Assay, properties, and regulation of rat ovarian δ-aminolevulinic acid synthetase activity

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 216, No. 2, July, pp. 593-599, 1982

Assay, Properties, ROBERT

and Regulation of Rat Ovarian Acid Synthetase Activity’

J. COOK, JAMES

Department of Phmmm~,

TSE, AND WALTER

&Aminolevulinic N. PIPER2

College of Medicine,University of Nebraska MedicalCater, Omaha, Nebraska 68105

Received November 17, 1981, and in revised form February 9, 1982

&Aminolevulinic acid synthetase (EC 2.3.1.37) has been detected in homogenates of rat ovaries. Optimal substrate and coenzyme concentrations, and parameters for assay of ovarian b-aminolevulinic acid synthetase have been determined. Subcellular fractionation studies have shown that enzyme activity is predominantly localized in the mitochondrial fraction. Fasting, which is known to increase enzyme activity in the adrenal and to have no effect on activity in the testis, had no effect on enzyme activity in the ovary. Administration of the hepatic inducer allylisopropylacetamide or the hormone progesterone failed to alter activity of the ovarian enzyme. The activity of the enzyme was significantly increased during the diestrus-1 phase of the estrus cycle, during pregnancy, and by human chorionic gonadotropin at 24 and 48 h, suggesting that ovarian d-aminolevulinic acid synthetase and the synthesis of heme may be under hormonal control.

The hemoprotein cytochrome P-450 serves as a terminal oxidase for various hydroxylation reactions that participate in steroidogenesis (l), and has been shown to be present in the ovaries (2-5). Rat ovarian microsomal cytochrome P-450 has been shown to increase during pregnancy, at a time when the rate of steroid biosynthesis is very high (5), and the concentration of rat ovarian mitochondrial cytochrome P-450 has been shown to increase lo-fold in immature superovulated rats (6). Another hemoprotein, peroxidase, has been shown to be present in rat ovary and to be induced by luteinizing hormone (7). These results suggest that the synthesis of ovarian heme and cytochrome P-450 may be under hormonal control. Investigations with rat testis have shown that testicular cytochrome P-450 is regulated by gonadotropins (g-10). Also, rat

testicular &aminolevulinic acid synthetase (ALAS)3 activity is increased by administration of human chorionic gonadotropin (HCG), suggesting that the regulation of testicular heme and cytochrome P-450 synthesis may be closely associated with the control of androgen biosynthesis (11). Since the ovaries are also under the control of the pituitary gonadotropins and are a site of cytochrome P-450-mediated steroidogenesis, an investigation was initiated into the regulation of heme synthesis in the rat ovary. The initial aims of this study were to develop an assay for ovarian ALAS, to define optimal conditions for assay of enzymatic activity, to study regulation of the enzyme by pharmacologic agents, and to determine whether activity changes during the estrus cycle and pregnancy. ’ Abbreviations used: AIA, allylisopropylacetamide; ALA, I-aminolevulinic acid, ALAS, 6-aminolevulinic acid synthetase; HCG, human chorionic gonadotropin; Hepes, N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid, LH, luteinizing hormone.

’ This research was supported by Grant ES-62423 from the National Institutes of Health. ’ To whom reprint requests should be sent. 593

0003-9861/82/080593-07$02.00/O Copyright All rights

Q 1982 by Academic Prans. Inc. of reproduction in any form reserved.

594

COOK, TSE, AND PIPER EXPERIMENTAL

PROCEDURES

Materials Succinyl-CoA synthetase (EC 6.2.1.4; porcine heart), pyridoxal-5’-phosphate, glycine, human chorionic gonadotropin (HCG), progesterone, hemin, albumin (human fraction V), and coenzyme-A were obtained from the Sigma Chemical Company, St. Louis, Missouri. Succinic acid, GTP (trilithium salt), and 6aminolevulinic acid (ALA) were purchased from Calbiochem, La Jolla, California. AG 5OW-X8-H+ cation-exchange resin (200-400 mesh) and AG l-X8acetate anion-exchange resin were obtained from Bio-Rad Laboratories, Richmond, California. 6-[414C]-Aminolevulinic acid (51.5 mCi/mmol) and [2,3“C]-succinic acid (68.1 mCi/mmol) were obtained from New England Nuclear, Boston, Massachusetts. Allylisopropylacetamide (AIA) was a generous gift from Hoffman-La Roche, Nutley, New Jersey. All other chemicals were of the highest purity available from commercial sources.

