Purification and characterization of ferro- and cobalto-chelatases.
Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40,000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240,000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h, but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 microM for the metal and 30.3 microM for mesoporphyrin, and for cobaltochelatase activity, 27 and 45.5 microM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.
Cánepa
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ET
Departmento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Llambías
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EB
D000818:Animals; D001426:Bacterial Proteins; D002413:Cations, Divalent; D002784:Cholesterol / Q000494:pharmacology; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D005294:Ferrochelatase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D013552:Swine
D001426:Bacterial Proteins; D002413:Cations, Divalent; D005230:Fatty Acids, Nonesterified; D010743:Phospholipids; D002784:Cholesterol; D008190:Lyases; C033211:cobaltochelatase; D005294:Ferrochelatase