Characterization of Brain and Liver Aromatase of Gilthead Seabream (Sparus aurata L., 1758) using Dibenzylfluorescein

Year 2013, Volume: 17 Issue: 1, 5 - 14, 12.07.2014

Demet Hançer Aydemir Alaattin Şen

Abstract

Aromatase catalyzes the conversion of androgens into estrogens. Data on native aromatase enzyme kinetics and thus actual catalytic activity are scarce in fish, impeding comparison of aromatase activity (AA) from different organs within and between species. In the present study, fluorescence aromatase assay was optimized to measure AA in the gilthead seabream (Sparus aurata L., 1758) using dibenzylfluorescein (DBF) as a fluorometric substrate in brain and liver microsomes. Brain and liver AA have showed linearity until 20 and 40 µg protein concentration throughout 30 minutes, respectively. In brain and liver optimum pH of the enzyme was found to be 6.50 and 8.25, respectively and optimum temperature was found to be 30°C for both tissues. It has been observed that brain and liver AA have saturated at and above 2 µM DBF concentrations. Determined Vmax and Km values of brain and liver aromatase using LineweaverBurk graph have calculated 8,054±0,550 and 8,389±0,543 pmol/min/mg protein and 0,840±0,161 µM and 0,959±0,152 µM, respectively. Testosterone appeared to competitively inhibit the Sparus aurata brain and liver aromatase. In conclusion, the parameters of this assay that are reported for brain and liver aromatase in gilthead seabream could be useful to measure AA in other species

Keywords

Aromatase, Gilthead seabream, Brain, Liver, Dibenzylfluorescein

Çipura (Sparus aurata L., 1758) Beyin ve Karaciğer Aromataz Enziminin Dibenzilfloresein Kullanılarak Karakterize Edilmesi

Abstract

Aromataz, androjenlerin ösrojenlere dönüşümünü katalizlemektedir. Doğal aromatazın katalitik aktivitesi ve enzim kinetikleri hakkında bilgi balıklarda çok azdır ve bu tür içi ve türler arasında farklı organlardan elde edilen aromataz aktivitesinin (AA) karşılaştırılmasını engellemektedir. Bu çalışmada, floresans aromataz metodu, florometrik bir substrat olan dibenzilfloresein (DBF)
kullanılarak çipura (Sparus aurata L., 1758) beyin ve karaciğer mikrozomlarında AA’ni ölçmek için karakterize edilmiştir. Beyin ve karaciğer AA 30 dak reaksiyon zamanı boyunca sırasıyla 20 μg ve 40 μg protein miktarına kadar doğrusallık göstermiştir. Beyin ve karaciğerde enzimin optimum pH’sı sırasıyla 6,50 ve 8,25 olarak ve optimum sıcaklığı her iki doku için 30 ˚C olarak bulunmuştur. Beyin ve karaciğer AA’inin 2 µM DBF konsantrasyonu üzerinde doyuma ulaştığı gözlenmiştir. Lineweaver-Burk grafiği kullanılarak yapılan saptamalarda beyin ve karaciğer aromataz enzimi için Vmax ve Km değerleri belirlemişi ve sırasıyla 8,054±0,550 pmol/dak/mg protein, 8,389±0,543 pmol/dak/mg protein ve 0,840±0,161 µM, 0,959±0,152 µM olarak hesaplanmıştır. Testosteronoun Sparus aurata beyin ve karaciğer aromataz enzimini yarışmalı bir şekilde inhibe ettiği gözlenmiştir. Çipurada beyin ve karaciğer aromatazı için ilk defa rapor edilmiş bu metodun parametreleri diğer türlerde de AA’ni ölçmek için faydalı olabilecektir.

Keywords

Aromataz, Çipura, Beyin, Karaciğer, Dibenzilfloresein

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