Methods Treatment

of Animals

Female Sprague-Dawley rats (175-250 g) were obtained from Sasco Inc., Omaha, Nebraska. Animals were permitted food and water ad l&&urn, except during fasting experiments in which food was withheld for 24 h prior to sacrifice. The rate were housed in pairs in hanging cages in a room maintained at 23°C and illuminated 12 h per day (lights on from 06:00-18~00 h) with midnight as the midpoint of the dark cycle. HCG was dissolved in 0.9% NaCl and administered subcutaneously (100 units; 2.5 ml/kg; 12-h intervals). AIA was dissolved in acetone:corn oil 1:3 (v/v), and administered subcutaneously (300 mg/ kg; 4.5 ml/kg). Progesterone was suspended in corn oil and administered intraperitoneally (20 mg/kg; 2.5 ml/kg; 24-h intervals). Hemin was dissolved in 0.1 N NaOH-1% albumin, the pH adjusted to 7.4 with HCl, and the solution administered intraperitoneally (16 mg/kg; 10 ml/kg; 12-h intervals). Control animals received equal volumes of vehicle, which did not alter the activity of ALAS. Only animals showing two consecutive a-day estrus cycles were used for experiments involving measurement of ALAS activity during the estrus cycle. The estrus cycle was followed by taking vaginal smears daily between O&O0 and 10~00 h. Timed pregnant Sprague-Dawley rate were obtained from Holtzman, Madison, Wisconsin, or animals showing two consecutive 4-day estrus cycles were mated with mature males.

Preparation

of Tisues

Animals were killed by cerebral concussion followed by decapitation. The ovaries were rapidly excised and washed in 0.9% NaCl-10 mM Hepes (pH

7.6) at 4°C. Ovaries were weighed and minced, and homogenates (lo%, w/v) were prepared in 0.9% NaCl-10 mM Hepe.s (pH 7.6) at 4°C using a motordriven glass Potter-Elvehjem homogenizer with a Teflon pestle (0.15 mm clearance). Homogenates were centrifuged at 850 g (r,,) for 20 set at 4°C which precipitated fibrous and connective tissue and facilitated subsequent assay for ALAS and subcellular fractionation of the homogenates.

Subcelhlar

Fractionation

of Rat Ovaries

(a) Fractionation. Ovaries were fractionated by a modified method of Cook and Blair (12) to give the following fractions: Mitochondrial fraction 1, mitochondrial fraction 2, microsomes, and cytosol. Homogenates were prepared in 4 vol of 0.25 Y sucrose10 m!d Hepes (pH 7.6) and centrifuged at 85Og (T-) for 20 s to remove fibrous and connective tissue. The resultant supernatant was designated as the homogenate. All centrifugation steps were performed at 4°C in either a Beckman Model J-21C centrifuge (speeds up to 16,000 rpm) or a Beckman Model L8-70 ultracentrifuge for speeds above 16,000 rpm. All g forces are expressed for r,,. The homogenate was centrifuged at 2500 rpm in a Beckman JA-17 rotor (860~) for 10 min. The supernatant S1 was recovered by aspiration and used for the isolation of mitochondrial fraction 2, microsomes, and cytosol. The pellet R1 was used for the isolation of mitochondrial fraction 1 and nuclei. R1 was resuspended in 4 ml of 0.25 M sucrose-10 rnM Hepes (pH 7.6), and 2-ml aliquots were then layered over 2.4 M sucrose-10 mM Hepes (pH 7.6). The tubes were centrifuged in a Type 40 rotor at 39,500 rpm (140,OOOg). Mitochondria formed a “button” at the 2.4 ~-0.25 M sucrose interface. The 0.25 M sucrose was removed by aspiration and the mitochondrial “button” freed from the tube by a spatula and poured with a minimum volume of 2.4 M sucrose into a homogenizer tube. Mitochondria were resuspended in 10 ml of 0.25 M sucrose-10 mM Hepes (pH 7.6) and centrifuged at 16,000 rpm for 10 min (35,OOOg). The resulting pellet was resuspended in 3 ml of 0.25M sucrose-10 mM Hepes (pH 7.6) and is referred to as mitochondrial fraction 1. No white nuclear pellet was detected after the high-speed spin of Ftl (heavy mitochondrial fraction). Fraction S1 was centrifuged at 16,000 rpm for 10 min (35,OOOg). The supernatant S, was removed by aspiration and used to isolate microsomes and cytosol. The pellet, h was resuspended in 10 ml of 0.25 M sucrose-10 rnM Hepes (pH 7.6) and centrifuged for 10 min at 16,000 rpm (35,OOOg).The supernatant was discarded and the pellet was resuspended in 3 ml of 0.25 sucrose-10 mM Hepes (pH 7.6) and is referred to as mitochondrial fraction 2 (light mitochondrial fraction). Fraction S, was centrifuged at 39,500 rpm in a Beckman Type 40 rotor for 60 min (14O,OOOg).The

