EXPERIMENTAL
AND
Subcellular
MOLECULAR
Localization HANS
Department
of Steroid GLAIJMANN
of Pathology II, Karolinska Receiued
May
27,
PATHOLOGY
( 1977)
Hormone
Metabolism
AND JAN-AKE
GUSTAFSSON
Huddinge Institutet,
17,1976,
221-234
Hospital and Department Stockholm, Sweden
and in reoised
form
December
in Rat Liver
of Chemistry,
15, 1976
The metabolism of 4-[4-%]androstene-3,17-dione was studied in smoothand rough-surfaced microsomes, plasma membranes, mitochondria, Golgi apparatus, and lysosomes isolated from rat liver. 5a-Reductase, 17P-hydroxysteroid reductase, 16~, 6/3-, and ‘la-hydroxylase activities were measurable in all isolated subcellular fractions except in lysosomes which did not metabolize 4-androstene-3,17-dione. Steroid metabolism patterns in smooth and rough microsomes were very similar. The extent of microsomal contamination in the plasma membrane, Golgi apparatus, and mitochondrial and lysosomal fractions was measured both by ultrastructural (morphometric) analysis and by distribution analysis of “marker enzymes” and was less than 15, 3, 16, and 30/o, respectively. Whereas it could not be excluded that the metabolism of steroids in the Golgi apparatus enriched fraction occurred due to microsomal contamination, the data indicate that the steroid metabolism in isolated plasma membranes and mitochondria occurred due to the presence of steroid reducing and hydroxylating enzyme systems in these cell organelles. Indirectly, such a similarity in enzymatic makeup between different cell organelles would tend to support “the membrane flow hypothesis.”
INTRODUCTION The endoplasmic reticulum (ER) is the main site for metabolism of drugs, steroid hormones, fatty acids, and polycyclic hydrocarbons (Mannering, 1972). Generally, low-molecular weight compounds are hydroxylated by an NADPHlinked electron transport chain with cytochrome P-450 as the terminal oxidase (Gillette et al., 1968). Wh ereas much interest has been focused on the distribution of hydroxylating activities in various microsomal subfractions, especially rough and smooth microsomes (Gram et al., 1968; Glaumann, 1970), relatively little interest has been devoted to the possible participation of other cell organelles such as the plasma membranes, Golgi apparatus, and lysosomes in the metabolism of low-molecular weight compounds. This is especially true for steroid hormones, which are considered to be the natural substrates for the microsomal NADPH-dependent hydroxylating chain (cf. Hamberg et al., 1974). The introduction of the membrane flow hypothesis has changed the previous static view on cell organelles as isolated entities of the cell to a more dynamic notion (Franke et al., 1971). The membrane flow hypothesis implies that the biogenesis of a specific membrane is accomplished by a migration of a membrane piece or membrane component from one cell organelle to another. The close morphological interrelation between the ER and the Golgi apparatus has long
Copyright All rights
@ 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0014-4800
222
GLAUMANN
AND
GUSTAFSSON
been known. Based on kinetic studies on the synthesis and degradation of ER membranes, Golgi apparatus membranes, and plasma membranes, Franke et al. (1971) postulated a flow of membranes from the ER to the Golgi apparatus and to the plasma membrane. If this is true, one could expect that these different types of cellular membranes might show similarities in their enzyme contents and function. Since steroid hormones are hydroxylated and reduced by several liver enzyme systems, we have investigated the distribution of these enzyme activities in some detail among various isolated subcellular fractions. A prerequisite for any conclusion regarding the participation of various cell organelles in the metabolism of steroids is that the purity and the degree of contamination of the isolated fraction be known. Therefore, a thorough morphological and biochemical characterization was performed to evaluate the actual composition of each fraction. MATERIALS
AND METHODS
Animals Male Sprague-Dawley rats weighing 150-180 g were used. Before sacrif?ce, the animals were starved for 15 hr. Chemicals 4-[4-‘C]Androstene-3,17-dione (specific activity, 60 mCi/mmoIe) was purchased from the Radiochemical Centre (Amersham, England) and purified by thin-layer chromatography. Unlabeled 4-androstene-3,17-dione was kindly given by Dr. J. Babcock (Upjohn Co., Kalamazoo, Michigan). Other chemicals were of reagent grade and obtained from Sigma Chemical Co. (St. Louis, Missouri). Isolation of Cell Organelks For preparation of total microsomes, 20% (w/v) liver homogenate in 0.29 M sucrose was centrifuged at 10,OOOgfor 20 min to remove cell debris, nuclei, mitochondria, and lysosomes. The supernatant was sedimented at 100,OOOgfor 60 min to obtain a microsomal fraction, which was suspended in 0.29 M sucrose. For isolation of rough and smooth microsomes, the method of Dallner (1963) and Glaumaml and Dallner (1968) was used with some modification. CsCl was added to the 10,OOOgsupernatant to a final concentration of 15 mM; 20 ml was layered over 10 ml of 1.30 M sucrose containing 13 mM CsCl and centrifuged at 27,000 rpm (131,000g) for 2 hr in a Christ Omega SW-27 rotor. The entire fluffy layer at the gradient boundary was collected and sedimented at 100,OOOg for 60 min to obtain a pellet of smooth microsomes. Distilled water was added to bring the sucrose concentration to 0.29 M, and the pellet containing the rough microsomal fraction was resuspended. The microsomes were then washed in 0.29 M sucrose. The use of an SW rotor, instead of an angle-head rotor, for the separation of rough and smooth microsomes gave purer fractions due to less pronounced sidewall impaction (Glaumann et al., 1975a). For isolation of a mitochondrial fraction, the livers were transferred to icecold 0.29 M sucrose, minced, and homogenized with four up-and-down strokes, using a Teflon-glass homogenizer (chamber clearance 0.15 to 0.23 mm) set at 300 rpm.
