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Stimulation of steroid C-21 hydroxylase activity in adrenal microsomes by serum protein
SHORT COMMUNICATIONS
I8 9
The author wishes to thank Professor G. E. BLACKMANand Dr. B. C. LOUGHMAN for valuable advice and encouragement during the course of this work.
Department of Agriculture, University of Oxford, Oxford (GreatBritain)
JOHN M. PALMER*
, j . M. PALMSR AND B. C. LOUG~MAN, New Phytologist, in the press. t W. G. ROSEN, Proc. So¢. ExptL Biol. Med., 85 (x954) 385. 8 W. G. RosEN, Plant Physiol., 32 (z957) Suppl. viii. 4 j . T. O. KIRK, Biochim. Biophys. Acta, 56 (1962) I39. 5 R. E. CLXCXXND D. P. HACKETT, Proc. Natl. Acad. Sci. U.S., 5 ° (i963) 243. 6 T. ERDOS AND A. UU-MAN, Nature, 183 (I959) 618. 7 M. DE DEXEN-GRENSO~, Biochim. Biophys. Acta, r 7 (1955) I57.
Received January
27th, i964
* Present Address: Department of Biochemistry, UCR, Riverside, Calif. (U.S.A.).
Biochim. Biophys. Acta, 90 (1964) z86-189
sc 23025
Stimulation of steroid C-21 hydroxylase activity in adrenal microsome$ by serum protein The ability of rat serum to stimulate corticosteroidogenesis in surviving rat-adrenal tissue and in adrenal homogenates has been described1-s. The active material was present in serum of normal, adrenalectomized, and hypophysectomized rats, was firmly associated with the serum proteins, and appeared to be distinct from other known stimulators of adrenocortical activity. Addition of small amounts of rat serum to adrenal homogenates (o.r ml/xoo mg adrenal tissue) markedly enhanced the conversion of progesterone to xI-deoxycorticosterone and corticosterone and suppressed the formation of IIfl-hydroxyprogesterone3. These results suggested that the serum factor stimulated microsomal C-2x hydroxylation* of progesterone at the expense of mitochondrial C-Ixfl hydroxyiition s of this substrate. The present study was undertaken to determine whether the active material was capable of stimulating C-2x hydroxylation of progesterone in isolated microsomes from rat adrenals. Adrenal glands were obtained under light Nembutal (sodium pentobarbital) anesthesia from young male rats (12o-15o g) of an inbred Sprague-Dawley strain. The glands were dissected free of fat and connective tissue, cut into eighths, and subjected to a preliminary incubation for x h in a medium composed of io 9 parts o.I54 M NaC! and 21 parts o.r54 M NaHCOs s. Preformed steroid hormones were removed by this procedure. The adrenals were then blotted, weighed, and homogenized in a loose-fitting glass homogenizer for x rain in 2 ml of o.25 M sucrose at o °. The homogenate was diluted to 8 ml with sucrose and the microsomal fraction was prepared essentially as described by RYAN AND ENGEL4. Nuclei and cell debris were removed by centrifugation at o ° and 700 × g for io rain. The crude mitochondrial fraction was sedimented by centrifugation at xoooo × g for 20 min, resuspended in 6 ml of 0.25 M sucrose, and recentrifuged at xoooo × g for IO rain. The two Biochim. Biophys. Acta, 90 (1964) 189--192
190
SHORT COMMUNICATIONS
supernatants were combined and centrifuged for 9 ° min at 105000 X g. The sedimented microsomal pellet was washed with o.25 M sucrose and then resuspended in this solution at a concentration equivalent to 125 mg wet weight of tissue per ml for the incubation experiments. The methods employed for the separation, identification and measurement of the steroidal incubation products will be described in detail s. Steroids were chromatographed on paper in the E 4 system of EBERLEIN AND BONGIOVANNI~. Radioactivity was measured in a windowless gas-flow strip scanner. The chromatogram was then sprayed with tetrazolium blue. The Ii-deoxycorticosterone spot was eluted and the steroid present estimated from the intensity of the colored complex. Recovery of corticosteroids carried through these procedures averaged 80 %. Serum preparations were added to the incubation medium in amounts which substituted for an equivalent volume of o.154 M KC1. Rat serum was prepared from blood fleshly drawn from the dorsal aorta. Separation into "protein" and "nonprotein" fractions was accomplished by dialysis with shaking for 24 h at 4 ° against 3 changes of distilled water, each 6o ml in volume. The non-dialysable fraction was assayed for adrenal-stimulating activity by addition to the incubation medium without further treatment; the dialysates were lyophilized and dissolved in distilled water before addition.
