The functional size of ferrochelatase determined in situ by radiation inactivation.
Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.
Straka
J G
JG
Department of Medicine, University of Minnesota Medical School, Minneapolis 55455.
Bloomer
J R
JR
Kempner
E S
ES
D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005294:Ferrochelatase / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D008930:Mitochondria, Liver / Q000201:enzymology*; D011487:Protein Conformation
D005294:Ferrochelatase