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 aft er 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 (Eα), activation enthalpy (ΔH), and Q10 (the difference in activity of enzyme caused by the increment of 10 oC) 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.