Ay. Jeng et al., MOUSE MACROPHAGE METALLOELASTASE EXPRESSED IN BACTERIA ABSOLUTELY REQUIRES ZINC FOR ACTIVITY, Journal of Biochemistry, 117(1), 1995, pp. 216-221
Mouse macrophage metalloelastase was expressed in Escherichia coli. Th
is recombinant enzyme (rMME) was present in the inclusion bodies that
were solubilized in 7 M guanidine HCl, After removal of guanidine HCl,
rMME was purified with a Q-Sepharose column. Degradation of [H-3]elas
tin by rMME absolutely required Ca2+; the optimal Ca2+ concentration w
as 5 mM. NaCl stimulated the enzyme activity; maximal stimulation was
obtained at 400 mM, The rMME activity was inhibited by metalloprotease
inhibitors, but not by serine, aspartyl, or thiol protease inhibitors
. Among the divalent cations tested, only Ba2+ and Sr2+ exhibited marg
inal stimulation of rMME activity in the absence of Ca2+. Cu2+, Zn2+,
or Cd2+ strongly inhibited rMME activity with IC50 values between 68 a
nd 180 mu M, while Mg2+, Ba2+, Mn2+, Co2+, and Sr2+ had no effect. The
requirement of Zn2+ for rMME activity was determined. Significant enz
yme activity was present in rMME treated with EDTA followed by Q-Sepha
rose column chromatography. Only when the inclusion bodies were solubi
lized in the presence of 20 mM EDTA, did an enzyme preparation which w
as absolutely dependent on exogenous Zn2+ for activity result, The opt
imal Zn2+ concentration for rMME activation was 100 mu M. These result
s indicate that Zn2+ is tightly bound to rMME.