Abstract
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.
Highlights
Ferrochelatase (EC 4.99.1.1) catalyzes the final step SDS’-polyacrylamide gel electrophoresis [5,6,7] and gel filof heme biosynthesis, the insertionof iron(I1) into pro- tration chromatography in the presence of 2 1 5 mM sodium toporphyrin, It is an integral protein of the inner mi- cholate [6,7] have yielded estimates forbovine ferrochelatase tochondrial membrane
Pre- acts witha polypeptide, a largeamount of energy is deposited, vious studies using sodium dodecyl sulfate-polyacryl- covalent bonds within thepolymer are broken, and the “hit”
The present study applies the techniqueof radiation inactivation and target size analysis to evaluate the functional size of ferrochelatase as it exists within the mitochondrial
Summary
Vol 266, No 36, Issue of December 25, pp. 2P4r6i3n7te-2d4i6n41U, .1S9.9A1. The FunctionalSize of Ferrochelatase Determinedin Situ by Radiation Inactivation*. The functional unit is likely that the larger sizes are artifacts of low detergent required for enzymic activity in intact mitochondria concentration [7].when titratewd ith inhibitory was found to havea mass of 82 f 13 kDa. In contrast, sulfhydryl reagents, there appears bteo one sulfhydry1/40,000 the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 f 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. This enzyme, which is located in or at the PAGE, estimates of the size of both the functional and strucinner face of the inner mitochondrial membrane, catalyzes tural unit were obtained. The human disease is inherited as an autosomal dominant trait, whereas in cattle it is autosomalrecessive [3, 4] Ferrochelatase from both normal and protoporphyric bovine liver has been isolated and partially characterized [5,6,7].
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