Hormonal regulation and characterization of MHG30 gene, a desaturase-like gene of hamster harderian gland.

The harderian gland (HG) is an orbital gland of the vast majority of land vertebrates. In the Syrian hamster these glands display a marked sexual dimorphism. Here we present data on a male specific clone named MHG30. The MHG30 cDNA (1470 bp) has significant sequence homologies with human #15µ10#Δ6-desaturase enzymes. The expression of MHG30 has been found in male HG and in the liver of both sexes, no other tissue showing the presence of MHG30 mRNA. Castration brings the MHG30 levels below detectable level in about 7 days. In in vitro cultures of male hamster HG cells, androgens (A) determine an enhancement of MHG30 expression in a time-dependent manner. Conversely, a continuous decrement has been observed in control cells and in cells treated with A plus flutamide (F) or with A and cycloheximide (Cy). Incubation of cells in cultures supplemented with desamethason (Dex) or thyroid hormone (T3) also increases MHG30 expression while 17ß-estradiol prevents the stimulatory effect exerted by A, Dex and T3. Findings strongly suggest that the MHG30 gene could be involved in supporting the sexual dimorphism and its expression is likely triggered by a series of hormonal interactions.

Carbonic anhydrases in the mouse harderian gland.

The harderian gland is located within the orbit of the eye of most terrestrial vertebrates. It is especially noticeable in rodents, in which it synthesises lipids, porphyrins, and indoles. Various functions have been ascribed to the harderian gland, such as lubrication of the eyes, a site of immune response, and a source of growth factors. Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyse the reaction CO2 + H2O <--> H+ + HCO3. They are involved in the adjustment of pH in the secretions of different glands. Thirteen enzymatically active isozymes have been described in the mammalian α-CA family. Here, we first investigated the mRNA expression of all 13 active CAs in the mouse harderian gland by quantitative real-time PCR. Nine CA mRNAs were detectable in the gland. Car5b and Car13 showed the highest signals. Car4, Car6, and Car12 showed moderate expression levels, whereas Car2, Car3, Car7, and Car15 mRNAs were barely within the detection limits. Immunohistochemical staining was performed to study the expression of Car2, Car4, Car5b, Car12, and Car13 at the protein level. The epithelial cells were intensively stained for CAVB, whereas only weak signal was detected for CAXIII. Positive signals for CAIV and CAXII were observed in the capillary endothelial cells and the basolateral plasma membrane of the epithelial cells, respectively. This study provides an expression profile of all CAs in the mouse harderian gland. These results should improve our understanding of the distribution of CA isozymes and their potential roles in the function of harderian gland. The high expression of mitochondrial CAVB at both mRNA and protein levels suggests a role in lipid synthesis, a key physiological process of the harderian gland.

Light pulse induces ALA-S gene expression in the rat Harderian gland.

The rodent Harderian glands (HGs) are large paired orbital organs with highest porphyrinogenic rates. We have previously shown that continuous light exposure abolished the day/night variations of the delta-aminolevulinate synthase (ALA-S; the rate-limiting enzyme for porphyrin biosynthesis) gene expression observed under standard light: dark cycles (LD 12:12) in the rat HGs. This study was designed to examine whether the ALA-S changes were actually associated directly with light. The response of ferrochelatase (enzyme that converts protoporphyrin IX into heme) to light was also examined. Male Wistar rats were acclimatized to light: dark cycles regimen of 12:12 for 2 weeks. At the end of the 2 weeks, a 1 h-light pulse was applied in the middle of the dark phase. Animals were sacrificed immediately after the end of the light pulse. HGs were collected and stored at -80 degrees C until processed for quantitative RT-PCR. A 1 h-light pulse applied during mid-dark caused a significant increase of ALA-S gene expression (3-fold higher than in controls), whereas it was without effect on ferrochelatase gene expression. Our results suggest that light per se may regulate ALA-S gene expression in the rat HGs, and reveal that the ALA-S gene expression, and so heme biosynthesis, is under a photodynamic control.

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.

Inhibition of urethane-induced carcinogenicity in cyp2e1-/- in comparison to cyp2e1+/+ mice.

