Non-michaelian behavior of 5α-reductase in human prostate

doi:10.1016/0022-4731(89)90289-6

Journal of Steroid Biochemistry

Volume 33, Issue 2, August 1989, Pages 155-163

https://doi.org/10.1016/0022-4731(89)90289-6 Get rights and content

Abstract

An in-depth analysis of the kinetics of 5α-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH5.5, 5α-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this “hysteretic” behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5α-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5α-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.

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Differences in steroid 5α-reductase iso-enzymes expression between normal and pathological human prostate tissue
1999, Journal of Steroid Biochemistry and Molecular Biology
We studied the expression level and cell-specific expression patterns of 5α-reductase (5α-R) types 1 and 2 iso-enzymes in human hyperplastic and malignant prostate tissue by semi-quantitative RT-PCR and in situ hybridisation analyses. In situ hybridisation established that 5α-R1 mRNA is preferentially expressed by epithelial cells and little expressed by stromal cells whereas 5α-R2 mRNA is expressed by both epithelium and stroma. Semi-quantitative RT-PCR has been performed on total RNA from different zones of normal prostate, BPH tissues and liver. We found that 5α-R1 and 5α-R2 mRNAs expression was near the same in all zones of normal prostate. In BPH tissue, 5α-R1 and 5α-R2 mRNAs expression was slightly but significantly increased, when it was compared to the levels recorded for normal prostate. In cancer samples, 5α-R1 mRNA expression was higher than in normal and hyperplastic prostate but the level of 5α-R2 mRNA was not statistically different from that observed in the different zones of normal prostate. In liver, 5α-R2 mRNA level was similar to that measured in BPH but 5α-R1 mRNA expression was ten times higher. The increase observed in 5α-R isoenzymes expression in BPH tissue could play an important role in the pathogenesis and/or maintenance of the disease.
Kinetic analysis of rat steroid 5α-reductase activity in prostate and epididymis homogenates at neutral pH: Evidence for type I activity in epididymis
1996, Journal of Steroid Biochemistry and Molecular Biology
Immunocytochemical studies and mRNA measurements have shown that the rat epididymis—like the rat prostate—expresses both rat steroid 5α-reductase isozymes, i.e. type I and II. So far, enzyme activity measurements in rat epididymis homogenates, however, do not support the presence of type I 5α-reductase activity. Incubating homogenates of both tissues with a wide range of substrate concentrations, we were able to detect activity of both isozymes in rat prostate and epididymis tissues at neutral pH. In rat prostate the amount of type I activity, as measured by the V_max at pH 7.0, exceeds that of type II 5α-reductase 50-fold. The efficiency ratio, $V_{max} K_{m}$ , of the type I isozyme accounts for 25% of the total in vivo potential activity. A possible anabolic role for the type I isozyme in rat prostate was thus surmised. In rat epididymis the V_max of type I and type II 5α-reductase at pH 7.0 were similar. Comparison of the efficiency ratio $V_{max} K_{m}$ of either isozyme in the rat epididymis, however, suggested that the type II isozyme would play the major role in the 5α-reduction of testosterone at physiological concentrations and at neutral pH. The specific localization of the isozymes should be considered to allow for correct quantification of their in vivo contribution to dihydrotestosterone formation.
Rat steroid 5α-reductase kinetic characteristics: Extreme pH-dependency of the type II isozyme in prostate and epididymis homogenates
1995, Journal of Steroid Biochemistry and Molecular Biology
Reevaluating the assay for rat steroid 5α-reductase isozymes in prostate and epididymis homogenates we encountered an extreme pH-dependency of the type II isozyme. The time-course of the metabolism of testosterone (T) to 17β-hydroxy-5α-androstan-3-one (DHT) at acidic pH shows an initial burst when the homogenate is not brought to pH before the start of the incubation. Therefore, the rat type II 5α-reductase isozyme does not follow Michaelian law under these conditions making a single time point measurement invalid. Assessing the pH-optimum of 5α-reduction in both rat prostate and epididymis homogenates we found a strong substrate dependency: at high substrate concentrations a pH-optimum for the type II isozyme of pH 5.0 was found, whereas at lower concentrations pH 5.5 is optimal. Establishing V_max (maximum velocities) and K_m (affinity constants) for the 5α-reduction of T at pH 4.5–8.0, the efficiency optimum $V_{max} K_{m}$ appeared to be pH 5.5 in both prostate and epididymis homogenates. Specifically at acidic pH these kinetic characteristics of the type II isozyme vary many-fold. Discrepancies in literature concerning 5α-reductase characteristics can, at least in part, be attributed to the choice of optimal pH, or to pH shifts during the assay.
Formation of 5α steroids by biotranformation involving the 5α-reductase activity of Penicillium decumbens
1994, Steroids