300C
20OC c
8
ooc
IOOC
0
10
Distance (cm)
20
Fig. z. Influence of whole rat serum on the conversion of [4-14C]progesterone to radioactive corticosteroids in rat adrenal microsomes. The incubation mixture contained 0. 4 m l of microsoma] suspension (equivalent to 48 mg adrenal tissue), 4o/~moles NaHCO s, 2.z/~moles N A D P H , and I1.6. Io -=/*moles [4-z6C]progesterone (o.o5/~C ) in o.oz25 m195 ~o ethmzol, made up to afina] volume of 2 m l w i t h o.z54 IV[ KCl. I n c u b a t i o n w a s carried o u t for I h a t 37.5 ° u n d e r a n a t m o s p h e r e of 95 ~o O s - 5 ~o COs. Steroidal p r o d u c t s in t h e i n c u b a t i o n m e d i a were e x t r a c t e d , c o m b i n e d f r o m 3 identical i n c u b a t i o n vessels, a n d c h r o m a t o g r a p h e d for 5 - 6 h; t h e c h r o m a t o g r a p h i c origin is i n d i c a t e d as o on t h e abscissa. T h e c o u n t s / m i n are corrected for b a c k g r o u n d b u t n o t for abs o r p t i o n o n t h e p a p e r (about 5 o % ) . Q - - O , no s e r u m a d d e d ; O - - O , o.i m l fresh r a t s e r u m p e r beaker. A b b r e v i a t i o n s u s e d are B (eortieosterone), D O C (i z-deoxycorticosterone), P (progesterone).
Biochim. Biophys. Acta, 90 (I964) 189-192
SHORT COMMUNICATIONS
I9I
Enzymic and cofactor requirements of the steroid c-2i hydroxylating system of beef adrenal microsomes were described by RYAN AND ENGEL4. Maximal activity was obtained under aerobic conditions upon addition of "substrate" quantities of NADPH to the rnicrosomal fraction sedimenting between 15ooo × g and 1o5ooo × g. No other extramicrosomal cofactor appeared to be necessary (see also ref. 8). In the present investigations, incubation of rat adrenal microsomes with [4-x4C]progesterone and NADPH, in the absence of any other additive, resulted in the appearance of radioactivity in the ii-deoxycorticosterone area of the chromatogram (Fig. i, solid circles). The identity of this material was established by its chromatographic mobility, ultraviolet and sulfuric acid spectra, staining properties with tetrazolinm blue, and formation of an acetylated product with properties identical to those of II-deoxycorticosterone-zI-acetate s. The relative absence of radioactive material in the corticosterone region of the chromatogram indicated that the microsomal C-2I hydroxylating system was essentially free of C-lip hydroxylase (EC 1.99.1.7) activity 5. TABLE I EFFECT OF PROTEIN AND NON-PROTEIN FRACTIONS OF RAT SERUM ON CONVERSION OF [4-14C]PROGESTERONE TO CORTICOSTEROIDS BY EAT ADRENAL MICROSOMES C o n d i t i o n s were s i m i l a r t o t h o s e o u t l i n e d in t h e l e g e n d t o Fig. I. E a c h a d d i t i v e w a s p r e s e n t i n a n a m o u n t e q u i v a l e n t to a p p r o x , o.i m l of serum. R e s u l t s a re r e p r e s e n t a t i v e of t h o s e o b t a i n e d in 6 s e p a r a t e e x p e r i m e n t s . Radioaai~y recovered*
Additive
None Fresh serum Lyophilized serum Non-dialysable fraction Dialysate
l:~ogesterona (%)
77.8 5 °.6 52.4 60. 5 91.8
z z-Deozycorticost~one z z-Deoxycoaiprodt~d eos~eTo~ Co~i¢ostm.on¢ (pg/.,roomg (%) (%) adrena)
16.9 41.5 39.0 33.0 5.8
3.8 5 .0 5.5 4.0 o.7
8 25 23 28 --
* P e r c e n t of t o t a l r a d i o a c t i v i t y on t h e c h r o m a t o g r a m ( a b o u t 13 ooo c o u n t s / m i n ) r e c o v e r e d u n d e r t h e r e s p e c t i v e peaks.