Urethane is an established animal carcinogen and has been classified as "reasonably anticipated to be a human carcinogen." Until recently, urethane metabolism via esterase was considered the main metabolic pathway of this chemical. However, recent studies in this laboratory showed that CYP2E1, and not esterase, is the primary enzyme responsible for urethane oxidation. Subsequent studies demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1-/- compared to Cyp2e1+/+ mice. Using Cyp2e1-/- mice, current studies were undertaken to assess the relationships between urethane metabolism and carcinogenicity. Urethane was administered via gavage at 1, 10, or 100 mg/kg/day, 5 days/week, for 6 weeks. Animals were kept without chemical administration for 7 months after which they were euthanized, and urethane carcinogenicity was assessed. Microscopic examination showed a significant reduction in the incidences of liver hemangiomas and hemangiosarcomas in Cyp2e1-/- compared to Cyp2e+/+ mice. Lung nodules increased in a dose-dependent manner and were less prevalent in Cyp2e1-/- compared to Cyp2e+/+ mice. Microscopic alterations included bronchoalveolar adenomas, and in one Cyp2e1+/+ mouse treated with 100 mg/kg urethane, a bronchoalveolar carcinoma was diagnosed. Significant reduction in the incidence of adenomas and the number of adenomas/lung were observed in Cyp2e1-/- compared to Cyp2e1+/+ mice. In the Harderian gland, the incidences of hyperplasia and adenomas were significantly lower in Cyp2e1-/- compared to Cyp2e+/+ mice at the 10 mg/kg dose, with no significant differences observed at the high or low doses. In conclusion, this work demonstrated a significant reduction of urethane-induced carcinogenicity in Cyp2e1-/- compared to Cyp2e1+/+ mice and proved that CYP2E1-mediated oxidation plays an essential role in urethane-induced carcinogenicity.

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.

Antioxidant activity in Spalax ehrenbergi: a possible adaptation to underground stress.

The blind subterranean mole rat Spalax ehrenbergi superspecies has evolved adaptive strategies to cope with underground stress. Hypoxia is known to stimulate reactive oxygen species generation; however, mechanisms by which Spalax counteracts oxidative damage have not been investigated before. We studied in Spalax the oxidative status of the Harderian gland (HG), an organ which is particularly vulnerable to oxidative stress in many rodents. With regard to the sexual dimorphism found in this gland, differences between males and females were determined and compared to the surface-dwelling Syrian hamster. Our results show, for the first time, that Spalax exhibits remarkably low biomolecular damage, which implies the existence of physiological strategies to avoid oxidative damage under fluctuating O(2) and CO(2) levels existing in the mole rat's subterranean niche. Correspondingly, main antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione reductase (GR), exhibited high activities in both genders; in particular, remarkably high levels were measured in SOD. SOD and GR activities showed statistically significant differences between sexes. Melatonin, an important circadian agent is also a very important antioxidant molecule and is synthesized in the Harderian glands (HGs) of Spalax. Therefore, the possible interaction between antioxidant enzymes and melatonin is suggested.

Hematopoietic prostaglandin D2 synthase in the chicken Harderian gland.

The Harderian gland (HG), a sero-mucous secreting organ in the eye orbit, has long been recognized as immunologically important in chickens. During experimentation to characterize immune components of the gland, proteomics analysis revealed the presence of hematopoietic prostaglandin D synthase (H-PGDS). Extraction of total RNA followed by RT-PCR produced cDNA of 597 base pairs. DNA sequencing revealed nucleic acid and predicted amino acid sequences that were 99% aligned with the one published sequence for chicken H-PGDS of the spleen. Alignment with murine, rat, and human H-PGDS were 69, 69, and 66%, respectively. Ocular vaccination of chickens with a Newcastle Disease/Infectious Bronchitis vaccine (Mass.-Ark. Strain) induced an increase in H-PGDS expression determined by real-time PCR. Furthermore, immunohistochemistry of frozen HG sections showed positive stained cells for both H-PGDS and mast cell tryptase in the sub-epithelial cell layers of the HG ducts. Based on the potent vasoactive role of PGD(2), it appears that the chicken HG is a site of active mucosal immunity partially mediated by PGD(2) synthesized by H-PGDS in the gland.

Evaluation of lysozyme and lactoferrin in lacrimal and other ocular glands of bison and cattle and in tears of bison.