STEROID
METABOLISM
IN
CELL
ORGANELLES
223
A 10% homogenate in 0.29 M sucrose was spun at 600g for 10 min in an MSE-25 centrifuge. The resulting supernatant was centrifuged at 6000g for 10 min, and the pellet was gently suspended with a pipette. The 6000g run was then repeated twice as a washing procedure. For isolation of the Golgi apparatus, homogenization of livers from ethanoltreated rats was performed in 0.5 M sucrose-5 mM MgC12 at low speed (about with three complete strokes. The fractionation 200 rpm) (25% h omogenate) procedure was performed according to Ehrenreich et al. ( 1973). Plasma membranes were separated by the method of Coleman et al. (1967). A liver lysosomal fraction was isolated according to Glaumann et al. ( 1975b ) after repetitive injections of an iron-sorbitol-citric acid complex. Preparation
for Electron
Aliquots of the cacodylate buffer into l-mm pieces, 2 hr in 1% 0~0~. electron microscopy Biochemical
Microscopy
suspended fractions were fixed in 2% glutaraldehyde-0.1 M and centrifuged at 20,OOOg for 30 min. The pellets were cut washed in cacodylate buffer, and additionally fixed for 1 to Dehydration, embedding, cutting, and staining procedures for have been previously described (Glaumann et al., 1975a).
Assays
Protein was determined according to Lowry et al. ( 1951) with serum albumin as standard. The following enzymes were measured for estimation of contamination: cytochrome P-450 and cytochrome b, (Omura and Sato, 1964)) NADPHand NADH-cytochrome c reductase ( Dallner, 1963)) glucose-6-phosphatase ( G6Pase ), S-nucleotidase ( AMPase ), UDP-galactosyl transferase ( cf. Glaumann et al., 1975a), and cathepsin D (Bowers et al., 1967). Steroid Incubation Cell organelle suspensions corresponding to 2-10 mg of protein were incubated in 2.0 ml of Bucher medium (Bergstrom and Gloor, 1955) with 20, 50, 100, 200, and 400 pg of 4-[4-14C]androstene-3,17-dione (2 x lo6 dpm per incubation) at 37°C for 10 min in the presence of an NADPH-regenerating system 10.03 pmole of MnC&, 3.0 +oles of NADP, 125 pmoles of isocitrate, and 0.4 unit of isocitrate dehydrogenase type IV (Sigma Chemical Co.)]. The reactions were started by adding the substrate in 50 ,J of acetone and were stopped by adding 20 v01 of chloroform-methanol, 2:l (v/v). A 0.2-vol of a solution of sodium chloride (0.9%, w/v) was added. The chloroform phase was collected and reduced to dryness under vacuum. The residue was dissolved in a small volume of chloroform-methanol (2:1, v/v) and applied to a precoated silica gel plate (250~, Merck, Darmstadt, Germany). The thin-layer plates were developed once in the solvent system chloroform-ethyl acetate, 4: 1 (v/v). After autoradiography for 7 days, radioactive zones could be localized and scraped off separately. Aliquots of the methanol extracts of the silica gel zones were assayed for radioactivity in a Packard liquid scintillation spectrometer, Model 3003, using Instagel as the scintillator liquid. Several samples from corresponding thin-layer chromatographic zones from different incubations were pooled to give sufficient amounts of material to per-
224
CLAUMANN
AND
GUSTAFSSON
FIG. 1. Electron micrograph of the isolated mitochondrial fraction. The pellet consists almost exclusively of mitochondrial profiles in condensed conformation. X 10,450. FK. 2. Appearance of isolated lysosomes. The pellet consists of lysosomes filled with ferritin granules. Note the absence of mitochondrial profiles. X17,100. Inset shows a lysosome with intact bordering membrane. X57,000. The cellular origin of the lysosomes has been discussed recently (Glaumann et al., 197513).
