studies on the 14α-hydroxylation of progesterone in mucor piriformis

J. Steroid Biochem. Molec. Biol. Vol. 45, No. 6, pp. 563-569, 1993 0960-0760/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

STUDIES ON THE 14~-HYDROXYLATION OF PROGESTERONE IN MUCOR PIRIFORMIS

K. M. MADYASTHA* and THERESA JOSEPH

Department of Organic Chemistry, Bio-organic Section, Indian Institute of Science, Bangalore 560 012, India

(Received 3 December 1992; accepted 11 January 1993)

Sununary---Cell-free extracts with high 14~-hydroxylase activity were prepared from induced vegetative cell cultures of Mucor piriformis by grinding in potassium phosphate buffer (0.05 M, pH 8.0) containing glucose (0.25 M), KCI (1 mM), glutathione (I.0 mM) and glycerol (10%). Although the ideal pH for preparing the cell-free extract from vegetative cells was 8.0, the pH optimum of the hydroxylase was found to be 7.6. Microsomes (2.0 mg) prepared from the crude cell-free extract hydroxylated progesterone to 14~-hydroxyprogesterone in ~ 60% yields in 30 min in the presence of NADPH and 02. Microsomes prepared from the uninduced cells did not contain any 14~-hydroxylase activity. The hydroxylase activity was inhibited to a significant extent by CO and p-chloromercuribenzoate whereas moderate inhibition was noticed in the presence of SKF-525A, metyrapone and N-methylmaleimide indicating the possible involvement of the eytochrome P-450 system in the reaction. The membrane bound hydroxylase was solubilized using Triton X-100 and the solubilized fraction contained nearly 35% of the original hydroxylase activity.

INTRODUCTION

Mucor piriformis isolated in our laboratory transformed progesterone predominantly into 14~-hydroxyprogesterone [1]. Although organ- isms belonging to the order Mucorales are known for their unique ability to hydroxylate various C~9 and C2~ steroids at Cl4 [2-6], very little is known regarding the enzyme system involved in this hydroxylation reaction. In fact our knowledge regarding the steroid hydroxylating enzymes from various filamentous fungi is still limited. This could mostly be due to the failure encountered by investigators in their efforts to prepare functionally active cell-free extracts from fungal systems. How- ever, a few reports have appeared in the literature on the hydroxylation of steroids by cell free extracts prepared from various fungi [7-15].

Zuidweg et al. [7] reported the preparation of cell free ll/3-hydroxylase from C. lunata capable of hydroxylating 17~, 21- dihydroxyprogesterone mainly at the 11/3- and 14~-positions. The 25,000g supernate of the cell-free extract contained both 11/3- and 14~- hydroxylase activities. Transformation of 19- nortestosterone into 10/3-, 11 fl-, and

*To whom correspondence should be addressed.

14ct-monohydroxylated derivatives have also been demonstrated using cell-free extracts from C. lunata [8]. These studies suggested that a single hydroxylase system may be reponsible for the hydroxylation of 19-norsteroids at different positions [8]. However, in all these cases, the sub-cellular localization as well as the nature of the hydroxylase system have not been established.

So far two fungal cell-free steroid hydroxylation systems have been studied: the l l~-hydroxylase of Aspergillus and Rhizopus species [9-14] and 7~-hydroxylase of P. blakesleeanus[15]. Both these hydroxylases have been shown to be inducible and are medi- ated by a cytochrome P-450 system.

We initiated this work with a view to establish the intracellular site and the nature of the 140t-hydroxylase in Mucor piriformis. Earlier we had developed a procedure to prepare active cell-free extract from steroid induced vegetative cell cultures of Aspergillus ochraceus with high l l~-hydroxylase activity[12,13]. This procedure was partially modified to prepare active cell-free extract from induced cells of Mucor piriformis.

We report 14~-hydroxylation of progesterone by cell-free extracts of Mucor piriformis. Con- ditions for the isolation of active cell-free extract from the vegetative mycelial cells were opti-

563

564 K.M. M~a)~hsrrtA and THERESA JOSEPH

mized and the sub-cellular localization of the hydroxylase and its properties were studied.

