the measurement
of 1dA0ase activity . References G .E . PALADE and P . SiEKEVITZ, J . BlOU ve . Bioahem . Cyt. é, 171 (1956) . G . DALLNER, P . SIEKEVITZ and G .E . PALADE, J . Cell Biol . ~, 73, 97
(1966) " 3" 4"
G . DALLNER, Aata Path . e t ~ücrobiol . Scand ., Suppl . 166 (1963) . G .L . SOTTCCASA, B . KUYLENSTIERNA, L . ERNSTER and A . BERGSTR6ND,
Vol. 7, No . 17
SMOOTH MICROSOMAL MEMBRANES
911
5"
P . E6II~LOT, C .J . BOS, E .L . BENEDETTI and Ph . R~1~E, Biochem. Biophys . Aota .20, 126 (1964) .
6.
G .V . MARINETTI, J . Lipid . Rea . 6, 315 (1965) "
7.
A . YON DER DECKEN and P .H . CAMPBELL, Biochem . J . ~, 195 (1964) "
e.
P . GRABAR and C .A . WILLIAMS, Bioohim. Biophys . Acta ~, 67 (1955) "
9.
A . VON DER DECKEN, Anal . Biochem . ]8, 444 (1967) "
10 . C .S . SONG and 0 . BODAxSRY, J . Biol . Chem . 2e~2, 694 (1967) " 11 . C . SCHNAITMAN, Y .G . ERWIN and J .W . GREENAWALT, J . Cell Biol . ~, 719 (1967) " 12 . T . PETERS, J . Biol . Chern . ~, 1186 (1962) . 13 " G-. DALLNER and R . NILSSON, J . Cell Biol . ~, 181 (1966) . 14 . P .W . JIINGBLIIT, Biochem . ZeitsahriYt ~, 267, 285 and 297 (1963) " 15 . S . ORRENIIIS, J .L .E . ERICSSON and L . ERNSTER, J . Cell Biol . ~, 627 (1965) " 16 . A .L . JONES and D .T . ARMSTRONG, Proo . Soo . Ezp . Biol . Yed. 1 1~, (1965) "
, 1136
17 " A .A . KHAN and J . FOLCH-PI, J . Nenrochem . 1~e, 1099 (1967) . 18 . G .R . JANSEN, M .E . ZANETTI and C .F . HIITCHINSOH, Biochem . J . ~ , 811 (1966) . 19 . G . DALLNER, A . BERGSTRAND and R . NIL330N, Abstracts . Oslo, p . 132 (1967) "
FEHS 4th YeetinR ,