Characterization of brain dibenzylfluorescein o-dibenzylase in gilthead seabream (Sparus Aurata L., 1758)

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Tezin Türü: Yüksek Lisans

Tezin Yürütüldüğü Kurum: Pamukkale Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, Türkiye

Tezin Onay Tarihi: 2007

Tezin Dili: Türkçe

Öğrenci: Demet Hançer

Danışman: Alaattin Şen

Özet:

Aromatase is encoded by the CYP19 gene, a member of the cytochrome P450 superfamily, andcatalyzes the conversion of androgens into estrogens. Data on native aromatase enzyme kineticsand 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, fluorescencearomatase assay was optimized to measure DBFOD activity in the gilthead seabream (Sparusaurata L., 1758) using O-benzylfluorescein benzyl ester (DBF) as a fluorometric substrate in brainand liver microsomes. Optimized assay variables included amount of tissue, pH, incubationtemperature, incubation time and substrate concentration. Brain and liver DBFOD activity haveshowed lineerity until 20 and 40 ?g protein concentration throughout 30 minutes, respectively. Inbrain and liver optimum pH of the enzyme have found to be 6.50 and 8.25, respectively andoptimum temperature have found to be 30°C for both tissues. It has been observed that brain andliver enzyme activities have saturated at and above 2 ?M DBF concentrations. Vmax and Km valuesof brain and liver DBFOD enzyme was determined using Lineweaver-Burk graph, and calculated8.054±0.550 pmol/min/mg protein, 8.389±0.543 pmol/min/mg protein and 0.840±0.161 ?M,0.959±0.152 ?M, respectively. Testosterone appeared to inhibit the Sparus aurata brain and liverDBFOD enzyme competitively. The effects of a variety of Turkish traditional dietary plants onDBFOD activity was also determined and these results could lead us to identify the diets that couldbe potential dietary source for novel aromatase inhibitors. Western blot analysis performed usinganti-rat aromatase antibodies. In conclusion, the parameters of this assay that are reported for brainand liver aromatase in gilthead seabream could be useful to measure aromatase activity in otherspecies.