Cloning and differential expression of steroid 5 alpha-reductase type 1 (Srd5a1) and type 2 (Srd5a2) from the Harderian glands of hamsters.

In hamsters, the Harderian glands (HGs) exhibit a marked sexual dimorphism which is thought to depend on dihydrotestosterone (DHT); however, it is unclear whether hamster HGs contain one or more 5 alpha-reductases and whether these enzymes are differentially expressed in males and females. In this study, we isolated specific cDNAs for 5 alpha-reductase 1 (Srd5a1) and 5 alpha-reductase 2 (Srd5a2), determined their sequences and investigated their expression in the HG of both sexes. Isozyme 1, cloned from liver mRNA, encodes a protein of 255 amino acids (aa); isozyme 2 cDNA, isolated from the epididymis encodes a 254-aa protein. When assayed in transfected HEK-293 cells, the type 1 isozyme displayed activity over a broad pH range (6.5-8), while isozyme 2 had a pH optimum of 5.5. Both isoenzymes efficiently catalyzed the in vitro transformation of T into DHT, with apparent K(m) values of 7.1 and 1.9 micromol/L for Srd5a1 and Srd5a2, respectively. Real-time PCR analysis revealed higher mRNA levels for Srd5a1 than for Srd5a2. Expression of both isoenzymes increased slightly in HGs of castrated males and showed variations during the estrous cycle in females. Hormonal replacement with 17beta-estradiol administered to spayed females induced the up-regulation of Srd5a2 mRNA levels. Altogether, our results demonstrated that both Srd5a1 and Srd5a2 are expressed in HGs without clear differences between males and females. The biochemical characteristics and relative expression of these 5 alpha-reductases support the view that both isozymes may play a relevant role in modulating androgen signaling in HG.

The influence of sex steroid hormones on ferrochelatase gene expression in Harderian gland of hamster (Mesocricetus auratus).

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

Sequence and structure of the rat housekeeping PBG-D isoform.

Porphobilinogen deaminase (PBG-D), a key enzyme in the tetrapyrrole biosynthetic pathway, is encoded by a single gene containing two different promoters. The upstream promoter, found in all cell types, initiates the transcription of the housekeeping PBG-D isoform, whereas the downstream one is erythroid-specific. In this study, we provide the first full sequence of a 1086bp cDNA covering the coding region for the rat ubiquitous PBG-D and its primary amino acid sequence. The cDNA encodes a 39,361 Da protein composed of 361 amino acids. Nucleotide sequence comparison between both isoforms from rat shows similarities of 99.5%, with four changes (C/G) in exon 8 and only one (C/A) in exon 12. Secondary structure prediction reveals that 76.5% of the amino acids from exon 1 are located in a loop. Potential phosphorylation, glycosylation, and myristoylation sites were revealed through motif searches. Housekeeping PBG-D contains coiled-coil segments known to be involved in dynamic rearrangements in the active site.

Rat harderian gland porphobilinogen deaminase: characterization studies and regulatory action of protoporphyrin IX.

Properties of purified porphobilinogen deaminase (PBG-D; EC 4.3.1.8) from rat harderian gland are here presented. The enzyme behaves as a monomer of Mr 38 +/- 2 kDa and is optimally active at pH 8.0-8.2. Its activation energy, determined by an Arrhenius plot, is 76.1 kJ/mol. Initial velocity studies showed a linear progress curve for uroporphyringen I formation and a hyperbolic dependence of the initial rate on substrate concentration, indicating the existence of a sequential displacement mechanism. Apparent kinetic constants, Km and Vm, calculated at 37 degrees C and pH 8.0 were 1.1 microM and 170 pmol/min mg, respectively. The pH dependence of the apparent kinetic parameters revealed the ionization of residues with pKAES and pKBES of 7.4 +/- 0.1 and 8.6 +/- 0.1, respectively, and a pKE value of 8.0 +/- 0.1. Incubation of PBG-D with 5.0 mM N-ethylmaleimide and 5.0 mM 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 led to inhibitions of 70 and 50%, respectively. The effect of pH, as well as the effect of thiol reagents, on enzyme activity strongly suggests the involvement of cysteine residue(s) in the mechanism of uroporphyrinogen I biosynthesis, in both the catalytic reaction and the substrate binding. Rat harderian gland PBG-D activity decreased with increasing concentrations of protoporphyrin IX, reaching a 40% inhibition at the in vivo concentration of the porphyrin and 7 microM PBG. Even at saturating concentrations of substrate, inhibition by protoporphyrin was not completely reversed. So, accumulated porphyrin may act as an regulator of PBG-D activity in rat harderian gland.

Steroid 5 alpha-reductase activity in the harderian glands of male and female Syrian hamster (Mesocricetus auratus).

The activity of the enzyme steroid 5 alpha-reductase in Harderian glands of adult syrian hamsters was assessed by measuring the conversion of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). The optimal conditions for this reaction were determined in vitro using whole gland homogenates and [3H]testosterone as substrate. Enzyme activity was maximal at pH 5.5. The Michaelis-Menten constant of the Harderian enzyme for T was 4.6 +/- 1.2 x 10(-6) M in females and 4.2 +/- 0.39 x 10(-6) M in males, estimated by Eadie-Hofstee plots. On the basis of relative maximum velocity values, there was 9 or 10 times more 5 alpha-reductase in females (2.8 +/- 0.67 nmol/mg protein/hr) than in males (0.289 +/- 0.029 nmol/mg protein/hr). Consistently, glands of intact male hamsters had lower 5 alpha-reductase activities than those of females. Castration of males significantly increased the enzymatic activity, which within 4 weeks reached female-like values. The levels of 5 alpha-reductase mRNA also increased with castration. There was a direct correlation between activity and mRNA levels of the enzyme in castrated male glands. Further, the administration of T or DHT to ovariectomized hamsters led to intact male values in the enzymatic activity of the gland. The sex differences in 5 alpha-reductase activity may be of relevance to the differential regulation exerted by androgen upon the physiology of male and female glands. The results are consistent with the view that 5 alpha-dihydrotestosterone is the active androgen in the Harderian gland.