Purification and properties of coproporphyrinogenase

Abstract

1. Coproporphyrinogenase has been prepared from rat-liver mitochondria and its properties have been studied. The isoelectric point was found to be around pH15.0 and the molecular weight to be 80000 + 8000. The pH optimum of the enzymic reaction was 7-4 and the apparent Km was of the order 0 03mM. The enzyme was destroyed by boiling and irreversible inactivation occurred below pH3.5. It could be stored at -10°without loss of activity. The enzyme acts specifically on coproporphyrinogen III and does not form protoporphyrinogen from trans-2,4-diacrylicdeuteroporphyrin or its porphyrinogen. It was unaffected by prolonged dialysis and no cofactor requirement could be demonstrated. 2. Column chromatography of a partially purified enzyme preparation on Sephadex G-200 was found to be an improved method of purification, which gave a coproporphyrinogenase 58-fold purified. The purified enzyme was studied electrophoretically but no evidence was obtained to suggest that more than one enzyme was involved in the reaction. 3. The action was studied of various compounds added to the system. The presence of monothiol groups in the enzyme system was indicated, whereas vicinal dithiol groups were not involved in the reaction. Metalchelating agents did not inhibit the reaction and no requirement for the presence of any essential metal has been found. All attempts to demonstrate the presence of a prosthetic group, in particular flavines, failed. Neither pyridoxal phosphate nor ATP was involved in the reaction, nor was a mitochondrial electron-transport chain required for the activity of the enzyme. Some circumstantial evidence was obtained to suggest that ci8-2,4-diacryliedeuteroporphyrin is an intermediate in the reaction.

Use of Cross-linking to Assess Subunit Interaction of Recombinant Human Coproporphyrinogen Oxidase

“…These data also correlate with the report of Medlock and Dailey that human-cloned copro'gen oxidase has an apparent monomeric molecular weight of 37.5 kDa by SDS-PAGE. The findings of Martasek et al [9] showing an active tetramer form of copro'gen oxidase are supported by the apparent molecular weight bands migrating as 86 and 107 kDa, indicating that in [2] S. cerevisiae ~ 35 kDa-Monomer ~70-75 kDa-Dimer Homodimer X-ray Crystallography Philips et al [10] Ultrogel AcA 44 Gel Filtration Camadro et al [3] 75 kDa Monomer Sephadex Gel Filtration Poulson and Polglase [4] Mouse Liver 35kDa-Monomer 70 kDa-Dimer Homodimer Ultrogel AcA 44 Gel Filtration Bogard et al [5] Bovine Liver 71.6 kDa Monomer Sephadex Gel Filtration Yoshinaga and Sano [6] 37 kDa-Monomer 74 kDa-Dimer Homodimer SDS-PAGE Gel Filtration Kohno et al [7] Human 37.5 kDa SDS-PAGE Medlock and Dailey [8] 76 kDa-Dimer 39.7-Monomer Homodimer Ultrogel AcA 44 Gel Filtration Martasek et al [9] Fig. 2: Superimposed representative HPLC chromatograms following enzymatic assay.…”

“…Current literature supports a dimeric active form of mammalian copro'gen oxidase, although there have been some discrepancies in the past. Batlle et al [2] purified copro'gen oxidase from rat liver and determined it to be an 80 kDa monomer. Copro'gen oxidase purified from S. cerevisiae was shown to be a homodimer by Camadro et al [3] , whereas Poulson and Polglase [4] determined the protein from the same species to be a monomer.…”

Coproporphyrinogenase activities in extracts of Rhodopseudomonas spheroides

“…The enzyme responsible for the aerobic reaction seems similar to that described in liver mitochondria (Sano & Granick, 1961;Batlle, Benson &Rimington, 1965) where O2 is the hydrogen acceptor, and no cofactors are required. The results suggest that in the anaerobic reaction a flavoprotein and iron, either as a component of the flavoprotein or as a non-haem-iron protein, are involved in the removal of hydrogen from coproporphyrinogen, and that NAD(P)+ is the final hydrogen acceptor.…”

Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, δ-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts

“…It was of interest to see whether Mg-Proto (Me) formation from ALA was inhibited under anaerobic conditions because coproporphyrinogenase has been reported to require 02 (2,17,20), while Mg-Proto (Me) accumulation has been observed in plants grown in O2-poor environments (21). No effect of anaerobiosis was detected on the conversion ofALA to Mg-Proto (Me) when the reaction vessel was flushed with N2 for 5 min before the substrate was added through a serum cap with a hypodermic syringe.…”