OVARIAN

I?-AMINOLEVULINIC

supernatant was removed by aspiration and is referred to as cytosol. The pellet was washed with a small volume of 0.25 M sucrose-10 mM Hepes (pH 7.6) and then resuspended in 3 ml of the same buffer. This is designated as the microsomal fraction. (b/ subcellular marker enzylLe assays. Succinatecytochrome c reductase was used as the mitochondrial marker enzyme and was assayed by the method of Tisdale (13). The microsomal marker was rotenone-insensitive NADH-cytochrome c reductase and was assayed as for succinate-cytochrome c reductase using 0.1 M Tris-HCl buffer (pH 8.5), NADH (0.2%) as the substrate with rotenone (4 M in 4 pl ethanol) as the inhibitor. Lactate dehydrogenase was used as the cytosol marker enzyme and was measured by the method of Stolsenbach (14). The mitochondrial matrix marker, malate dehydrogenase, was used to check for mitochondrial leakage and was assayed by the method of Beaufay et al. (15).

d-Amirwlevulinic

Acid Synthetase Assay

The activity of ovarian ALAS was measured by the method of Tofilon and Piper (11). Maximum ALAS activity was obtained using an assay mixture containing (final concentrations): 80 mM Hepes buffer (pH 7.6), 100 rnM glycine, 5 rnM NasEDTA, 0.1 mPd pyridoxal-5’-phosphate, 5 rnM sodium malate, 10 rnrd sodium malonate, 5 mM sodium arsenite, 0.5 mM sodium succinate (containing 1.5-3.0 &i [2,3-“C$uccinic acid), and a succinyl-CoA generating system consisting of 0.02 unite of succinyl-CoA synthetase, 0.5 mrd GTP, 20 mrd MgCl,, 1.0 mM coenxyme A, and 2 rnre glutathione to a final volume of 2 ml. The reaction mixture was incubated for 20 min at 37°C prior to addition of ovarian homogenate in order to prime the system with succinyl-CoA. Reactions were initiated by the addition of enzyme (homogenate or subcellular fraction) to the reaction mixture (total volume 2 ml). Assays were conducted at 37’C for 10 min. Product formation was found to be linear for 15 min with up to 5.5 mg protein per 2 ml of reaction mixture. Reactions were terminated by the addition of 0.5 ml 25% trichloroacetic acid and protein precipitates were removed by centrifugation. The amount of [“CfALA produced was determined by the method of Tofilon and Piper (11) which involved purification of the product from the reaction mixture by ion-exchange chromatography using AG 5OW-X8-Na+ followed by formation of ALA-pyrrole and subsequent purification with ion-exchange chromatography using AG 1-X8-acetate. The ALA-pyrrole was eluted with 4 ml of methanol-l N acetic acid, 21 (v/v). The eluate was added to 12 ml of scintillation fluid composed of 4 g 2,5-diphenyloxazole and 50 mg pbi$2(5-phenyloxaxolyl)lbenzene per liter of toluene-Triton X-190,2:1 (v/v), and counted for radioactivity in a Beckman LS8000 liquid scintillation counter (83% efficiency).