EXPERIMENTAL

Materials

All steroids and biochemicals used in the present investigation were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). The authentic sample of 14~-hydroxyprogesterone was a generous gift from the Medical Research Council, London. Randomly labelled [3H]progesterone was obtained from Bhabha Atomic Research Centre (Bombay, India) SKF- 525 A was obtained from Smith, Kline and French Labs (Philadelphia, PA, U.S.A.). All other chemicals used were of chemically pure grade.

Microorganism

The organism used in the present study was isolated from soil. Based on various morpho- logical, cultural and biochemical characteristics, the organism was identified as Mucor piriformis. It was maintained either on potato dextrose agar (PDA) slants or modified Czapek-Dox [16] agar slants, stored at 4°C and sub-cultured periodically.

Shake culture growth of the organism

Flasks (500 ml) containing 100ml of sterile modified Czapek Dox medium [16] were inocu- lated with 1 ml of a spore suspension from a 5-day-old culture of Mucor piriformis and incubated at 29-30°C on a rotary shaker (220 rpm) for 36 h. After this growth period 100 mg of progesterone in 1.0 ml of acetone was added to each flask and the incubation was allowed to continue for a period of 12 h (induction period) before harvesting the mycelia. [In some experiments 10mg of progesterone (in 0.1ml of acetone) was added to each flask to induce the hydroxylase system.]

Preparation of the cell-free extract

All operations described were carried out between 0-4°C unless otherwise specified. After the induction period, the mycelia were filtered through cheese cloth, washed thoroughly with 0.5% NaCI solution and finally washed with distilled water [9]. The cells were then suspended in 0.05M phosphate buffer (pH8.0) and filtered. Potassium phosphate buffer (0.05 M, pH8.0) containing glucose (0.25M), KCI

(lmM), glutathione (1.0mM) and glycerol (10%, v/v) was used as the grinding buffer (buffer A). The mycelia (5 g wet wt) were mixed with pre-cooled acid washed glass powder (12.5 g) and 5.0 ml of buffer A. The mixture was ground well using a pre-cooled mortar and pestle for 5 min. To the finely ground mycelial suspension, buffer A (20 ml) was added, mixed well and filtered through cheese cloth. The crude cell-free preparation was centrifuged at 3000g for 20 min and the supernatant was designated as crude cell-free extract.

Preparation of microsomal suspension

The crude cell-free extract was centrifuged at 9600g for 20min. The post-mitochondrial supernatant was further centrifuged at 105,000g for 90 min. The pellet, designated as the microsomal fraction, was washed and resuspended in potassium phosphate buffer (0.025 M, pH 7.6) containing glutathione (0.5 mM), MgC12 (1 mM) and glycerol (20%, v/v) (buffer B) at a concentration of about 10 mg protein ml-1.

While establishing the ideal conditions required to prepare active cell-free extract from the mycelial mat, grinding was performed using different buffer compositions and pH.

Preparation of microsomes completely free from progesterone (inducer)

In experiments where 100 mg of progesterone (in 1.0 ml of acetone) was added to each flask for inducing the hydroxylase system, the microsomes prepared from the induced mycelia as described before, were suspended in distilled water and lyophilized. The lyophilized microsomes (50 mg) were suspended in chilled acetone (-30°C, 10 ml) and stirred for 5 min. The suspension was filtered at -10°C and the microsomal residue obtained was homogenized in buffer B (10 ml) and centrifuged at 3000g for 20 min. The supernatant containing microsomal protein is completely free from progesterone.

Solubilization of the microsomal hydroxylase system

Microsomal protein (30 mg) was suspended in buffer B (6 ml) and to this 10% Triton X-100 solution was added dropwise with stirring at 0°C keeping the detergent to protein ratio as 0.5: 1.0. The mixture was stirred for an ad- ditional period of 45 min and then centrifuged at 105,000g for 90min. The supernatant was assayed for its protein content and 14~-hydroxylase activity. The detergent present in the

14~-Hydroxylation of steroids 565

solubilized material was removed by passing through Biobead SM-2.

