Studies on porphyrinogen carboxy-lyase from chick embryo liver.
The purpose of the present work was to find the optimal conditions for the assay of chick embryo liver porphyrinogen carboxy-lyase. The enzyme activity was studied as a function of protein and substrate concentrations, time, pH value and incubation temperature. The effects of reduced glutathione (GSH), ethylenediaminetetra-acetate (EDTA), oxygen and several chemical compounds such as pyridoxal phosphate, sodium and potassium halogenated salts, sulph - hydryl reagents, chelating agents and ferrous iron were also investigated. The following results were found: (1) The optimal protein concentration was 1.25-2.5 mg/ml. For the substrate uroporphyrinogen III, the best concentration was 2 mumol/l; a slight inhibition was found at higher substrate concentrations. (2) The optimal pH value was 6.8 for both stages of uroporphyrinogen III decarboxylation ( octacarboxylic first stage----heptacarboxylic second stage---- tetracarboxylic porphyrinogen). (3) The activity increased with the incubation temperature (25-60 degrees C). (4) The enzyme activity was not enhanced by the addition of GSH or other sulph - hydryl reagents (cysteine and dithiothreitol), nor by the incorporation of EDTA or other chelating agents (Na-diethyldithiocarbamate, alpha,alpha'-bipyridyl and Na-pyrophosphate). (5) Oxygen diminished the second stage of decarboxylation. (6) Pyridoxal phosphate seems not to be a cofactor necessary in the decarboxylation. (7) NaCl or KCl diminished the activity in higher degree than did NaF or KF; the second stage was, in all cases, more affected than the first. (8) FeSO4 slightly diminished the second stage of uroporphyrinogen III decarboxylation at concentrations of 0.05 and 0.15 mmol/l, but both stages were significantly decreased at 0.3 mmol/l.
D000818:Animals; D002262:Carboxy-Lyases / Q000032:analysis* / Q000378:metabolism; D002642:Chick Embryo; D004492:Edetic Acid / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D010100:Oxygen; D013696:Temperature
D004492:Edetic Acid; D002262:Carboxy-Lyases; C022430:porphyrinogen carboxy-lyase; D005978:Glutathione; D010100:Oxygen