Purification and Characterization of Spermidine/Spermine N¹-Acetyltransferase from Rat Liver

An enzyme catalyzing the acetylation of the polyamines, spermidine or spermine, has been purified 112000-fold to homogeneity from livers of rats treated with carbon tetrachloride. Major purification steps involved affinity chromatography on sym-norspermidine-Sepharose, from which the enzyme was eluted by spermidine, and affinity chromatography on Cibacron blue agarose, from which the enzyme was released by coenzyme A. Similar final specific activities were obtained by using either method as a final purification step, providing strong evidence for homogeneity. The enzyme preparation gave a single band on Polyacrylamide gel electrophoresis carried out under native or denaturing conditions. It had an apparent molecular weight of about 115 000 made up of two subunits of 60000. The acetyltransferase acted on spermidine to form only N¹-acetylspermidine. Spermine was also a substrate, giving N¹-acetylspermine, and N¹-acetylspermine could be acetylated to form N¹,N¹²-diacetylspermine. The V_max values for spermidine, spermine, and N¹-acetylspermine were 8, 1.8, and 1.2 µmol of product min⁻¹ mg⁻¹, respectively, when assays were carried out in the presence of saturating concentrations of acetyl-CoA. The K_m for acetyl-CoA was 1.5 µM. and the for spermidine, spermine, and N¹-acetylspermine were 130 µM, 35 µM, and 30 µM, respectively. These results suggest that both spermidine and spermine would be physiological substrates for this enzyme. Coenzyme A was quite strongly inhibitory, having a K_i of 40 µM, but an even more powerful inhibitor could be produced by reacting coenzyme A with methyl methanethiosulfate to form coenzyme A methyl disulfide. The purified enzyme had no deacetylase activity and the reaction was effectively irreversible. The name spermidine/spermine N¹-acetyltransferase is suggested for this enzyme, which may play an important role in the interconversion of polyamines.