Purification and Properties of a Catechol‐O‐Methyltransferase of Human Liver

An S‐adenosylmethionine: catechol‐O‐methyltransferase occurs in the 105000 ×g supernatant of human liver. As compared with the homogenate, the enzyme was purified 380‐fold by adjusting the 24000 ×g supernatant to pH 5, fractionation with (NH4)2SO4, gel filtration on Sephadex G‐200 and G‐100 columns and ion‐exchange chromatography on CM‐Sephadex and DEAE‐cellulose columns. The purified enzyme preparation was homogeneous when subjected to further chromatography on Sephadex G‐100 and DEAE‐cellulose, disc electrophoresis on polyacrylamide gel at pH 9.5 and analytical ultracentrifugation. The enzyme exhibited a s20, w of 3.6 S with a molecular weight of 29000. The final preparation of catechol‐O‐methyltransferase was very labile, but could be stabilised by the addition of EDTA and MgCl2 in equimolar concentrations. The activity of the enzyme was assayed in the presence of cysteine and MgCl2 with 2‐hydroxy‐oestradiol‐17β and radioactive S‐adenosylmethionine as substrates. The metabolites of 2‐hydroxyoestradiol‐17β formed during incubation were identical with 2‐methoxyoestradiol‐17β and 2‐hydroxyoestradiol‐17β 3‐methyl ether. Throughout the purification procedure, the ratio of the 2‐ and 3‐methyl ethers, respectively, remained constant at 2.1.