Effect of mercury on rabbit myelin CNP-ase in vitro.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) catalyzes hydrolysis of 2',3'-cyclic nucleotides to form the corresponding 2'-monophosphates. Rabbit myelin fraction with CNPase specific activity between 30-40 mumoles/min/mg protein was incubated in the presence of various inorganic and organic heavy metal compounds: HgCl2; (CH3Hg)OH; Pb(NO3)2; Pb(C2H302)2.3H20; (C2H5)2Pb; (C2H5)3SnCl. The enzyme has been shown to be almost exclusively sensitive to mercurials in microM concentration range. This would arise from the high solubility of mercurials in organic solvents, which allows them to penetrate into hydrophobic regions of the enzyme to react with active sulfhydryl groups. CNP-ase inhibition by methylmercury was biphasic: A reversible, non-competitive inhibition with an apparent Ki = 1 microM occurred after a 5 min preincubation time of the enzyme with the inhibitor. In the case of longer preincubation time, as well as in the presence of HgCl2, the graph of enzyme activity versus protein concentration intercepted the abscissa to the right of the origin, indicating that mercurials are irreversible inhibitors of the enzyme. After 45 min of preincubation of the inhibitors with the enzyme 1 nmol of HgCl2 completely blocks CNP-ase activity equivalent to 15.6 micrograms of myelin protein, whereas 1 nmole of Met-Hg blocks activity in 9.9 micrograms proteins. This apparently irreversible inhibition of CNP-ase activity by HgCl2 could be fully restored by the use of an excess of hydrophobic low molecular weight thiols, lipoic acid being the most efficient. Dithiothreitol, a hydrophilic complexing agent, was potent to reverse the inhibition caused by Met-Hg only during the short time experiments. Both low molecular weight thiols, and also EDTA in the case of inorganic mercury could prevent the inhibition of CNP-ase by mercurials, if preincubated for 15 min with the inhibitors, prior to the addition of the enzyme. The irreversible type of inhibition of CNP-ase by Met-Hg was only partially reversed in the presence of low molecular weight thiols. This suggests that the formation of a metal-mercaptide complex is not the only mechanism of inhibition by methylmercury. The possibility of lipid peroxidation triggered by methylmercury with subsequent inhibition of the enzyme activity was not supported by the experimental results. In fact, myelin associated CNP-ase activity appears to be very resistant to the structural membrane alterations caused by lipid peroxidation.