Assay and incubation condition

The 14~-hydroxylase assay mixture contained potassium phosphate (0.025 M, pH 7.6), [all]progesterone (0.032 #tool, 100,000 cpm), glutathione (0.5 mM), MgC12 (1 raM), glucose- 6-phosphate (2.5 gmol), NADPH (0.25 gmol), glucose-6-phosphate dehydrogenase (0.4 U), glycerol (20%, v/v) and microsomal protein (2-3 mg) in a total volume of 1.5 ml. The reaction was initiated by the addition of NADPH- generating system and the mixture was incubated aerobically for 30 min at 30°C on a rotary shaker. At the end of the incubation period, the reaction was terminated by the addition of 1 ml of chloroform. The enzymatic product formed was extracted with 2ml of chloroform (3 times). The organic layer was dried over Na2SO4 and evaporated to dryness. The residue was dissolved in a known volume of chloroform and an aliquot was subjected to thin layer chromatography (TLC; silica gel) using benzene-isopropanol (90:10) as the solvent system. Authentic 14~-hydroxyprogesterone was used as a marker. The spots were visualized by exposing the plate to I2 vapours. The area corresponding to progesterone, 14~-hydroxyprogesterone and the remaining portion of the TLC plate were scraped into vials containing 5 ml of scintillation cocktail and the tritium content determined.

Effect of inhibitors on the hydroxylation was determined as follows. Each inhibitor was pre- incubated with the microsomes at 30°C for 5 min, after which the hydroxylase assay was carried out as described above. Metyrapone and SKF-525 A were added as a solution in aceteone. Other inhibitors were added in a phosphate buffer (0.025 M, pH 7.6). The concentration of each inhibitor used is as men- tioned in Table 5. Carbon monoxide (CO) inhibition experiments were carried out by bub- bling CO into the assay mixture for 1-2 rain before the addition of substrate and NADPH- generating system. In control assays, N 2 was bubbled for 1-2 min.

Substrate specificity of the hydroxylase was carried out using acetone treated microsomes. The assays were conducted as described previously using 5 mg microsomal protein and various steroids (50 #g in 5 #1 acetone). The assay mixture was incubated at 30°C for 1 h and the hydroxylated product formed was identified by

comparing the high pressure liquid chromatography (HPLC) profile (retention time) with that of the authentic compounds.

Protein was estimated by the method of Lowry et al. [17]. The NADPH-cytochrome c reductase activity was measured at 550 nm as reported previously [18].

HPLC analyses were carried out on a Waters Associate instrument with a fixed wavelength (254 nm) u.v. detector. The analyses were performed on a micro porasil normal phase column using a chloroform-methanol (95:5) solvent system. Solvents were pumped isocratically at a flow rate of 0.8 ml/min. The levels of substrate and the 14~t-hydroxylated product formed in the assay mixture were determined on the basis of the area under the respective peaks and compared with a standard graph obtained by injecting a known amount of 14~t-hydroxylated products under identical conditions.

RESULTS

In the present studies it was noticed that grinding carried out in potassium phosphate buffer (0.1 M, pH 8.0) resulted in a cell-free extract with higher hydroxylase activity (~ 50%) than the extract prepared in Tris-HCl medium. In fact phosphate buffer and glycerol (10%) appear to be critical for isolating cell-free extracts with high 14~-hydroxylase activity. A marginal increase (~ 10%) in the hydroxylase activity was noticed when glucose (0.25 M) and KC1 (1 raM) were included in the grinding medium whereas the presence of EDTA (10mM) was not essential for isolating active cell-free extracts. Glutathione (1 mM) appeared to be marginally better than dithiothreitol (DTT) (1 raM) whereas mercaptoethanol (1 mM) was a poor substitute. Cell-free extracts prepared at pH8.0 had maximum 14~t-hydroxylase activity whereas extracts prepared at pH 7.0 showed only 30% of the optimal activity. A gradual decrease in the activity was noticed when the pH of the grinding medium was maintained above 8.0.

Formation of 14~-hydroxyprogesterone (14~-OHP) in the assay mixture was established by HPLC analysis as described under Exper- imental. The peak corresponding to 14~t-OHP was enhanced (Rt 4 min 3 s) when mixed with authentic compound. The enzymatic product formed (14~t-OHP) was further identified by performing a large scale incubation using [3H]progesterone. The identity of the enzymatic

