Stimulation of myosin light-chain kinase by Cd²⁺ and Pb²⁺

Abstract

The effect of Cd²⁺ on myosin light chain kinase (MLCK) reported in the literature is controversial, apparently because the level of Ca²⁺ contaminating the reaction mixture could not be accurately controlled by the addition of a metal chelator when Cd²⁺ was also present. In the present study, we have reduced the contaminating Ca²⁺ to a trace level that did not interfere with the enzyme activity; thus the use of a metal chelator was not necessary. We showed that Cd²⁺, or Pb²⁺ had a biphasic effect on MLCK isolated from chicken gizzard: stimulation at low and inhibition at high concentrations. (The stimulatory effect of on the enzyme activity isolated from chicken gizzard: stimulation at low and inhibition at high concentrations). The stimulatory effect of Cd²⁺ or Pb²⁺ on MLCK activity was not seen in the absence of calmodulin, and was abolished by trifluoperazine, a calmodulin antagonist, indicating that the heavy metals exert their activation via calmodulin. The inhibition of the enzyme activity by Cd²⁺ or Pb²⁺ at higher concentrations was also seen with the calmodulin-independent catalytic fragment of MLCK, suggesting that the inhibition is probably through their binding to sulfhydryl groups that are essential for catalytic activity. Pb²⁺ was more effective than Cd²⁺ in stimulating the enzyme activity, but less potent in inhibition. The extent of stimulation by heavy metals most likely resulted from a combination of the biphasic effects. Dithiothreitol and N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine selectively chelated Cd²⁺ and Pb²⁺ over Ca²⁺, and reversed their stimulatory or inhibitory effect on MLCK. Thus, they are useful agents to discriminate between the action of Ca²⁺ from that of these heavy metal cations. Cd²⁺ and Pb²⁺ not only activited MLCK in vitro, but also triggered smooth muscle contraction in an isolated rabbit aortic strip.