OBJECTIVE: To evaluate lactoferrin and lysozyme content in various ocular glands of bison and cattle and in tears of bison. SAMPLE POPULATION: Tissues of ocular glands obtained from 15 bison and 15 cattle and tears collected from 38 bison. PROCEDURE: Immunohistochemical analysis was used to detect lysozyme and lactoferrin in formalin-fixed, paraffin-embedded sections of the ocular glands. Protein gel electrophoresis was used to analyze ocular glands and pooled bison tears by use of a tris-glycine gel and SDS-PAGE. Western blotting was used to detect lactoferrin and lysozyme. RESULTS: Immunohistochemical staining for lactoferrin was evident in the lacrimal gland and gland of the third eyelid in cattle and bison and the deep gland of the third eyelid (Harder's gland) in cattle. Equivocal staining for lactoferrin was seen for the Harder's gland in bison. An 80-kd band (lactoferrin) was detected via electrophoresis and western blots in the lacrimal gland and gland of the third eyelid in cattle and bison, Harder's glands of cattle, and bison tears. An inconsistent band was seen in Harder's glands of bison. Lysozyme was not detected in the lacrimal gland of cattle or bison with the use of immunohistochemical analysis or western blots. Western blots of bison tears did not reveal lysozyme. CONCLUSIONS AND CLINICAL RELEVANCE: Distribution of lactoferrin and a lack of lysozyme are similar in the lacrimal gland of cattle and bison. Differences in other tear components may be responsible for variability in the susceptibility to infectious corneal diseases that exists between bison and cattle.

Effects of delta-aminolevulinic acid and melatonin in the harderian gland of female Syrian hamsters.

Effects of delta-aminolevulinic acid (ALA) and melatonin were investigated in the female Syrian hamster Harderian gland. This is an organ physiologically exposed to strong oxidative stress due to the highest porphyrinogenic rates known in nature. Enzyme activities of porphyrin biosynthesis and of antioxidative protection, oxidative protein modification, and histological integrity were studied. In the porphyrin biosynthetic pathway, ALA and melatonin acted synergistically by downregulating ALA synthase (ALA-S) and stimulating product formation from ALA; the combination of ALA and melatonin suppressed ALA-S activity, down to about 1% of that in controls. While ALA effects on porphyrinogenesis can be interpreted in terms of homeostasis, melatonin's actions may be seen in relation to seasonality and/or reduction of oxidative stress. Among antioxidant enzymes, superoxide dismutase (SOD) and glutathione reductase (GR) activities were diminished by ALA, presumably due to the vulnerability of their active centers to free radicals, whereas melatonin moderately increased SOD. Both ALA and melatonin strongly stimulated catalase (CAT), thereby counteracting the oxidative stress induced by ALA and its metabolites. Nevertheless, exogenous ALA caused a strong net rise in protein carbonyl and considerable damage of tissue. When given together with ALA, melatonin antagonized these effects and largely protected the integrity of glandular structures.

Melatonin protects against delta-aminolevulinic acid-induced oxidative damage in male Syrian hamster Harderian glands.

Effects of the prooxidant delta-aminolevulinic acid (ALA) and the antioxidant melatonin (MEL) were investigated in the male Syrian hamster Harderian gland (HG). Rodent Harderian glands are highly porphyrogenic organs, which may be used as model systems for studying damage by delta-aminolevulinic acid and its metabolites, as occurring in porphyrias. Chronic administration of delta-aminolevulinic acid (2 weeks) markedly decreased activities of the porphyrogenic enzymes delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) and of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), whereas porphobilinogen deaminase (PBG-D) remained unaffected. This treatment led to increased lipid peroxidation (LPO) and oxidatively modified protein (protein carbonyl) as well as to morphologically apparent tissue damage. Melatonin also caused decreases in delta-aminolevulinate synthase, delta-aminolevulinate dehydratase, superoxide dismutase, glutathione reductase and catalase. Despite lower activities of antioxidant enzymes, lipid peroxidation and protein carbonyl were markedly diminished. The combination of delta-aminolevulinic acid and melatonin led to approximately normal levels of delta-aminolevulinate dehydratase, glutathione reductase, catalase and protein carbonyl, and to rises in superoxide dismutase and porphobilinogen deaminase activities; lipid peroxidation remained even lower than in controls and the appearance of the tissue revealed a protective influence of melatonin. These results suggest that melatonin may have profound effects on the oxidant status of the Harderian gland.

Physiological oxidative stress model: Syrian hamster Harderian gland-sex differences in antioxidant enzymes.