566 K . M . MADYASTHA and THERESA JOSEPH

Table 1. 14~-Hydroxylase activity in different sub-cellular fractions

Sub-cellular fractions

14~-Hydroxylase activity

14a-OHP Specific formed activity Total

(%) (nmol. min - i m g - 1) activity

3000g supernatant 28.0 0.10 21.00 9600g pellet 9.0 0.03 0.10 105,000g pellet

(microsomes) 48.0 0.17 10.2 I 105,000g supernantant 0 - - - - 105,000g pellet from

uninduced mycelia 0 - - - -

These experiments were carried out with mycelia where 10 mg of progesterone was added during the induction period. Grinding of the mycelia (5 g), preparation of different subcellular fractions, 14~-hydroxylase assay were carried out as described in Experimental. Organism grown in the absence of progesterone is referred to as uninduced mycelia.

product as [3H]14~-OHP was established by crystallization to constant specific activity after addition of carrier 14~-OHP (5 mg). In a repre- sentative experiment, the specific activity of [3H]14~-OHP in the third, fourth and fifth re- crystallization were 14128, 14303 and 14387 dpm/mg, respectively. The mass spectrum of enzymatically formed 14~-OHP was identical to that of authentic 14~-OHP.

Intracellular site

Differential centrifugation studies (Table 1) carried out with the active cell-free extract indicated that most of the hydroxylase activity was associated with the membrane fraction sedi- menting at 105,000g (microsomes). Very little activity was associated with the 9600g pellet whereas the 105,000g supernatant (cytosol) and microsomes prepared from uninduced cells did not contain any hydroxylase activity (Table 1).

pH optimum

The pH of the incubation medium markedly affected the formation of 14~-OHP. Although the optimum pH for the grinding medium was 8.0, the hydroxylase has a pH optimum of 7.6 (Table 2). A marked decline in the activity was noticed above pH 7.8. The hydroxylase activity was significantly reduced at pH values below

Table 2. Effect of varying pH on the microsomal 14ct-hydroxylase activity

Specific activity pH of the incubation medium (nmol .min- i mg 1)

6.6 0.13 7.0 0.38 7.3 0.43 7.6 0.45 7.8 0.36 8.0 0.29

The hydroxylase assays were conducted in potassium phosphate buffer (0.025 M) of indicated pH. The details of the assays are as described in Experimental.

7.0, being 30% of the optimum activity at pH 6.6 (Table 2).

Requirements for 14~-hydroxylase

Both NADPH and 02 were necessary for the hydroxylase activity (Table 3). NADH was a very poor substitute for NADPH. Addition of NADH along with NADPH did not produce any synergistic effect. A considerable increase in the activity was noticed when NADPH was replaced by a NADPH generating system. Hy- droxylase activity could not be detected when the assay was conducted in the absence of Oz, indicating the requirement for molecular 02.

Time course and temperature effect

The formation of 14a-OHP by microsomes at 30°C at different intervals of time was studied (Table 4). The formation of 14~-OHP was linear during the first 30 min and during this period 50-60% of the substrate was converted into 14~-OHP. Maximal conversion was seen between 45 and 60 min and the level almost re- mained constant up to 180 min. During this period no other hydroxymetabolites were formed. The hydroxylation reaction was significantly affected by temperature. The optimum temperature for hydroxylation was found to be 30°C. However, about 60% of the activity was lost when the assays were carried out at either 15 or 40°C.

Induction of the 14~-hydroxylase system

Microsomes prepared from uninduced cells did not contain any hydroxylase activity (Table 1). However, progesterone added during the induction period was shown to induce the hydroxylase system. Maximum microsomal hydroxylase activity was noticed when 100 mg of progesterone was added during the induction

14a -Hydroxy la t ion o f steroids

Table 3. Requirements of microsomal 14a-hydroxylation of progesterone

140t-OHP formed Deletions Additions (nmol. rain = t. mg- i)

None (complete) - - 0.46 NADPH-generating system - - - - NADPH-generating system NADPH (1 #tool) 0.24 NADPH-generating system NADH (1/~mol) 0.02 NADPH-generating system NADPH (1/~mol) +

NADH (1/Jmol) 0.27 Complete minus 02 - - - -

Assays were conducted in triplicate with 2.0 mg of microsomal protein and [~H]progesterone (0.032/~mol, 100,000cpm) as described in Experimental. Septum capped test tubes were used to conduct assays anaerobically. The tubes were deaerated with N 2 and the residual 02 was removed by pre-incu- bating the assay mixture with glucose (0.5 mM) and glucose oxidase (200 U) for 10min and after that the reaction was initiated by the addition of the NADPH-generating system.