ACID SYNTHETASE

ACTIVITY

Recovery and Ident$cation

595 of [‘X’JALA

ALA recovery was determined by addition of 2 mmol of ALA (0.026 pCi of [4-“C]ALA) to reaction mixtures devoid of [2,3-“Clsuccinate and was found to be 91%. “C-Labeled material was identified as [“C]ALA-pyrrole by thin-layer chromatography using authentic ALA-pyrrole as a standard. The methanol-l N acetic acid eluate from the AG l-X&acetate ion-exchange column, containing [“CJALA, was evaporatedto dryness at 60°C under nitrogen. The residue was dissolved in 0.1 ml of methanol-glacial acetic acid, 21 (v/v), and aliquots were applied to silica gel G plates (250 pm; Analtech) and chromatographed in n-butanol-water-glacial acetic acid, 45~1 (v/v/v; upper phase). The plates were dried and developed with modified Ehrlich’s reagent (16) to detect ALA pyrrole. Lanes of 1 cm above the origin were scraped into 0.5 ml Hz0 and counted for “C radioactivity using 10 ml ACS (Amersham) in a Beckman Model LS8000 liquid scintillation counter. Using this method, 93% of the applied radioactivity was recovered from the area of the pink spot corresponding to authentic ALA pyrrole (Rf 0.76).

Protein

Assays

Protein was determined by the method of Bradford (17) using bovine serum albumin (Fraction V, Sigma) as the standard.

Statistics The significance of differences between means was determined by subjecting the data to analysis by Student’s t test. RESULTS

Determination of Optimal Conditions Assay of Ovarian ALA Synthetase Activity

for

The concentration of succinate (2 mnn) required for optimal ALAS activity in ovarian homogenate was similar to that reported for the rat heart (18) and rat brain (19). Rat adrenal (20) and rat testicular (11) homogenate ALAS systems have been reported to require lower concentrations of succinate for maximal activity. The optimal glycine concentration required for the ovarian enzyme was similar to that previously reported for rat liver (21), adrenal (20), heart (18), and testis (11). The apparent K, and Vvalues for glycine were 22 mM and 413 nmol/g protein/h, respectively. It is not possible to determine the

596

COOK,

TABLE EFFECF

TSE,

AND

I

OF Succm-CoA

GENERATING

SYSTEM

ON RAT OVARIAN ALAS ACMVITYD

Reaction mixture

ALAS activity b (nmol ALA/g protein/h)

Without generating system With generating system

34f 6 214 + 14”

‘The components of the succinyl-CoA generating system were: 0.5 m&f GTP, 20 m?d MgCls, 1.0 mM CoA, and 0.02 units succinyl-CoA synthetase. ‘Values are the means + SEM of three determinations. ’ Significant difference from reaction mixture without succinyl-CoA generating system (P < 0.01).

PIPER

in rat liver (21) by inhibition of ALA dehydratase activity and inhibition of aminoacetone formation, had no apparent effect on the activity of ovarian ALAS when present at a concentration of 5 mM. However, EDTA (5 mM) was retained in the reaction mixture because of its reported inhibitory effects on ALA dehydratase activity. A succinyl-CoA generating system was essential for maximum ALAS activity in ovarian homogenates (Table I). Testicular homogenates also require a succinyl-CoA generating system (ll), whereas adrenal (20), heart (18), liver (21), and brain (19) homogenates do not. Subcellular

Km or V herein for succinyl

CoA, since this substrate was formed via a generating system employing succinate and succinic thiokinase. Pyridoxal-5’-phosphate (0.2 mM), a coenzyme for ALAS (22, 23), was required for maximal activity. In the absence of added pyridoxal-5’-phosphate, ovarian ALAS activity was decreased by 35%. This optimal concentration (0.2 mM) is identical to the reported requirements for rat adrenal (20) and rat brain (19) homogenates. EDTA, which increases ALA formation

The subcellular distribution of ovarian ALAS activity is shown in Table II. The results with marker enzymes show that the fractionation procedure was successful in producing relatively pure subcellular fractions. The majority of the ALAS activity was associated with the mitochondria (70.7%) as had been shown for the brain (19), liver (24), adrenal (20), and testis (11). The majority of activity is localized in the light mitochondrial fraction. The presence of ALAS activity in the cy-

TABLE SUECELLULAR

DISTRIBUTION

Distribution of Rat Ovarian ALAS Activity

OF OVARIAN

II ALAS

AND MARKER

ENZYMES”

Activity (% of the total activity recovered in the fractions) Activity recovered from homogenate (%I Protein ALAS Malate dehydrogenase Succinate-cytochrome c reductase Rotenone-insensitive NADH-cytochrome c reductase Lactate dehydrogenase

Mitochondrial Fraction 1 (heavy mitochondria)

Mitochondrial Fraction 2 (light mitochondria)

Microsomal fraction

Cytosol

100 91.0 87.4

10.5 18.1 24.8

11.6 52.6 66.4

12.4 3.9 1.1

65.5 25.4 8.7

61.1

16.8

83.2

N.D.b

N.D.”

