Studies on the reductive cleavage of canavanine and canavaninosuccinic acid

Ammonium bicarbonate extracts (pH 9.0) of water-washed liver tissue particles served to mediate the reductive cleavage of canavanine and canavaninosuccinic acid (CSA) by reduced lipoate. The active fraction migrated to the anode during electrophoresis at pH 8.9 on a polyvinyl chloride block.The reactions were followed by the increase in absorbance for oxidized lipoate at 333 nm; by a ferricyanide-nitroprusside reagent or diacetyl reaction for guanidine; the ninhydrin reaction, after paper electrophoresis, for homoserine; and the ferricyanide-nitroprusside or Sakaguchi reaction for guanidino-succinic acid. EDTA, BAL, Co²⁺, cyanide ion, and transferrin acted as inhibitors for the reaction. Dithiothreitol could be substituted for the dihydrolipoate but resulted in a slower rate.The activity of the liver extracts could be duplicated by ferrous ion. This was demonstrated by studying the course of the reaction in order to compare rate constants and Michaelis constants. These constants were essentially the same for Fe²⁺ and the liver extract. Optimum pH was the same for both systems. It is postulated that the active liver extract is an iron-carrying high-molecular weight substance which is probably a protein. This is based on its behavior on Sephadex, DEAE-cellulose, and in the electrophoretic field.