Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Field measurements of nitrogen-fixing activity of intact saplings ofAlnus maximowiczii in the subalpine zone of Mt Fuji

Nitrogen-fixing (acetylene-reducing) activity of intact saplings ofAlnus maximowiczii was measured under natural conditions in the subalpine zone of Mt Fuji. The nitrogen-fixing activity was detected from the middle of June when expansion of leaves had just begun to the end of October when the shedding of leaves was almost completed. Diurnal changes in the activity were almost parallel with those of ground temperature. The measured nitrogen-fixing activity was related to ground temperature and total leaf area. Using this relation, annual nitrogen fixation was estimated from the data of ground temperature and leaf area measured in the field. The amount of annual nitrogen fixation was almost the same as that of nitrogen used for annual growth. It was concluded that nitrogen fixation by nodules made a considerable contribution to the nitrogen economy in the saplings ofA. maximowiczii.     

Comparison of ecophysiological responses to heavy snow in two varieties ofAucuba japonica with different areas of distribution

Aucuba japonica varieties are common evergreen understory shrubs in Japan.Aucuba japonica var.borealis is distributed on the Sea of Japan side of Honshu and Hokkaido where heavy snow cover lasts for more than 3 months in winter.Aucuba japonica var.japonica is distributed in areas with shallow or no snow on the Pacific Ocean side of Honshu and Shikoku. The ecophysiological characteristics of var.borealis were compared with those of var.japonica to examine the effects of heavy and long-term snow cover on the life cycle of var.borealis. Shoots of both varieties were shaded in crushed ice for 110 days, but their photosynthetic activities, chlorophyll contents and the chlorophylla/b ratio was not affected. The leaves of var.borealis were no less frost tolerant than those of var.japonica. In spite of the difference in environmental factors, both varieties had similar characteristics in seasonal changes of photosynthesis, respiration and chlorophylla/b ratio. These results suggest that var.japonica could survive in areas with heavy snow where it does not normally occur. Leaf net production (LNP) was estimated based on the microclimatic data and seasonal photosynthetic and respiration rates. The difference in the annual LNP between the two varieties was equivalent to the difference in the LNP during the snow season. One of the major effects of snow cover is to interrupt and reduce the production period of var.borealis.     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

Search for substrate-based inhibitors fitting the S2' space of malarial aspartic protease plasmepsin II.

Plasmepsin (Plm) has been identified as an important target for the development of new antimalarial drugs, since its inhibition leads to the starvation of Plasmodium falciparum. A series of substrate-based dipeptide-type Plm II inhibitors containing the hydroxymethylcarbonyl isostere as a transition-state mimic were synthesized. The general design principle was provision of a conformationally restrained hydroxyl group (corresponding to the set residue at the P2' position in native substrates) and a bulky unit to fit the S2' pocket.     

Concanavalin A Inhibits Auxin-Induced Elongation and Breakdown of (1 -> 3), (1 -> 4)-{beta}-D-Glucans in Segments of Rice Coleoptiles

Concanavalin A (Con A) suppresses auxin-induced elongation ofsurface-abraded segments from both dicotyledonous and poaceousplants. In coleoptile segments of rice (Oryza sativa L.), theauxin-induced decrease in the minimum stress-relaxation timeand increase in the mechanical extensibility of the cell wallswere also inhibited by Con A, indicating that the lectin suppresseselongation by inhibiting the cell wall loosening. Auxin causeda decrease in the level of (1 3), (1 4)-ß-D-glucansin the cell walls of rice coleoptile segments, and this decreasewas also inhibited by the lectin. Con A suppressed the autolytichydrolysis of the glucans, as well as their breakdown in vitroby a protein fraction that had been extracted from the cellwalls of rice coleoptiles with 1 M NaCl. Furthermore, most ofthe glucan-hydrolyzing activity of the wall proteins bound toa Con A-Sepharose column, suggesting that glycoprotein enzymesare involved in the hydrolysis. Although Con A also affectedthe hydrolysis of other wall polysaccharides, the present data,when considered in combination with the inhibitory effects ofglucan-specific or glucanasespecific antibodies, support theview that the breakdown of (1 3),(1 4)-ß-D-glucansis associated with the cell wall loosening that is responsiblefor auxin-induced elongation in Poaceae. (Received August 17, 1994; Accepted February 15, 1995)     

Reorientation of Cortical Microtubules in the Sub-Apical Region during Tuberization in Single-Node Stem Segments of Potato in Culture

The initial events in tuberization were examined in single-nodestem segments of potato, in which the tuberization was easilyregulated in culture. The addition of 8% sucrose to the culturemedium caused the cessation of elongation of lateral shootsand the swelling of the sub-apical region of each shoot. Swellingwas first induced by lateral cell expansion, which was followedby periclinal cell division. The divided cells then expandedlaterally. The alteration in the direction of growth was accompaniedby the reorientation of arrays of cortical microtubules (MTs),which was monitored by immunofluorescence microscopy. Cellsin the sub-apical region of elongating shoots had prominenttransverse arrays of MTs. The MTs in swelling cells were orientedlongitudinally with respect to the axis of the shoot. Finally,the arrays of MTs became completely disorganized. By contrast,the elongation of lateral shoots continued in GA₃-treated segmentsand the cells in the sub-apical region of such shoots retainedconspicuous transverse arrays of MTs during culture, even inthe presence of a high concentration (8%) of sucrose. (Received July 2, 1994; Accepted May 19, 1995)     

Paralogous origin of the rhodopsinlike opsin genes in lizards

Rhodopsinlike opsins constitute a distinct phylogenetic group (Yokoyama 1994, Mol. Biol. Evol. 11:32–39). This RH2 group includes the green-sensitive opsins in chicken and goldfish and the blue-sensitive opsin in a nocturnal lizard gecko. In the present study, we isolated and sequenced the genomic DNA clones for the RH2 opsin gene, rh2 _Ac , of the diurnal lizard Anolis carolinensis. This single-copy gene spans 18.3 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Phylogenetic analysis strongly suggests that rh2 _Ac is more closely related to the chicken green opsin gene than to the gecko blue opsin gene. This gene tree differs from the organismal tree, where the two lizard species should be most closely related, implying that rh2 _Ac and the gecko blue-sensitive opsin genes have been derived from duplicate ancestral genes.Correspondence to: S. Yukiyama     

Characterization of a Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Comparison with a 6S-Form

Abstract: 6R-l -erythro-Tetrahydrobiopterin (6R-BH₄) is a cofactor for aromatic l -amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R-BH₄ acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S-BH₄, a diastereoisomer of 6R-BH₄, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R-BH₄. Perfusion of 6S-BH₄ or 6R-BH₄ through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S-BH₄, was one-sixth of that by 6R-BH₄. 6S-BH₄ increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l -amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R-BH₄. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S-BH₄-induced dopamine release was abolished, but most of the 6R-BH₄-induced increase persisted. Coadministration of 6S-BH₄ with 6R-BH₄ inhibited the increase in dopamine release induced by 6R-BH₄ alone. These results show that 6R-BH₄ stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S-BH₄ acts as an antagonist of 6R-BH₄ at this site, although it has cofactor activities.