The Syrian hamster Harderian gland, a juxtaorbital organ exhibiting marked gender-associated differences in contents of porphyrins and melatonin, was used as a model system for comparing strong (in females) and moderate (in males) physiological oxidative stress. Histological differences showing much higher cell damage in females were studied in conjunction with lipid peroxidation and activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Lipid peroxidation and enzyme activities were measured throughout the circadian cycle, revealing the importance of dynamical processes in oxidative stress. Especially in lipid peroxidation and in catalase, short-lasting rises exhibited strongest gender differences. Peaks of lipid peroxidation were about three times higher in females, compared to males. Catalase peaks of females exceeded those in males by several hundred-fold. Average levels of superoxide dismutase and glutathione peroxidase were about three or two times higher in females, respectively. A clear-cut diurnally peaking rhythm was found in glutathione peroxidase of females, which was not apparent in males. Glutathione reductase showed differences in time patterns, but less in average activities. The time courses of lipid peroxidation and of protective enzymes are not explained by circulating melatonin, whereas melatonin formed in the Harderian gland should contribute to differences in average levels. Neither damage nor antioxidative defense simply reflect the illumination cycle and are, therefore, not only a consequence of photoreactions.

beta-glucuronidase activity is elevated in the rat harderian gland after bromocriptine but decreased after cyproterone acetate treatment or castration. Postcastrational effects on the ultrastructure of the gland.

OBJECTIVE: To investigate the effect of castration, bromocriptine and cyproterone acetate treatment on lysosomal hydrolytic enzymes and ultrastructure of the rat Harderian gland. METHODS: Groups of rats were subjected to the treatment by bromocriptine, cyproterone acetate and castration. Harderian glands were dissected from each experimental animal cut into small pieces and immediately stored in liquid nitrogen. Only those from castrated animals were fixed in Karnovsky fluid and processed for electron microscopy. The activity of acid phosphatase and ss-glucuronidase were estimated by kits from Sigma (St. Louis, Mo, USA). Semi-thin sections were stained with 1-% toluidine blue and ultra-thin sections with uranyl acetate and counter-stained with lead citrate. The examination was performed by Joel JEM-1200 EX II electron microscope. RESULTS: The treatment of male rats with the prolactin release inhibitor bromocriptine induced a significant increase in ss-glucuronidase activity. On the contrary, such activity was significantly decreased after treatment with the testosterone receptor antagonist cyproterone acetate as well as after castration. However, these treatments did not alter the activity of lysosomal acid phosphatase. Castration induced dramatic changes mostly in type A cells such as the appearance of lipid vacuoles with irregular forms and the predomination of smooth endoplasmic reticulum (SER) throughout the cytoplasm. The most dramatic postcastrational change was the degeneration of the mitochondria. These changes in type A cells might be due to the uneven distribution of the testosterone receptors in the rat Harderian gland, which are more numerous in type A cells. CONCLUSIONS: the gland physiology is responsive to alteration in circulating prolactin and testosterone levels.

Effect of estrogenic perturbations on delta-aminolevulinic acid-induced porphobilinogen deaminase and protoporphyrin IX levels in rat Harderian glands, liver, and R3230AC tumors.

The Harderian gland in rodents highly expresses enzymes of the heme biosynthetic pathway that are responsible for porphyrin production. Interestingly, many of the steps in Harderian gland heme biosynthesis, including protoporphyrin production, are controlled hormonally. We hypothesized that estrogenic alterations, ovariectomy or tamoxifen administration, might also alter the response of porphobilinogen deaminase activity and/or protoporphyrin IX production to delta-aminolevulinic acid administration in the hormonally responsive R3230AC rat mammary adenocarcinoma. We also determined whether the response of the R3230AC tumor, borne on ovariectomized hosts, to delta-aminolevulinic acid-based photodynamic therapy was altered compared with tumors treated on intact hosts. Ovariectomy of female Fischer rats bearing the hormonally responsive R3230AC mammary adenocarcinoma caused a significant reduction in delta-aminolevulinic acid-induced protoporphyrin IX levels and porphobilinogen deaminase activity in tumors compared with levels in tumors from intact animals treated with delta-aminolevulinic acid. In contrast, although porphobilinogen deaminase activity in the Harderian gland from ovariectomized animals was reduced significantly compared with that in glands from intact animals, protoporphyrin IX levels were unaltered. Administration of the anti-estrogen tamoxifen to tumor-bearing rats resulted in a significant increase in porphobilinogen deaminase in both tumor and Harderian gland. Although administration of delta-aminolevulinic acid increased protoporphyrin IX levels in Harderian glands in tamoxifen-treated animals, tumor levels of protoporphyrin IX remained unaltered in the tamoxifen-treated rats. Treatment of R3230AC tumors with delta-aminolevulinic acid-based photodynamic therapy in ovariectomized rats resulted in a significantly reduced response compared with the same treatment regimen in intact animals, 4.9+/-0.39 versus 10.6+/-0.6 days to reach twice the initial tumor volume, respectively. These results indicate that the hormonal status of the host should be considered when treating hormonally sensitive tumors with delta-aminolevulinic acid-based photodynamic therapy.