567

period. This activity was almost 3-4 times that seen when 10mg progesterone was used for induction. It was difficult to remove all the adhering steroids from the mycelial mat. When 10 mg progesterone was used for inducing the hydroxylase system, the amount of progesterone present in the microsomes was almost negligible. However, when 100 mg progesterone was used for inducing the hydroxylase system, even after washing the mycelial mat following the reported procedure [9], the microsomes prepared still contained 5 #g progesterone per mg of microsomal protein. In all the assays carried out with such microsomes, the microsomal progesterone was estimated by solvent extraction and HPLC analysis. The amount of progesterone present per mg of protein was taken into account while calculating the specific activity of 14a-hydroxylase. The adhering progesterone can be completely eliminated from microsomes by acetone treatment (see Experimental). Acetone washed microsomes retained nearly 75% of the original hydroxylase activity.

Table 4. Time course of hydroxylation of progesterone by microsomes from Mucor

piriformis at 30°C

14a-OHP formed Time (nmol)

10 12.58 20 29.45 30 36.90 45 41.90 60 43.00 120 44.40 180 46.40

Assays were carried out for the indicated time period using 2.0 mg of microsomal protein and 0.064/~mol of progesterone. The details of the assay conditions are as described in Exper- imental.

Phenobarbital failed to induce the hydroxylase system.

Effect of inhibitors Using acetone washed microsomes, the 14a-

hydroxylase was inhibited to a significant extent in the presence of CO and p-chloromercuribcnzoate (p-CMB) (Table 5). Moderate inhibition was also noticed with SKF-525 A, metyrapone and N-methylmaleimide whereas KCN (1 mM) and NaN3 (1 mM) produced < 5% inhibition.

Substrate specificity of the hydroxylase system

Studies carried out with progesterone- induced microsomes clearly demonstrated that the hydroxylase system catalyzes the 14~-hydroxylation of testosterone, androstenedione, 16-dehydroprogesterone, 1,2-dehydrotestosterone besides progesterone (Table 6). However, 14~-hydroxylation was not observed when epitestosterone, 17a-hydroxyprogesterone, 17~t-ethynyl-19-nortestosterone and 7- keto-dehydroepiandrosterone were used as substrates.

Table 5. Effect of different inhibitors on the 14~z-hydroxylase system

Inhibitor used Inhibition (%)

None (control) a 0 SKF-525 A (0.5 mM) 24 Metyrapone (0.5 mM) 31 CO (bubbled for 2 min) 86 KCN (I mM) 4 p-CMB 0.1 mM 40

0.5 mM 98 N-Methylmaleimide (0.5 mM) 31 NaN~ (1 mM) 5

"One hundred percent activity (0% inhibition) represents 0.380 nmol 14~-OHP formed rain - ~ nag protein- ~. Inhibitors were pre-incn- bated with microsomes (2.0 mg) for 5 rain at 30°C before the substrate and cofactors were added. These experiments were carried out using acetone washed microsomes (for explanation, see Discussion). Microsomes were isolated from induced mycelia (100 mg of progesterone added during induction period).

568 K . M . MADYASTHA and THER~A JOSEPH

Table 6. Substrate specificity of the microsomal 14a-hydroxylase system

14~-Hydroxylated product Substrate tested formed (% conversion)

Progesterone 69 Testosterone 65 Androstenedione 30 16-Dehydroprogesterone 81 1,2-Dehydrotestosterone 45 17~-Hydroxyprogesterone Epitestosterone 17~-Ethynyl-19-nortestosterone 7-Ketodehydroepiandrosterone

Progesterone induced microsomes were prepared from mycelium grown in the presence of 100mg of progesterone during the induction period. These experiments were carried out with acetone washed microsomes. Assays were conducted with 5 mg of microsomal protein and 50 ;tg of substrate. The hydroxylated products formed were identified by comparing their HPLC profiles (retention time) with that of the authentic compounds. The % conversion of each substrate was determined as described under Experimental.

Stability

The microsomal 14a-hydroxylase retained more than 90% of its activity for 6 weeks when stored at -20°C.

Solubilization of microsomal 14~-hydroxylase

Treatment of the microsomal fraction with Triton X-100 resulted in solubilization of nearly 35% of the 14~-hydroxylase activity. The solubilized fraction contained considerable amounts of NADPH-cytochrome c reductase activity (37.6 nmol/mg/min). However, we were not able to get a proper reduced CO difference spectrum with the solubilized microsomes.