STEROID
METABOLISM
IN
CELL
ORGANELLES
225
This fractio of isolated rough surfaced microsomes. FIG. 3. Appearance or somewhat elongated profiles with several ribosomes attached to the outer surface. X36,10( FIG. 4. Appearance of isolated smooth-surfaced microsomes. This fraction contains on! smooth-surfaced vesicles and no contaminating rough-surfaced microsomes or plasma men brane sheaths. X38,000.
:rmit gas chromatographic-mass spectrometric analysis. The analyses were pe ra formed on an LKB 2091 instrument using a lo/O SE-30 column. Mass spect md were recorded on magnetic tape using the incremental mode of operation 2
226
GLAUMANN
AND
GUSTAFSSON
FIG. 5. Electron micrograph of Golgi apparatus enriched fraction. The fractionation procedure was performed after ethanol administration according to Ehrenreich et al. ( 1973). This fraction corresponds to the total Golgi fraction (GFI + GF, + GK). The fraction contains numerous Golgi vacuoles and cisternae filled with VLDL clusters. Small Golgi vesicular profiles containing single particles are also present. These vesicular profiles most likely rep-
STEROID
METABOLISM
IN
CELL
ORGANELLES
227
were then treated in a PDP ll/lO computer (Digital Equipment AB, Solna, Sweden). A compound was considered identified if it had the same mass spectrum and gas-liquid chromatographic behavior as the reference compound. RESULTS Ultrustruc-tural
Characterization
AND
DISCUSSION
of the Isolated Fractions
Mitochondrial fraction. Electron micrographs of the mitochondrial preparations revealed that the fractions consisted almost exclusively of mitochondria with intact outer membranes (Fig. 1). A few rough and smooth microsomes (
AND
Subcellular
MOLECULAR
Localization HANS
Department
of Steroid GLAIJMANN
of Pathology II, Karolinska Receiued
May
27,
PATHOLOGY
( 1977)
Hormone
Metabolism
AND JAN-AKE
GUSTAFSSON
Huddinge Institutet,
17,1976,
221-234
Hospital and Department Stockholm, Sweden
and in reoised
form
December
in Rat Liver
of Chemistry,
15, 1976
The metabolism of 4-[4-%]androstene-3,17-dione was studied in smoothand rough-surfaced microsomes, plasma membranes, mitochondria, Golgi apparatus, and lysosomes isolated from rat liver. 5a-Reductase, 17P-hydroxysteroid reductase, 16~, 6/3-, and ‘la-hydroxylase activities were measurable in all isolated subcellular fractions except in lysosomes which did not metabolize 4-androstene-3,17-dione. Steroid metabolism patterns in smooth and rough microsomes were very similar. The extent of microsomal contamination in the plasma membrane, Golgi apparatus, and mitochondrial and lysosomal fractions was measured both by ultrastructural (morphometric) analysis and by distribution analysis of “marker enzymes” and was less than 15, 3, 16, and 30/o, respectively. Whereas it could not be excluded that the metabolism of steroids in the Golgi apparatus enriched fraction occurred due to microsomal contamination, the data indicate that the steroid metabolism in isolated plasma membranes and mitochondria occurred due to the presence of steroid reducing and hydroxylating enzyme systems in these cell organelles. Indirectly, such a similarity in enzymatic makeup between different cell organelles would tend to support “the membrane flow hypothesis.”