88.0 56.8

14.9 10.2

10.6 4.7

74.5 12.1

N.D.b 73.0

a Subcellular fractions were obtained, and enzyme assays performed as described under Methods. ’ Not detected.

OVARIAN

6-AMINOLEVULINIC

ACID

SYNTHETASE

TABLE IV

tosol may rdlect thepresence ofacytosolic form that has been characterized by Hayashi et al. (25) in rat liver. It appears that leakage from mitochondria during fractionation was minimal as indicated by the low level of malate dehydrogenase (8.7%) detected in the cytosol. The Effect of Fasting on Rat Ovarian ALAS Activity Fasting for 24 h prior to enzyme assay had no effect on ovarian ALAS activity (Table III). This is consistent with results for other rat organs such as brain (19), testis (ll), and liver (20). Rat heart (18) ALAS activity is known to decrease and rat adrenal (20) ALAS activity to increase following 24-h fasting periods. Ovarian

ALAS Activity during Estrus Cycle

the

The effect of various stages of the estrus cycle on ALAS activity is shown in Table IV. There was a significant (P < 0.05) increase in ALAS activity between estrus and diestrus-1 which declined through diestrus-2 and proestrus. ALAS Activity in Rat Ovary during Pregnancy ALAS activity was significantly increased during pregnancy when compared to the activity at estrus (Day 0) as shown in Fig. 1. ALAS increased in activity up to Day 6, followed by a slight decrease at Day 9 after which time there was a steady increase reaching a peak at Day 15. The enzyme activity decreased at Day 18 and TABLE EFFECT

Treatment Fed Fasted

597

ACTIVITY

RAT OVARIAN ALAS ACTIVITY THE ESTRUS CYCLE“

Stage of cycle

DURING

ALAS activity b (nmol ALA/g protein/h)

Estrus Diestrus-1 Diestrus-2 Proestrus

233+28 344 + 46’ 313 + 29 287 + 28

a Assay of ALAS activity and monitoring of the estrus cycle were conducted as described under Methods. All assays were performed on ovarian homogenates obtained between 1190 and 1200 h. bValues are the means + SEM of 10 determinations. c Significant difference from estrus (P < 0.05).

then remained constant prior to parturition.

to Day 20 just

The Effect of Progesterone Treatment on Rat Ovarian ALAS Activity The increase of ALAS activity during diestrus reported herein coincides with an elevation of blood progesterone by the corpus luteum (26), which suggests that this hormone might regulate the activity of ovarian ALAS. However, administration of progesterone did not alter the activity of ovarian ALAS (Table V). The Effect of AIA Treatment on Rat Ovarian ALAS Activity AIA is known to be a potent inducer of rat hepatic ALAS activity. However, adwar

III

OF FASTING ON RAT OVARIAN ALAS ACTIVIW

ALAS activity b (nmol ALA/g protein/h) !&X+24 200 + 18

a Food was withheld from rats for 24 h. b Values are the means + SEM of five determina-

FIG. 1. Rat ovarian ALAS activity during pregnancy. Assay of ALAS activity is described under Methods. All assays were performed on ovarian homogenates obtained between 1090 and 1290 h. The results are expressed as means + SEM and the num-

COOK, TSE, AND PIPER TABLE EFFECT

OF

V

DISCUSSION

AIA, PROGESTERONE, OVARIAN

Treatment

ALAS

AND HCG ACTN~

ON RAT

ALAS activity b (nmol ALA/g protein/h)

Control AIA Progesterone

314 + 19 (28) 342 + 48 (5) 361 zk 26 (6)

HCG 24h 48h 48 h + hemin

426 f 54 (6) 489 f 34 (11) 416 + 35 (5)

‘AIA was administered subcutaneously (300 mg/ kg) 16 h prior to sacrifice. Progesterone was administered intraperitoneally (20 mg/kg) at 0 and 24 h, and animals were sacrificed at 48 h. HCG was administered subcutaneously (100 unite) at 12-h intervals, and animals were sacrificed at 24 or 48 h. Hemin was administered intraperitoneally (16 mg/kg) at 12-h intervals. bValues are the means f SEM, with numbers of replicate animals treated indicated in the parentheses. ’ Significant difference from control (P < 0.01).

