Function and structure of rat hepatic coproporphyrinogen oxidase.
Rat hepatic coproporphyrinogen oxidase, the sixth enzyme in the heme biosynthetic pathway, was purified 1340-fold with a yield of 39.7%. To obtain the soluble enzyme, different methods were applied to disrupt mitochondria, with sonication giving the highest yield (85%). The minimum catalytic form of enzyme was a dimer with a molecular mass of 77 +/- 4 kDa. The existence of aggregated forms was possible since in fractions of gel filtration elution activity was observed with higher molecular mass. We determined a Stokes radius of 36.3 A, a sedimentation coefficient (S20,w) of 5.06 S, and frictional ratio of 1.29, suggesting a nearly globular shape of the protein. Regardless of the type of salt, high ionic strength inhibits the enzyme, probably modifying its native structure. Experiments with amino acid modifiers showed that histidine, arginine, and tryptophan are involved in the catalytic process. Non-ionic detergents and phospholipids activated the enzyme, probably because they reproduce its natural hydrophobic environment. The present study describes a simple method for the purification of rat liver coproporphyrinogen oxidase, introducing for the first time data on the structure and function of the protein in a tissue often used as a laboratory model in biological studies, and contributing to the study of human hereditary coproporphyria.
Sorianello
E M
EM
Departamento tie Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Argentina.
Mazzetti
M B
MB
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003304:Coproporphyrinogen Oxidase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D003902:Detergents / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology*; D017830:Octoxynol / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011136:Polysorbates / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014867:Water / Q000378:metabolism
D003902:Detergents; D010743:Phospholipids; D011136:Polysorbates; D014867:Water; D017830:Octoxynol; D003304:Coproporphyrinogen Oxidase; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal