Anjan K. Bhattacharyya

Photoaffinity labeling of rat liver microsomal steroid 5α-reductase by 2-azido-NADP

Abstract Preincubation of female rat liver microsomal preparations with [2′- 32 P]2N 3 -NADP + followed by photolysis with UV lighjt (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32 P into at least 3 major protein bands in the molecular weight range of 14–197 Kd. Labeling of a 26 Kd band, the apparent molecular weight of 5α-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP + (100μM) completely inhibited the incorporation of [2′- 32 P]2N 3 -NADP + and preserved the enzyme activity, whereas excess NAD + (100μM) failed to protect 5α-reductase (5αR) activity. Similar results were obtained with the detergent-solubilized form of 5αR. Polyethylene glycol (PEG) fractionation of detergent-solubilized with the detergent-solubilized preparations of 5αR showed that all the 5αR activity could be recovered in the 6.5%, pellet with a 3—4-fold increase in the specific activity. Photolysis of this fraction with [2′- 32 P]2N 3 -NADP + resulted in ∼ 2-fold increase in 32 P labeling of the 5αR band. Increasing photolysis time and concentration of the [2′- 32 P]2N 3 -NADP + indicated that the half-life for photoincorporation and the apparent K d were 1.0 min and 2 μM, respectively. Theser results suggest that 2N 3 -NADP + is an effective probe of the NADP(H) binding site of 5αR, and is a useful marker during purification of the enzyme.

Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5α-reductase (isozyme-1) I: Analysis of aromatic and hydroxylated amino acid residues

Abstract Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5α-reductase isozyme-1 (r5αR-1) is in a highly conserved region of the polypeptide sequence (residues 160–190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5αR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an ∼40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5αR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an ∼5-fold decrease in Km for NADPH and a significant increase (∼18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5αR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.

Photoaffinity labeling of rat steroid 5α-reductase (isozyme-1) by a benzophenone derivative of a 4-methyl-4-azasteroid

[1,2-3H]N-4(Benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha-androstane-17 beta-carboxamide ([3H]-4MABP) has been synthesized as a photoaffinity probe of the steroid-binding domain of rat steroid 5 alpha-reductase isozyme-1 (5 alpha R-1). Reversible binding of the probe to 5 alpha R-1 in microsomal preparations yielded a reversible dissociation constant (Kd) of -3 nM, whereas inhibition experiments indicated that the probe had a 50% inhibition concentration of 4.4 nM and was a competitive inhibitor of the enzyme (Ki approximately 3 nM) with respect to testosterone. SDS-PAGE analysis of microsomal, detergent-solubilized, and (6.5%) polyethylene glycol-precipitated fractions of 5 alpha R-I photolyzed with [3H]4MABP in the presence of NADPH showed that the radioactivity was incorporated into a single protein band with a mass of 26 kDa (apparent molecular weight of 5 alpha R-1). UV photolysis was accompanied by an irreversible loss in enzyme activity, consistent with its covalent modification. Increasing the time of UV irradiation and concentration of [3H]4MABP indicated that the half-life and apparent Kd for its photo insertion were approximately 3 min and 7.5 nM, respectively. Photolysis in the presence of a 20-fold excess of N,N-diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide or the 3-carboxysteroid SKF-105111 resulted in partial protection of 5 alpha R-1 from the probe, whereas minimal incorporation of radioactivity was observed in the absence of NADPH or in the presence of NADP+. The results indicate that [3H]4MABP is an effective probe of the steroid (D-ring) binding domain of 5 alpha R-1.

Inhibition of rat steroid 5α-reductase (isozyme 1) by suramin

Abstract In this study, we show the inhibition of rat steroid 5α-reductase (isozyme 1) by suramin. The enzyme activity decreased in a dose-dependent manner as suramin concentrations increased with the calculated drug dose required for 50% inhibition (at 5 μM testosterone and 200 μM NADPH) being 13 μM. Suramin showed non-competitive inhibition of 5α-reductase with respect to testosterone (KTI = 2.4 μM) and competitive inhibition with respect to NADPH (KNADPHi = 220 nM). Furthermore, suramin and NADP+, but not NAD+, protected 5α-reductase from labeling by 2-azido-NADP+, a photoactive probe which has recently been used to identify the NADPH binding domain of 5α-reductase. These results suggest that suramin inhibits rat steroid 5α-reductase (isozyme 1) at the level of NADPH binding to the enzyme.

Analysis of the steroid binding domain of rat steroid 5α-reductase (isozyme-1) the steroid D-ring binding domain of 5α-reductase

Abstract We have previously shown that the photoactive 4-azasteroid, [1,2 3 H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5α-androstan-17β-carboxamide is an effective probe of rat steroid 5α-reductase (isozyme-1) (5αR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5αR-1 activity were ultraviolet (UV)-photolyzed with [ 3 H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5αR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55–56 min. Rechromatography of this fraction using a modified gradient (elution 54–55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15–18 of the 5αR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an ∼12-fold increase in the K m for testosterone, whereas the K m for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.

Expression of rat steroid 5α-reductase (isozyme-1) in Spodoptera Frugiperda, SF21, insect cells: Expression of rat steroid 5α-reductase

Abstract The enzyme steroid 5α-reductase (5αR) catalyzes the reduction of testosterone (T) to 5α-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5αR type 1 isozyme (r5αR 1). The full length of r5αR1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda . Sf21, insect cells. The expressed recombinant r5α-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K m values for NADPH and T were 17 μM and 2.7 μM, respectively. Inhibition of the recombinant r5αR1 by N,N diethyl-4-aza-4-methyl-3-oxo-5α-androstane-17β-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5αR1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5αR1. Photoaffinity labeling by [2′-32P]-2-azido-NAD P + ([2′- 32 P]2N 3 -NAD P + ) and [1,2 3 H] N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5α androstane-17β-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.