Ultrastructural localization of acetylcholinesterase (AChE) activity in the chicken Harderian gland.

Localization of acetylcholinesterase (AChE) was investigated in the chicken Harderian gland at the electron microscopic level. Nerve cells in the pterygopalatine ganglion showed AChE activity. They had a pale and large nucleus which was round or oval in shape. Reaction product of AChE was detected between the nuclear envelopes; in the cisterna of rough endoplasmic reticulum and the lumen of the Golgi lamellae, and on the plasma membrane of the nerve cell. In the interstitium of the gland, nerve fibers showing AChE activity were easily found. They were often seen in the perivascular space and between plasma cells. These nerve fibers had varicosities in contact with plasma cells and the endothelium or the smooth muscle fiber of the blood vessels. AChE-positive varicosities or terminals contained many small clear vesicles (about 50nm in diameter) and a few large dense-cored vesicles (about 100 nm in diameter). No contacts of nerve fibers with acinar cells or the ductal epithelium were observed in the present study. Our data indicate that cholinergic nerves play distinct roles in the regulation of the immune function of the chicken Harderian gland.

Northern analysis of type II iodothyronine deiodinase mRNA in rat Harderian gland.

It has been known that type II iodothyronine deiodinase activity is present in rat Harderian gland and the activity is significantly increased by isoproterenol administration. We have performed Northern analyses to study whether the transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and whether the isoproterenol stimulation of type II iodothyronine deiodinase activity in rat Harderian gland is due to the change in its mRNA level. Northern analyses have demonstrated that type II iodothyronine deiodinase mRNA, approximately 7.5 kb in size, is expressed in rat Harderian gland, and the mRNA levels as well as the deiodinase activities are greater in hypothyroid rats than those in euthyroid rats. Type II iodothyronine deiodinase mRNA levels and the deiodinase activities in Harderian gland were increased by isoproterenol administration, and the increase in the mRNA levels preceded that in the deiodinase activities. These results indicate that 7.5 kb transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and beta-adrenergic stimulation of type II iodothyronine deiodinase activity is due at least in part to the increase in its mRNA level.

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.

Novel regulation of delta-aminolevulinate synthase in the rat harderian gland.

The mode of expression of delta-aminolevulinate synthase (ALAS), as well as that of mRNAs for other heme pathway enzymes, was examined in the rat Harderian gland. Northern blot and in situ hybridization analyses demonstrated that the non-specific ALAS (ALAS-N) mRNA is highly expressed in this tissue, whereas the erythroid-specific ALAS (ALAS-E) mRNA is not. Immunoblot analysis of ALAS also confirmed this finding at the protein level. ALAS-N mRNA was maximally induced in the Harderian gland and was not increased further by treatment of animals with 2-allyl-2-isopropylacetamide (AIA). The levels of mRNAs for other heme pathway enzymes, i.e., delta-aminolevulinate dehydratase, porphobilinogen deaminase, uroporphyrinogen decarboxylase, and coproporphyrinogen oxidase, also were increased markedly in the Harderian gland and not influenced by AIA treatment. The level of ferrochelatase (FeC) mRNA in the gland was, however, lower than that in the liver. The gland contained an extremely high level of protoporphyrin, while heme was undetectable. Microsomal heme oxygenase-1 (HO-1) mRNA levels were significantly higher in the Harderian gland than in the liver. When isolated glands were incubated with hemin in vitro in organ cultures, the level of HO-1 mRNA was increased, whereas the ALAS-N mRNA level was not. These findings indicate that markedly elevated levels of protoporphyrin and extremely low levels of heme in the Harderian gland are the results of both decreased expression of FeC and markedly increased expression of ALAS-N and HO-1. The constitutive expression of the ALAS-N gene in the Harderian gland suggests a novel transcriptional control mechanism of this gene.