Addition of a small amount of fresh rat serum to the incubation medium (I : 20, v/v) resulted in marked stimulation of the conversion of progesterone to II-deoxycorticosterone (Fig. I, open circles). The active material in rat serum retained its full potency on lyophilization and appeared to be associated quantitatively with the serum protein fraction after dialysis (Table I). The dialysate m a y have inhibited progesterone utilization by rat adrenal microsomes, as it did in adrenal homogenates a. Only small amounts of radioactivity could be detected in corticosterone in any of the situations studied. The mechanism by which small quantities of protein or protein-bound material in rat serum stimulate adrenal corticosteroidogenesis in vitro is obscure. The observation that this substance is active in adrenal whom cell preparations1, ~, as well as in homogenates 8 and isolated microsomes, indicates that it may be capable of entering the cell and is not dependent upon cellular integrity for its action. COOPER et a2.8-10 recently presented evidence which suggests that other microsomal oxidases compete with steroid C-2I hydroxylase (EC 1.99.1.11 ) for reducing equivalents originating in Biochim. Biophys. Aaa, 90 (1964) 189- 92
i92
SHORT COMMUNICATIONS
NADPH. C-2I steroid hydroxylation was stimulated in adrenal microsomes* and homogenatesx° by catecholamines, in amounts far in excess of blood levels, and by certain inhibitors (e.g., cyanide) which appear to act by blocking competing oxidations. However, these materials did not concomitantly alter C-1iE hydroxylation in adrenal homogenate#°, whereas serum or serum protein completely obliterated the formation of ii~-hydroxyprogesterone under similar conditionss. These results suggest that the serum factor may function in the selective channeling of substrate and cofactor to microsomal sites, possibly via a mechanism which involves association of these reactants with the stimulator. The possibility exists that the serum stimulator of adrenal steroid C-2I hydroxylation may serve a significant physiological role in the maintenance of the basal rate of corticosteroidogenesis over which pituitary ACTH action is superimposed. This investigation was supported by a research grant (GM-o3869) from the National Institutes of Health, U.S. ]Public Health Service, and by a contract between the Office of Naval Research, Department of the Navy and the University of California (NR 11o-4o2).
Department of Biological Chemistry, School of Medicine and the Brain Research Institute University of California, Center for the Health Sciences, Los Angeles, Calif. (U.S.A.)
RHODA MAKOFF* SIDNEY ROBERTS
1 S. ROBERTS, Ciba Found. Colloq. on Endocrinol., II (1957) 167. R. MAKOFF AND S. ROBERTS, Federation Proc., 19 (196o) i6o. 8 t{. MAKOFF, S. ROBERTS AND D. D. FOWLER, in p r e p a r a t i o n . 4 K. J. RYAN AND L. L. ENGEL, J. Biol. Chem., 225 (1957) IO3. 5 M. L. SWEAr, J. Am. Chem. Sot., 73 (I95 I) 4056. s S. KORITZ AND F. G. PgRON, J. Biol. Chem., 234 (1959) 3122. W. EBERLEIN AND A. BON~IOVANNI, Arch. Biochem. Biophys., 59 (1955) 90. s D. Y. COOPER AnD O. ROSENTHAL, Arch. Biochem. Biophys., 96 (I962) 33I. 9 D. Y. COOPER, R. W. ESrABROOK AND O. ROSENTHAL, J. Biol. Chem., 238 (I963) I32O. 10 D. Y. COOPER AND O. ROSE~THAL, Arch. Biochem. Biophys., 96 (1962) 327.
Received March 7th, 1964 * P r e d o c t o r a l Trainee, M e n t a l H e a l t h T r a i n i n g P r o g r a m (5 T I MH-6415), U.S. Public H e a l t h Service. P r e s e n t a d d r e s s : D e p a r t m e n t of Biological C h e m i s t r y , H a r v a r d U n i v e r s i t y Medical School, B o s t o n , Mass. (U.S.A.).
