The enzymatic conjugation of m-aminophenol.

It has been demonstrated by many workers (l-5) that phenols may be conjugated with glucuronic acid or inorganic sulfate in vitro by liver slices and by cell-free preparations. By using a system developed in this laboratory, together with a modification of the analytical procedure described by Levvy and Storey (3), the synthesis of the conjugated sulfate, and possibly of the glucuronide, has been accomplished in the presence of rat liver homogenates when m-aminophenol and potassium sulfate or glucuronate were present in the appropriate incubation mixture. The work reported here deals primarily with the synthesis of m-aminophenylsulfuric acid. Synthetic Reaction-If m-aminophenol and potassium sulfate are incubated aerobically in the presence of a KC1 homogenate of rat liver in an appropriate incubation medium, the synthesis of m-aminophenylsulfuric acid, of the order of 150 rnpM per mg. of enzyme nitrogen, is observed. The amount of synthesis is dependent upon the concentration of the inorganic sulfate present in t,he incubation mixture, with saturation at about 0.01 M sulfate (Fig. 1). This aerobic synt.hesis with whole rat liver homogenate was studied extensively by varying the experimental conditions and the components of the medium. The detailed conditions for maximum synthesis are presented in the experimental section. The enzyme system has a broad pH optimum (6.5 to 7.4), and at least 0.001 M Mg++ is necessary. Location of Enzyme System-In order to determine the location of the enzyme system involved, the rat liver homogenate was separated into various fractions by differential centrifugation at 2000 X g for 10 minutes and 18,000 X g for 90 minutes, the successive fractions being termed the residue, high speed residue, and supernatant fluid, respectively. Each of the fractions was tested individually and in various combinations for its synthetic ability (Fig. 2). None of the fractions are active alone. Recombination of the low speed