ministration of AIA failed to alter the activity of ovarian ALAS (Table V). AIA appears to be a specific inducer for hepatic tissue, since the activity of ALAS is not altered by this agent in the adrenal (20), brain (19), heart (18), or testis (11). The Effect of HCG Treatment Ovarian ALAS Activity

on Rat

The levels of the hemoprotein, cytochrome P-450, have been reported to be increased in the rat ovarian mitochondrial fraction following administration of HCG (6). Thus, it is possible that the synthesis of ovarian heme might be regulated through gonadotropin control of ALAS activity. Thus, rats were pretreated with HCG and activity of ovarian ALAS was measured. HCG pretreatment caused an increased ALAS activity of 36% at 24 h and 56% at 48 h, respectively, as shown in Table V. Administration of hemin did not produce any significant impairment of the increase of ovarian ALAS caused by HCG.

The hemoprotein, cytochrome P-450, is known to participate in the biosynthesis of various steroid hormones. Thus, knowledge of the regulation of the synthesis of heme is important for our understanding of how various therapeutic drugs, hormones, or toxicants regulate steroidogenesis in various endocrine organs, such as the ovary. Optimal assay conditions for measurement of homogenate ALAS activity and regulation of the enzyme appear to differ for tissues, even for various endocrine organs. For example, a succinyl-CoA regenerating system is required for optimal activity in the ovary as reported herein, and for the testis (11) but not for the adrenal (20), brain (19), heart (18), or liver (21). Also, pyridoxal-5’-phosphate is required for maximal ovarian, adrenal (20), and brain (19) activity but not for the testicular enzyme (11). Furthermore, fasting has no effect on the activity of the brain (19) or testicular (11) enzyme, but increases adrenal (20) and decreases cardiac (18) ALAS activity. As reported herein, fasting does not alter ovarian ALAS activity. Conditions modifying the cellular activity of ALAS probably depend upon the functional role of the organ. The synthesis of heme in organs such as the ovary or testis are not likely to undergo regulatory modifications for coping with stress like the heart or adrenal. The increase in ALAS activity during the estrus cycle was not coincident with the elevations of blood LH or l?‘@estradiol which occur during proestrus, nor with the increased progesterone release from the rat ovarian interstitial cells during late proestrus and early estrus. The significant increase in ALAS activity occurred 2 days after proestrus during diestrus-1. This correlates with the findings of Klinken and Stevenson (4) and Naumoff and Stevenson (6) who showed that there are significant increases in rat ovarian mitochondrial cytochrome P-450 and cholesterol side-chain cleavage activity and plasma progesterone levels during the transition from the immature follicle to active steroid-producing

OVARIAN

&AMINOLEVULINIC

ACID

corpora lutea. During diestrus-1 there is an increase in progesterone secretion by the corpora lutea. Unless pregnancy or pseudopregnancy ensues there is a decline in progesterone secretion during the early hours of diestrus-2 which is correlated with the regression of the corpora lutea (26). During pregnancy or pseudopregnancy the corpora lutea continues to secrete progesterone. During pregnancy ALAS activity increased and reached a peak at Day 15. This increase correlates well with the increase in microsomal cytochrome P-450 and bs in the rat ovary during pregnancy (5). The increase in ovarian ALAS activity during diestrus-1 may be in response to a need for increased synthesis of heme for cytochrome P-450-mediated synthesis of steroids by the corpora lutea in preparation for pregnancy. Administration of HCG to rats has been reported to elevate the levels of ovarian mitochondrial cytochrome P-450 (6). As described herein, the activity of ovarian ALAS also is increased after treatment of rats with HCG. The increased activity of ovarian ALAS at 24 h precedes the reported elevation of mitochondrial cytochrome P-450 (6), which suggests that ovarian heme synthesis may be controlled by pituitary gonadotropins. The administration of hemin did not appreciably alter the increase of ALAS activity caused by HCG. Hemin is known to prevent increases of hepatic ALAS caused by various drugs. The differential effect of hemin on hepatic and ovarian ALAS activity could be explained merely by an inability of heme to reach the cells of the ovary. Sedman and Tephly (27) have recently shown that rat cardiac ALAS activity is not modified by administration of hemin. Determination of optimal assay parameters for measurement of ovarian ALAS activity is an initial step in a series of studies which are being conducted in order to learn how heme synthesis and steroidogenesis are regulated by hormones, drugs, and toxicants.

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