Biochim. Biophys. Acta, 90 (I964) 189-129
I8 9
The author wishes to thank Professor G. E. BLACKMANand Dr. B. C. LOUGHMAN for valuable advice and encouragement during the course of this work.
Department of Agriculture, University of Oxford, Oxford (GreatBritain)
JOHN M. PALMER*
, j . M. PALMSR AND B. C. LOUG~MAN, New Phytologist, in the press. t W. G. ROSEN, Proc. So¢. ExptL Biol. Med., 85 (x954) 385. 8 W. G. RosEN, Plant Physiol., 32 (z957) Suppl. viii. 4 j . T. O. KIRK, Biochim. Biophys. Acta, 56 (1962) I39. 5 R. E. CLXCXXND D. P. HACKETT, Proc. Natl. Acad. Sci. U.S., 5 ° (i963) 243. 6 T. ERDOS AND A. UU-MAN, Nature, 183 (I959) 618. 7 M. DE DEXEN-GRENSO~, Biochim. Biophys. Acta, r 7 (1955) I57.
Received January
27th, i964
* Present Address: Department of Biochemistry, UCR, Riverside, Calif. (U.S.A.).
Biochim. Biophys. Acta, 90 (1964) z86-189
sc 23025
Stimulation of steroid C-21 hydroxylase activity in adrenal microsome$ by serum protein The ability of rat serum to stimulate corticosteroidogenesis in surviving rat-adrenal tissue and in adrenal homogenates has been described1-s. The active material was present in serum of normal, adrenalectomized, and hypophysectomized rats, was firmly associated with the serum proteins, and appeared to be distinct from other known stimulators of adrenocortical activity. Addition of small amounts of rat serum to adrenal homogenates (o.r ml/xoo mg adrenal tissue) markedly enhanced the conversion of progesterone to xI-deoxycorticosterone and corticosterone and suppressed the formation of IIfl-hydroxyprogesterone3. These results suggested that the serum factor stimulated microsomal C-2x hydroxylation* of progesterone at the expense of mitochondrial C-Ixfl hydroxyiition s of this substrate. The present study was undertaken to determine whether the active material was capable of stimulating C-2x hydroxylation of progesterone in isolated microsomes from rat adrenals. Adrenal glands were obtained under light Nembutal (sodium pentobarbital) anesthesia from young male rats (12o-15o g) of an inbred Sprague-Dawley strain. The glands were dissected free of fat and connective tissue, cut into eighths, and subjected to a preliminary incubation for x h in a medium composed of io 9 parts o.I54 M NaC! and 21 parts o.r54 M NaHCOs s. Preformed steroid hormones were removed by this procedure. The adrenals were then blotted, weighed, and homogenized in a loose-fitting glass homogenizer for x rain in 2 ml of o.25 M sucrose at o °. The homogenate was diluted to 8 ml with sucrose and the microsomal fraction was prepared essentially as described by RYAN AND ENGEL4. Nuclei and cell debris were removed by centrifugation at o ° and 700 × g for io rain. The crude mitochondrial fraction was sedimented by centrifugation at xoooo × g for 20 min, resuspended in 6 ml of 0.25 M sucrose, and recentrifuged at xoooo × g for IO rain. The two Biochim. Biophys. Acta, 90 (1964) 189--192
190
SHORT COMMUNICATIONS
supernatants were combined and centrifuged for 9 ° min at 105000 X g. The sedimented microsomal pellet was washed with o.25 M sucrose and then resuspended in this solution at a concentration equivalent to 125 mg wet weight of tissue per ml for the incubation experiments. The methods employed for the separation, identification and measurement of the steroidal incubation products will be described in detail s. Steroids were chromatographed on paper in the E 4 system of EBERLEIN AND BONGIOVANNI~. Radioactivity was measured in a windowless gas-flow strip scanner. The chromatogram was then sprayed with tetrazolium blue. The Ii-deoxycorticosterone spot was eluted and the steroid present estimated from the intensity of the colored complex. Recovery of corticosteroids carried through these procedures averaged 80 %. Serum preparations were added to the incubation medium in amounts which substituted for an equivalent volume of o.154 M KC1. Rat serum was prepared from blood fleshly drawn from the dorsal aorta. Separation into "protein" and "nonprotein" fractions was accomplished by dialysis with shaking for 24 h at 4 ° against 3 changes of distilled water, each 6o ml in volume. The non-dialysable fraction was assayed for adrenal-stimulating activity by addition to the incubation medium without further treatment; the dialysates were lyophilized and dissolved in distilled water before addition.
