Mouse macrophage metalloelastase expressed in bacteria absolutely requires zinc for activity.
Mouse macrophage metalloelastase was expressed in Escherichia coli. This 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 [3H]elastin by rMME absolutely required Ca2+; the optimal Ca2+ concentration was 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 marginal stimulation of rMME activity in the absence of Ca2+. Cu2+, Zn2+, or Cd2+ strongly inhibited rMME activity with IC50 values between 68 and 180 microM, while Mg2+, Ba2+, Mn2+, Co2+, and Sr2+ had no effect. The requirement of Zn2+ for rMME activity was determined. Significant enzyme activity was present in rMME treated with EDTA followed by Q-Sepharose column chromatography. Only when the inclusion bodies were solubilized in the presence of 20 mM EDTA, did an enzyme preparation which was absolutely dependent on exogenous Zn2+ for activity result. The optimal Zn2+ concentration for rMME activation was 100 microM. These results indicate that Zn2+ is tightly bound to rMME.
Jeng
A Y
AY
Research Department, Pharmaceuticals Division, Ciba-Geigy Corp., Summit, NJ 07901, USA.
Wong
M
M
Duelfer
T
T
Shapiro
S D
SD
Kramer
R A
RA
Hu
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli; D017737:Macrophages, Peritoneal / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000097:blood / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000096:biosynthesis / Q000097:blood / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015032:Zinc / Q000494:pharmacology*
D011994:Recombinant Proteins; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; D015032:Zinc