Heterogeneity of rat type I 5 alpha-reductase cDNA: cloning, expression and regulation by pituitary implants and dihydrotestosterone.

Primer extension analysis reveals the presence of different forms of mRNA species for rat type I 5 alpha-reductase. Using a 5 alpha-reductase cDNA probe to screen the rat liver lambda gt11 cDNA library, we isolated cDNA clones that have 4 additional amino acids in the NH2-terminal region as compared with the previously reported sequence for rat type I 5 alpha-reductase. These four additional amino acids elongate the rat type I 5 alpha-reductase amino acid sequence to 259 amino acids, the same number as in human type I 5 alpha-reductase, with which it shares 60% identity. Expression of the long and short rat type I 5 alpha-reductase by transfection in human adrenal adenocarcinoma cells, SW-13 cells, indicated that the long cDNA encoded a protein with a higher affinity for the substrate than the short cDNA. To determine the effect of pituitary hormones and dihydrotestosterone (DHT), the mRNA levels in the livers of rats treated with pituitary implants, hypophysectomized, castrated, and castrated coupled with DHT treatment were quantified by dot-blot hybridization assay using rat type I 5 alpha-reductase cDNA as probes. The results demonstrated that rat type I 5 alpha-reductase mRNA is stimulated by pituitary hormones and castration but is decreased by DHT and hypophysectomy.

Cations inhibit specifically type I 5 alpha-reductase found in human skin.

Steroid 5 alpha-reductase catalyzes the reduction of testosterone into the very potent androgen dihydrotestosterone. Previously, we showed that human type I 5 alpha-reductase is expressed mainly in the skin, whereas a type II 5 alpha-reductase is more specifically expressed in the prostate. To assess the possible differential effects of various cations on the two types of 5 alpha-reductase, we constructed expression vectors and transfected them into SW-13 cells, a human adrenal carcinoma cell line containing negligible endogenous 5 alpha-reductase activity. The expressed 5 alpha-reductases were analyzed for their sensitivity to Li, Ca, Cd, Cu, Mg, Mn, Ni, Zn, and Fe. The results showed that type I 5 alpha-reductase was strongly inhibited by Cd, Cu, and Zn and moderately inhibited by Ni and Fe, with 50% inhibitory concentration values of 0.9, 1.9, 2.0, 169.2, and 174.3 microM, respectively. In contrast, type II 5 alpha-reductase activity was inhibited only by Cu, with a 50% inhibitory concentration value of 19.2 microM. The data showed that cations could specifically control 5 alpha-reductase activity expression, which is more strongly inhibited in a target tissue, especially the skin.

Characterization, expression, and immunohistochemical localization of 5 alpha-reductase in human skin.

Human skin has been shown to contain a high level of 5 alpha-reductase activity, the enzyme that catalyses the conversion of the weak androgen testosterone into dihydrotestosterone, the most potent androgen. Because two types of 5 alpha-reductase genes have been characterized in humans, we have cloned 5 alpha-reductase cDNAs from adult human keratinocyte and skin fibroblast cDNA libraries to identify and gain better knowledge of the 5 alpha-reductase expressed in normal human skin. Nucleotide sequence analysis shows that the clones obtained correspond to the type I 5 alpha-reductase. RNase protection analysis using (poly A)+ RNA obtained from human skin and prostate also confirms that type I 5 alpha-reductase is the predominant type expressed in normal skin, whereas type II 5 alpha-reductase is the major form found in the prostate. Following polymerase chain reaction amplification of human keratinocyte and skin fibroblast cDNA, a low level of type II 5 alpha-reductase cDNA has been detected. Using antipeptide antibodies raised in rabbits against the peptide sequence covering amino acids 227 -240 to perform immunohistochemical localization of 5 alpha-reductase, we have found that 5 alpha-reductase is distributed in sweat and sebaceous glands, as well as in the epidermal cell layers, thus providing the basis for the important role of androgens in human skin and its appendages.

Cloning and expression of cDNA encoding human placental estrogen sulfotransferase.

Using two oligoprimers derived from the bovine placental estrogen sulfotransferase sequence, we amplified a probe for human placental estrogen sulfotransferase. Using this probe to screen a human placental cDNA library constructed in lambda gt11, we isolated a cDNA clone of 1.3 kb encoding human estrogen sulfotransferase. DNA analysis predicts a protein of 295 amino acids with a calculated molecular weight of 34,199. Alignment of the amino acid sequence with other sulfotransferases indicates that human placental estrogen sulfotransferase shares 68.6, 68.2 and 65.9% similarity with bovine placental, guinea pig adrenocortical, and rat liver estrogen sulfotransferase, respectively. It shows also 95.6, 57.6, 85.3, and 54.2% similarity to human phenol, human DHEA, rat phenol, and rat hydroxysteroid sulfotransferase, respectively. Transfection of expression vectors encoding human estrogen sulfotransferase and dehydroepiandrosterone (DHEA) sulfotransferase in human adrenal adenocarcinoma SW-13 cells indicates that estrogen sulfotransferase transforms estrone more specifically, whereas DHEA sulfotransferase is more specific for DHEA and pregnenolone.

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas.

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.

17 beta-hydroxysteroid dehydrogenase activity in canine pancreas.

The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.

Periodate-oxidized NADP+ is a powerful inhibitor of human placental estradiol-17 beta dehydrogenase.

Periodate-oxidized NADP+ inhibits the NAD+-linked activity of human placental estradiol-17 beta dehydrogenase (EC 1.1.1.62). The inhibition appears to be competitive with respect to NAD+ and can be reversed by dialysis or gel filtration. The apparent inhibitor constant for the periodate-oxidized analogue is 0.047 microM. The presence in the incubation mixture of NAD+ protects the enzyme against inhibition. No inhibitory effects of the coenzyme analogue are observed on the NADP+-linked activity of the enzyme.