300C
20OC c
8
ooc
IOOC
0
10
Distance (cm)
20
Fig. z. Influence of whole rat serum on the conversion of [4-14C]progesterone to radioactive corticosteroids in rat adrenal microsomes. The incubation mixture contained 0. 4 m l of microsoma] suspension (equivalent to 48 mg adrenal tissue), 4o/~moles NaHCO s, 2.z/~moles N A D P H , and I1.6. Io -=/*moles [4-z6C]progesterone (o.o5/~C ) in o.oz25 m195 ~o ethmzol, made up to afina] volume of 2 m l w i t h o.z54 IV[ KCl. I n c u b a t i o n w a s carried o u t for I h a t 37.5 ° u n d e r a n a t m o s p h e r e of 95 ~o O s - 5 ~o COs. Steroidal p r o d u c t s in t h e i n c u b a t i o n m e d i a were e x t r a c t e d , c o m b i n e d f r o m 3 identical i n c u b a t i o n vessels, a n d c h r o m a t o g r a p h e d for 5 - 6 h; t h e c h r o m a t o g r a p h i c origin is i n d i c a t e d as o on t h e abscissa. T h e c o u n t s / m i n are corrected for b a c k g r o u n d b u t n o t for abs o r p t i o n o n t h e p a p e r (about 5 o % ) . Q - - O , no s e r u m a d d e d ; O - - O , o.i m l fresh r a t s e r u m p e r beaker. A b b r e v i a t i o n s u s e d are B (eortieosterone), D O C (i z-deoxycorticosterone), P (progesterone).
Biochim. Biophys. Acta, 90 (I964) 189-192
SHORT COMMUNICATIONS
I9I
Enzymic and cofactor requirements of the steroid c-2i hydroxylating system of beef adrenal microsomes were described by RYAN AND ENGEL4. Maximal activity was obtained under aerobic conditions upon addition of "substrate" quantities of NADPH to the rnicrosomal fraction sedimenting between 15ooo × g and 1o5ooo × g. No other extramicrosomal cofactor appeared to be necessary (see also ref. 8). In the present investigations, incubation of rat adrenal microsomes with [4-x4C]progesterone and NADPH, in the absence of any other additive, resulted in the appearance of radioactivity in the ii-deoxycorticosterone area of the chromatogram (Fig. i, solid circles). The identity of this material was established by its chromatographic mobility, ultraviolet and sulfuric acid spectra, staining properties with tetrazolinm blue, and formation of an acetylated product with properties identical to those of II-deoxycorticosterone-zI-acetate s. The relative absence of radioactive material in the corticosterone region of the chromatogram indicated that the microsomal C-2I hydroxylating system was essentially free of C-lip hydroxylase (EC 1.99.1.7) activity 5. TABLE I EFFECT OF PROTEIN AND NON-PROTEIN FRACTIONS OF RAT SERUM ON CONVERSION OF [4-14C]PROGESTERONE TO CORTICOSTEROIDS BY EAT ADRENAL MICROSOMES C o n d i t i o n s were s i m i l a r t o t h o s e o u t l i n e d in t h e l e g e n d t o Fig. I. E a c h a d d i t i v e w a s p r e s e n t i n a n a m o u n t e q u i v a l e n t to a p p r o x , o.i m l of serum. R e s u l t s a re r e p r e s e n t a t i v e of t h o s e o b t a i n e d in 6 s e p a r a t e e x p e r i m e n t s . Radioaai~y recovered*
Additive
None Fresh serum Lyophilized serum Non-dialysable fraction Dialysate
l:~ogesterona (%)
77.8 5 °.6 52.4 60. 5 91.8
z z-Deozycorticost~one z z-Deoxycoaiprodt~d eos~eTo~ Co~i¢ostm.on¢ (pg/.,roomg (%) (%) adrena)
16.9 41.5 39.0 33.0 5.8
3.8 5 .0 5.5 4.0 o.7
8 25 23 28 --
* P e r c e n t of t o t a l r a d i o a c t i v i t y on t h e c h r o m a t o g r a m ( a b o u t 13 ooo c o u n t s / m i n ) r e c o v e r e d u n d e r t h e r e s p e c t i v e peaks.