D I S C U S S I O N

Earlier we had developed a procedure for the preparation of active cell-free extract with high 1 l~-hydroxylase activity from induced vegetative cell cultures of A. oehraceus. According to that procedure, to obtain optimal hydroxylase activity, EDTA (10raM), glycerol (10%) and DTT (5 mM) were required in the grinding medium[12, 13]. Grinding of the mycelium should be carried out at pH 8.3 in Tris-HC1 medium to get optimal hydroxylase activity in the cell-free extract[12, 13]. However, in the present studies, it was observed that grinding at pH 8.0 in phosphate buffer was necessary to realize optimal 14~-hydroxylase activity. Although glycerol (10%) appears to be a critical component of the grinding medium, omission of EDTA in the grinding medium did not affect the hydroxylase activity in the cell-free extract. Pre- viously phosphate buffer has been used for the preparation of microbial cell-free steroid hydroxylases [7, 9, 11, 19].

Mucor piriformis isolated in our laboratory was shown to transform progesterone predominantly into 14~-OHP [1]. In the present study we have clearly established for the first time that the 14~-hydroxylase activity is solely localized in the microsomal fraction. Microsomes (2 mg) isolated following our method converted nearly 50% of the added progesterone into 14~-OHP in 30 min at 30°C. During this period no other hydroxylated metabolites were formed. Micro- somes isolated from cells grown in the absence of progesterone was devoid of hydroxylase activity indicating the inducible nature of the 14~-hydroxylase system. Steroid hydroxylases studied so far from various fungi have been shown to be inducible[7-15]. Progesterone (100 rag) added to the growth medium induced the microsomal hydroxylase activity to a significant extent. Phenobarbital failed to induce the microsmal hydroxylase activity. Earlier we have demonstrated that A. ochraceus grown in the presence of phenobarbital failed to induce the microsomal l l~-hydroxylase activity[13]. Steroids such as testosterone, androstenedione, 16-dehydroprogesterone were not as efficient as progesterone in inducing 14~-hydroxylase activity (data not shown). Sur- prisingly, 16-dehydroprogesterone was found to be a very good substrate for the 14~-hydroxylase system (Table 6), but not a good inducer of the hydroxylase when it was added during the induction period.

Microsomal 14~-hydroxylase activity was inhibited to a significant extent by CO and p-CMB whereas moderate inhibition was seen with SKF-525 A, metyrapone and N- methylmaleimide but not by cyanide and azide (Table 5). These inhibition studies indicate the possible involvement of cytochrome P-450 in the hydroxylation reaction. Inhibition studies were carried out using acetone-washed microsomes. It is interesting to note that when un- washed microsomes were used, CO failed to inhibit the 14~-hydroxylation reaction. This could be possible due to the presence of large amounts of inducer (progesterone) still adhere- ing to the microsomes which might have completely blocked the CO-binding sites. However, washing the microsomes with acetone completely removes the progesterone adhering to microsomes. Although the hydroxylation reaction was inhibited by some of the typical inhibi- tots of cytochrome P-450, the microsomal fraction as well as the detergent solubilized microsomes failed to give a proper cytochrome

14a-Hydroxylation of steroids 569

P-450 spectrum, but the detergent-solubilized microsomes contained considerable NADPH- cytochrome c reductase activity (37.6 nmol/min/ mg). Many of the steroid hydroxylases isolated from various filamentous fungi have been shown to contain the cytochrome P-450 system [11-15].

Microsomes isolated from progesterone- induced mycelia were found to carry out the 14~-hydroxylation of various C19 and C2~ steroids (Table 6). However, 17~-hydroxyprogesterone, epitestosterone, and 17~-ethynyl-19- nortestosterone were not accepted as substrates (Table 6) supporting the earlier report that a bulky substitution at C-17~ would prevent hydroxylation at C-14~ by Mucorales [3]. How- ever, steroids with a 17fl-hydroxy group as in testosterone were readily hydroxylated [20].

Acknowledgement--Financial assistance for T.J. from C.S.I.R., New Delhi, and I.I.Sc., Bangalore, is gratefully acknowleged.

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studies on the 14α-hydroxylation of progesterone in mucor piriformis

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