The biotransformation of a series of Δ⁴-3-ketosteroids by the Penicillium decumbens ATCC 10436 has been investigated. Conversion to the 5α-dihydrosteroid was observed substrates of the androsterone and pregne series: the reaction is tolerant of non-polar substituents (Cl and CH₃) at C-4 of the substrate, but does not occur in the presence of a 4-hydroxyl group, or with additional unsaturation at the Δ¹orΔ⁶ positions. A-nor, B-nor, 3-deoxy-, and 3,5-cycloandrostanes are not reduced, but 6-methylenestestosterone is converted to a 6-methylene-5α-dihydro derivative. Several biotransformations are reported which involve oxidoreductase activity at C-3 and/or C-17, either concomitant or independent of Δ⁴ reduction: the substrate specificity of the oxidoreductase processes has been examined and defined by the use of 3α-hydroxy, 3β-hydroxy, 3-keto, 17β-keto substituted steroids. In this way, the existence in P. decumbens of 3β-hydroxy-3-keto and 17β-hydroxy-17-keto oxidoreductases has been demonstrated.
Inhibition of the activity of 'basic' 5α-reductase (type 1) detected in DU 145 cells and expressed in insect cells
1994, Journal of Steroid Biochemistry and Molecular Biology
The purpose of this study was 2-fold: (1) to identify the 5α-reductase (5α-R) isozyme(s) present in DU 145 cells, a human cell-line of low androgen sensitivity derived from a cerebral metastasis of an epithelial prostate cancer; and (2) to compare the inhibitory potencies of three compounds on the ‘basic’ 5α-R isozyme expressed in a baculovirus-directed insect cell system. Conversion of testosterone (T) into 5α-dihydrotestosterone (DHT) in DU 145 cells was measured by HPLC coupled to a Flo-one HP radioactivity detector. DU 145 cells exhibited 5α-R activity (21 pmol DHT/min/mg protein) at pH 7.4 which disappeared at pH 5.5 suggesting that, of the two genomically distinct human isozymes identified so far, type 1 5α-R is expressed in DU 145 cells. This was confirmed by at least two observations: first, 5α-R activity in DU 145 cells was inhibited with much higher potency by 4-MA than by finasteride which is known to be a very poor competitor of the ‘basic’ enzyme (IC₅₀s=2.8 ± 0.2 and 264 ± 55 nM, respectively). Second, only the type 1 5α-R cDNA and not type 2 5α-R cDNA hybridized with DU 145 RNA. A high potency differential was also recorded for the inhibition of ‘basic’ type 1 5α-R expressed in a baculovirus-directed-insect cell system by these two compounds, 4-MA being considerably more active than finasteride (K_i = 8.4 ± 2.3 and 330 ± 9 nM, respectively). This inhibition was competitive. On the other hand, inhibition by an n-hexane lipid/sterol extract of Serenoa repens (LSESr) was non-competitive and, when expressed in terms of recommended therapeutic doses, was 3-fold greater for LSESr than for finasteride. These studies suggest that LSESr might exert a regulatory inhibitory activity due to its specific lipid/sterol composition.
Baculovirus-directed expression of human prostatic steroid 5α-reductase 1 in an active form
1993, Journal of Steroid Biochemistry and Molecular Biology
In the human prostate, the enzyme steroid 5α-reductase (h5αR) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. Two distinct cDNAs coding for h5αR in the human prostate have been previously characterized. Enzyme h5αR1 shows a maximum activity at basic pH whereas h5αR2 has an acidic pH optimum activity. We report here the expression of the human steroid h5αR1 in a eukaryotic expression system : the baculovirus-directed-insect cell expression system. The full length cDNA was inserted into the Autographa californica nuclear polyhedrosis virus genome and expressed in Spodoptera frugiperda, Sf9, insect cells. Sf9 cells were infected with the recombinant baculovirus and homogenates used in h5αR activity assays by high pressure liquid chromatography showed that a catalytically active enzyme was produced. The recombinant enzyme showed an apparent K_m for testosterone of 2.07 μM and a V_max of 10.1 nmol of dihydrotestosterone/min/mg of protein. Recombinant h5αR1 activity was inhibited by specific h5αR inhibitors such as 4-MA (K_i = 2.6 nM). Subcellular distribution in Sf9 cells demonstrated that the enzyme was associated with the nuclear membrane.

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Non-michaelian behavior of 5α-reductase in human prostate

Abstract

J. steroid Biochem.

J. steroid Biochem.

J. steroid Biochem.

J. biol. Chem.

Steroids

J. steroid Biochem.

Meth. Enzymol.

Biochim. biophys. Acta

J. biol. Chem.

J. biol. Chem.

J. biol. Chem.

J. biol. Chem.

J. steroid Biochem.

J. steroid Biochem.

J. steroid Biochem.

Steroids

Binding of testosterone, dihydrotestosterone and progesterone to rat prostate cytosol: application of microcalorimetry

Endocr. Exp.

Selective retention of dihydrotestosterone by prostatic nuclei

Nature

The dihydrotestosterone (DHT) hypothesis of prostate cancer and its therapeutic implications

Prostate

Biochemical endocrinology of human prostatic tumors

Progr. Cancer Res. Ther.

5α-reductase activity in human prostate cancer is related to the histological differentiation of the tumour

Clin. Endocr.

Design of antiandrogens and their mechanism of action: a case study (Anandron)

Horm. Res.