INTRODUCTION The endoplasmic reticulum (ER) is the main site for metabolism of drugs, steroid hormones, fatty acids, and polycyclic hydrocarbons (Mannering, 1972). Generally, low-molecular weight compounds are hydroxylated by an NADPHlinked electron transport chain with cytochrome P-450 as the terminal oxidase (Gillette et al., 1968). Wh ereas much interest has been focused on the distribution of hydroxylating activities in various microsomal subfractions, especially rough and smooth microsomes (Gram et al., 1968; Glaumann, 1970), relatively little interest has been devoted to the possible participation of other cell organelles such as the plasma membranes, Golgi apparatus, and lysosomes in the metabolism of low-molecular weight compounds. This is especially true for steroid hormones, which are considered to be the natural substrates for the microsomal NADPH-dependent hydroxylating chain (cf. Hamberg et al., 1974). The introduction of the membrane flow hypothesis has changed the previous static view on cell organelles as isolated entities of the cell to a more dynamic notion (Franke et al., 1971). The membrane flow hypothesis implies that the biogenesis of a specific membrane is accomplished by a migration of a membrane piece or membrane component from one cell organelle to another. The close morphological interrelation between the ER and the Golgi apparatus has long
Copyright All rights
@ 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0014-4800
222
GLAUMANN
AND
GUSTAFSSON
been known. Based on kinetic studies on the synthesis and degradation of ER membranes, Golgi apparatus membranes, and plasma membranes, Franke et al. (1971) postulated a flow of membranes from the ER to the Golgi apparatus and to the plasma membrane. If this is true, one could expect that these different types of cellular membranes might show similarities in their enzyme contents and function. Since steroid hormones are hydroxylated and reduced by several liver enzyme systems, we have investigated the distribution of these enzyme activities in some detail among various isolated subcellular fractions. A prerequisite for any conclusion regarding the participation of various cell organelles in the metabolism of steroids is that the purity and the degree of contamination of the isolated fraction be known. Therefore, a thorough morphological and biochemical characterization was performed to evaluate the actual composition of each fraction. MATERIALS
AND METHODS
Animals Male Sprague-Dawley rats weighing 150-180 g were used. Before sacrif?ce, the animals were starved for 15 hr. Chemicals 4-[4-‘C]Androstene-3,17-dione (specific activity, 60 mCi/mmoIe) was purchased from the Radiochemical Centre (Amersham, England) and purified by thin-layer chromatography. Unlabeled 4-androstene-3,17-dione was kindly given by Dr. J. Babcock (Upjohn Co., Kalamazoo, Michigan). Other chemicals were of reagent grade and obtained from Sigma Chemical Co. (St. Louis, Missouri). Isolation of Cell Organelks For preparation of total microsomes, 20% (w/v) liver homogenate in 0.29 M sucrose was centrifuged at 10,OOOgfor 20 min to remove cell debris, nuclei, mitochondria, and lysosomes. The supernatant was sedimented at 100,OOOgfor 60 min to obtain a microsomal fraction, which was suspended in 0.29 M sucrose. For isolation of rough and smooth microsomes, the method of Dallner (1963) and Glaumaml and Dallner (1968) was used with some modification. CsCl was added to the 10,OOOgsupernatant to a final concentration of 15 mM; 20 ml was layered over 10 ml of 1.30 M sucrose containing 13 mM CsCl and centrifuged at 27,000 rpm (131,000g) for 2 hr in a Christ Omega SW-27 rotor. The entire fluffy layer at the gradient boundary was collected and sedimented at 100,OOOg for 60 min to obtain a pellet of smooth microsomes. Distilled water was added to bring the sucrose concentration to 0.29 M, and the pellet containing the rough microsomal fraction was resuspended. The microsomes were then washed in 0.29 M sucrose. The use of an SW rotor, instead of an angle-head rotor, for the separation of rough and smooth microsomes gave purer fractions due to less pronounced sidewall impaction (Glaumann et al., 1975a). For isolation of a mitochondrial fraction, the livers were transferred to icecold 0.29 M sucrose, minced, and homogenized with four up-and-down strokes, using a Teflon-glass homogenizer (chamber clearance 0.15 to 0.23 mm) set at 300 rpm.
STEROID
METABOLISM
IN
CELL
ORGANELLES
223
A 10% homogenate in 0.29 M sucrose was spun at 600g for 10 min in an MSE-25 centrifuge. The resulting supernatant was centrifuged at 6000g for 10 min, and the pellet was gently suspended with a pipette. The 6000g run was then repeated twice as a washing procedure. For isolation of the Golgi apparatus, homogenization of livers from ethanoltreated rats was performed in 0.5 M sucrose-5 mM MgC12 at low speed (about with three complete strokes. The fractionation 200 rpm) (25% h omogenate) procedure was performed according to Ehrenreich et al. ( 1973). Plasma membranes were separated by the method of Coleman et al. (1967). A liver lysosomal fraction was isolated according to Glaumann et al. ( 1975b ) after repetitive injections of an iron-sorbitol-citric acid complex. Preparation
for Electron
Aliquots of the cacodylate buffer into l-mm pieces, 2 hr in 1% 0~0~. electron microscopy Biochemical
Microscopy
suspended fractions were fixed in 2% glutaraldehyde-0.1 M and centrifuged at 20,OOOg for 30 min. The pellets were cut washed in cacodylate buffer, and additionally fixed for 1 to Dehydration, embedding, cutting, and staining procedures for have been previously described (Glaumann et al., 1975a).
