Abstract
The resolution and reconstitution of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system into a supernatant and a pellet fraction was accomplished by a procedure involving salt treatment followed by osmotic shock. Recombination of pellet and supernatant fractions was required for cyclase activity. This restoration effect could be demonstrated using either Mg-protoporphyrin IX or Mg-protoporphyrin IX monomethyl ester as the cyclase substrate in the presence or absence of S-adenosylmethionine. Pretreatment of the pellet fraction with either 8-hydroxyquinoline or desferal mesylate inhibited cyclase activity, indicating that there is a heavy-metal-ion requirement in this fraction. The cyclase supernatant protein(s) was not internalized by Sephadex G-50 and did not bind to Blue Sepharose, suggesting that it has a molecular mass of over 30 kDa and that it does not bind the cofactor NADPH. The cyclase supernatant protein did bind to MgProtoMe2-bound Sepharose and could be eluted by raising the pH to 9.7 in the presence of 4 mM-n-octyl glucoside. The pH optimum of the cyclase was 9.0. About a 40-fold purification of the cyclase supernatant protein was achieved by a combination of (NH4)2SO4 fractionation and phenyl-Sepharose chromatography.