Addition of a small amount of fresh rat serum to the incubation medium (I : 20, v/v) resulted in marked stimulation of the conversion of progesterone to II-deoxycorticosterone (Fig. I, open circles). The active material in rat serum retained its full potency on lyophilization and appeared to be associated quantitatively with the serum protein fraction after dialysis (Table I). The dialysate m a y have inhibited progesterone utilization by rat adrenal microsomes, as it did in adrenal homogenates a. Only small amounts of radioactivity could be detected in corticosterone in any of the situations studied. The mechanism by which small quantities of protein or protein-bound material in rat serum stimulate adrenal corticosteroidogenesis in vitro is obscure. The observation that this substance is active in adrenal whom cell preparations1, ~, as well as in homogenates 8 and isolated microsomes, indicates that it may be capable of entering the cell and is not dependent upon cellular integrity for its action. COOPER et a2.8-10 recently presented evidence which suggests that other microsomal oxidases compete with steroid C-2I hydroxylase (EC 1.99.1.11 ) for reducing equivalents originating in Biochim. Biophys. Aaa, 90 (1964) 189- 92
i92
SHORT COMMUNICATIONS
NADPH. C-2I steroid hydroxylation was stimulated in adrenal microsomes* and homogenatesx° by catecholamines, in amounts far in excess of blood levels, and by certain inhibitors (e.g., cyanide) which appear to act by blocking competing oxidations. However, these materials did not concomitantly alter C-1iE hydroxylation in adrenal homogenate#°, whereas serum or serum protein completely obliterated the formation of ii~-hydroxyprogesterone under similar conditionss. These results suggest that the serum factor may function in the selective channeling of substrate and cofactor to microsomal sites, possibly via a mechanism which involves association of these reactants with the stimulator. The possibility exists that the serum stimulator of adrenal steroid C-2I hydroxylation may serve a significant physiological role in the maintenance of the basal rate of corticosteroidogenesis over which pituitary ACTH action is superimposed. This investigation was supported by a research grant (GM-o3869) from the National Institutes of Health, U.S. ]Public Health Service, and by a contract between the Office of Naval Research, Department of the Navy and the University of California (NR 11o-4o2).
Department of Biological Chemistry, School of Medicine and the Brain Research Institute University of California, Center for the Health Sciences, Los Angeles, Calif. (U.S.A.)
RHODA MAKOFF* SIDNEY ROBERTS
1 S. ROBERTS, Ciba Found. Colloq. on Endocrinol., II (1957) 167. R. MAKOFF AND S. ROBERTS, Federation Proc., 19 (196o) i6o. 8 t{. MAKOFF, S. ROBERTS AND D. D. FOWLER, in p r e p a r a t i o n . 4 K. J. RYAN AND L. L. ENGEL, J. Biol. Chem., 225 (1957) IO3. 5 M. L. SWEAr, J. Am. Chem. Sot., 73 (I95 I) 4056. s S. KORITZ AND F. G. PgRON, J. Biol. Chem., 234 (1959) 3122. W. EBERLEIN AND A. BON~IOVANNI, Arch. Biochem. Biophys., 59 (1955) 90. s D. Y. COOPER AnD O. ROSENTHAL, Arch. Biochem. Biophys., 96 (I962) 33I. 9 D. Y. COOPER, R. W. ESrABROOK AND O. ROSENTHAL, J. Biol. Chem., 238 (I963) I32O. 10 D. Y. COOPER AND O. ROSE~THAL, Arch. Biochem. Biophys., 96 (1962) 327.
Received March 7th, 1964 * P r e d o c t o r a l Trainee, M e n t a l H e a l t h T r a i n i n g P r o g r a m (5 T I MH-6415), U.S. Public H e a l t h Service. P r e s e n t a d d r e s s : D e p a r t m e n t of Biological C h e m i s t r y , H a r v a r d U n i v e r s i t y Medical School, B o s t o n , Mass. (U.S.A.).
Biochim. Biophys. Acta, 90 (I964) 189-129