Assays
Protein was determined according to Lowry et al. ( 1951) with serum albumin as standard. The following enzymes were measured for estimation of contamination: cytochrome P-450 and cytochrome b, (Omura and Sato, 1964)) NADPHand NADH-cytochrome c reductase ( Dallner, 1963)) glucose-6-phosphatase ( G6Pase ), S-nucleotidase ( AMPase ), UDP-galactosyl transferase ( cf. Glaumann et al., 1975a), and cathepsin D (Bowers et al., 1967). Steroid Incubation Cell organelle suspensions corresponding to 2-10 mg of protein were incubated in 2.0 ml of Bucher medium (Bergstrom and Gloor, 1955) with 20, 50, 100, 200, and 400 pg of 4-[4-14C]androstene-3,17-dione (2 x lo6 dpm per incubation) at 37°C for 10 min in the presence of an NADPH-regenerating system 10.03 pmole of MnC&, 3.0 +oles of NADP, 125 pmoles of isocitrate, and 0.4 unit of isocitrate dehydrogenase type IV (Sigma Chemical Co.)]. The reactions were started by adding the substrate in 50 ,J of acetone and were stopped by adding 20 v01 of chloroform-methanol, 2:l (v/v). A 0.2-vol of a solution of sodium chloride (0.9%, w/v) was added. The chloroform phase was collected and reduced to dryness under vacuum. The residue was dissolved in a small volume of chloroform-methanol (2:1, v/v) and applied to a precoated silica gel plate (250~, Merck, Darmstadt, Germany). The thin-layer plates were developed once in the solvent system chloroform-ethyl acetate, 4: 1 (v/v). After autoradiography for 7 days, radioactive zones could be localized and scraped off separately. Aliquots of the methanol extracts of the silica gel zones were assayed for radioactivity in a Packard liquid scintillation spectrometer, Model 3003, using Instagel as the scintillator liquid. Several samples from corresponding thin-layer chromatographic zones from different incubations were pooled to give sufficient amounts of material to per-
224
CLAUMANN
AND
GUSTAFSSON
FIG. 1. Electron micrograph of the isolated mitochondrial fraction. The pellet consists almost exclusively of mitochondrial profiles in condensed conformation. X 10,450. FK. 2. Appearance of isolated lysosomes. The pellet consists of lysosomes filled with ferritin granules. Note the absence of mitochondrial profiles. X17,100. Inset shows a lysosome with intact bordering membrane. X57,000. The cellular origin of the lysosomes has been discussed recently (Glaumann et al., 197513).
STEROID
METABOLISM
IN
CELL
ORGANELLES
225
This fractio of isolated rough surfaced microsomes. FIG. 3. Appearance or somewhat elongated profiles with several ribosomes attached to the outer surface. X36,10( FIG. 4. Appearance of isolated smooth-surfaced microsomes. This fraction contains on! smooth-surfaced vesicles and no contaminating rough-surfaced microsomes or plasma men brane sheaths. X38,000.
:rmit gas chromatographic-mass spectrometric analysis. The analyses were pe ra formed on an LKB 2091 instrument using a lo/O SE-30 column. Mass spect md were recorded on magnetic tape using the incremental mode of operation 2
226
GLAUMANN
AND
GUSTAFSSON
FIG. 5. Electron micrograph of Golgi apparatus enriched fraction. The fractionation procedure was performed after ethanol administration according to Ehrenreich et al. ( 1973). This fraction corresponds to the total Golgi fraction (GFI + GF, + GK). The fraction contains numerous Golgi vacuoles and cisternae filled with VLDL clusters. Small Golgi vesicular profiles containing single particles are also present. These vesicular profiles most likely rep-
STEROID
METABOLISM
IN
CELL
ORGANELLES
227
were then treated in a PDP ll/lO computer (Digital Equipment AB, Solna, Sweden). A compound was considered identified if it had the same mass spectrum and gas-liquid chromatographic behavior as the reference compound. RESULTS Ultrustruc-tural
Characterization
AND
DISCUSSION
of the Isolated Fractions
Mitochondrial fraction. Electron micrographs of the mitochondrial preparations revealed that the fractions consisted almost exclusively of mitochondria with intact outer membranes (Fig. 1). A few rough and smooth microsomes (
