Characterization of ferrochelatase in kidney and erythroleukemia cells.
Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.5 and 12.7 microM, respectively. The Km values for iron and zinc with mesoporphyrin IX were 3.51 and 3.17 microM, respectively. The purified enzyme showed a single band with an apparent molecular mass of 42,000 daltons (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbit antibody against the purified enzyme markedly inhibited activities of the enzyme from both the kidney and liver. Immunoblot analysis showed that the antibody reacted with the renal as well as the hepatic enzymes showing the same molecular weight. Peptide mapping with trypsin or alpha-chymotrypsin showed that digested peptides of renal enzyme were similar to those of hepatic enzyme. Ferrochelatase activity in mouse erythroleukemia (MEL) cells increased in parallel with an increase of heme synthesis by treatment with dimethylsulfoxide. Using immunoblotting techniques, the amount of the enzyme in the MEL cells has been shown to increase by the induction, showing a molecular mass of 41 kDa which was the same as that of the mouse hepatic enzyme. Comparative structural analysis of the enzyme of MEL cells and that of mouse liver by peptide mapping showed that the partial digestive peptides of both enzymes exhibited a similar pattern. These results strongly suggest that ferrochelatase in kidney, liver and erythroid cells can be of one type.
Nakahashi
Y
Y
Third Department of Internal Medicine, Kansai Medical University, Osaka, Japan.
Taketani
S
S
Sameshima
Y
Y
Tokunaga
R
R
D000818:Animals; D002417:Cattle; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005294:Ferrochelatase / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D007501:Iron / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008190:Lyases / Q000378:metabolism*; D008652:Mesoporphyrins / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D011524:Protoporphyrins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D008652:Mesoporphyrins; D011524:Protoporphyrins; C031468:mesoporphyrin IX; C028025:protoporphyrin IX; D007501:Iron; D008190:Lyases; D005294:Ferrochelatase; D004121:Dimethyl Sulfoxide