Synthesis of divinyl protochlorophyllide. Enzymological properties of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system.
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.
Walker
C J
CJ
Department of Botany, University of California, Davis 95616.
Castelfranco
P A
PA
Whyte
B J
BJ
D000645:Ammonium Sulfate; D002614:Chelating Agents; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D009249:NADP / Q000378:metabolism; D009997:Osmotic Pressure; D010105:Oxygenases / Q000378:metabolism*; D015125:Oxyquinoline / Q000494:pharmacology; D011521:Protochlorophyllide / Q000031:analogs & derivatives* / Q000096:biosynthesis; D011524:Protoporphyrins / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000494:pharmacology; D012995:Solubility; D013010:Sonication; D013379:Substrate Specificity; D014675:Vegetables / Q000187:drug effects / Q000201:enzymology
D002614:Chelating Agents; D011524:Protoporphyrins; C039629:magnesium protoporphyrin; C033434:protoporphyrin IX monomethyl ester; C020301:2,4-divinylprotochlorophyllide; D011521:Protochlorophyllide; D009249:NADP; D015125:Oxyquinoline; D012436:S-Adenosylmethionine; D010105:Oxygenases; C053161:magnesium protoporphyrin monomethyl ester oxidative cyclase; C041104:dimethyl protoporphyrin IX; D000645:Ammonium Sulfate