MOUSE MACROPHAGE METALLOELASTASE EXPRESSED IN BACTERIA ABSOLUTELY REQUIRES ZINC FOR ACTIVITY

Citation
Ay. Jeng et al., MOUSE MACROPHAGE METALLOELASTASE EXPRESSED IN BACTERIA ABSOLUTELY REQUIRES ZINC FOR ACTIVITY, Journal of Biochemistry, 117(1), 1995, pp. 216-221
Citations number
24
Categorie Soggetti
Biology
Journal title
ISSN journal
0021924X
Volume
117
Issue
1
Year of publication
1995
Pages
216 - 221
Database
ISI
SICI code
0021-924X(1995)117:1<216:MMMEIB>2.0.ZU;2-5
Abstract
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.