Hakan SÖYÜT, Şükrü BEYDEMİR, Saltuk Buğrahan CEYHUN

Changes in carbonic anhydrase activity and gene expression of Hsp70 in rainbow trout (Oncorhynchus mykiss) muscle after exposure to some metals

The effects of some heavy metals Co(II), Cu(II), Zn(II), and Ag(I) on carbonic anhydrase (CA) activity from rainbow trout (RT) muscle were investigated. Moreover, the effects of these metals on the expression of heat shock protein 70 (Hsp70) gene in muscle tissue were examined by real-time quantitative PCR (RT-PCR) in muscle tissue after exposure to the metals at the end of 6, 12, 24, and 48 h. CA was purified with a specific activity of 2300 EU mg-1, a yield of 19%, and 1080-fold. The molecular weights (Mw) of subunit and native enzyme were approximately 30 and 31 kDa, respectively. Optimum pH, stable pH, optimum temperature, activation energy (Ea), activation enthalpy (DH), and Q10 (the difference in activity of enzyme caused by the increment of 10 °C) value were determined. Apparent Michaelis constant (Km), maximum reaction rate (Vmax), and turnover rate of the enzyme (Kcat) values were 1.29 mM, 0.17 µmol min-1, and 28.8 s-1, respectively. The catalytic efficiency (Kcat/Km) was 22.3. The heavy metals decreased in vitro CA activity. Inhibition mechanisms of the metal ions were noncompetitive, except for the Co(II) ion, which was competitive. The expression of the Hsp70 gene was increased in the presence of the metal ions. The expression level at the end of 48 h was the highest for all of the metals. Consequently, the in vitro inhibition rank order was determined as Co(II) > Zn(II) > Cu(II) > Ag(I). Interestingly, Ag(I) was the most effective metal ion on Hsp70 gene expression.

Anahtar Kelimeler:

Key words: Carbonic anhydrase, characterization, muscle, rainbow trout, heavy metals, heat shock protein 70

Changes in carbonic anhydrase activity and gene expression of Hsp70 in rainbow trout (Oncorhynchus mykiss) muscle after exposure to some metals

Keywords:

Key words: Carbonic anhydrase, characterization, muscle, rainbow trout, heavy metals, heat shock protein 70,

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Soyut and Beydemir, 2008 Co2+ 032 05 Competitive Cu2+ 0 95 Uncompetitive Zn2+ 1 035 Competitive Brain Co2+ 05 4 × 10–2 Competitive Soyut et al., 2008 Zn2+ 31 15 Uncompetitive Cu2+ 0 6 Noncompetitive Ag+ 8 Competitive Cd2+ 5 5 Competitive Muscle Co2+ 099 065 Competitive Present study Zn2+ 330 790 Noncompetitive Cu2+ 90 15 Noncompetitive Ag+ 4 0 Noncompetitive metal exposure resulted in higher Hsp70 expression in fi sh tissues, including hepatocytes (17,48). Th e results of another study clearly demonstrated an elevated Hsp70 accumulation of trout hepatocytes
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