Characterization of human spermidine/spermine N1-acetyltransferase purified from cultured melanoma cells

Abstract

Extreme inducibility of spermidine/spermine acetyltransferase (SSAT) by bis-ethyl derivatives of spermine in human large cell lung carcinoma and melanoma cells has prompted biochemical characterization of the purified enzyme. Treatment of human MALME-3 melanoma cells with 10 μ m N 1,N 11 -bis(ethyl)norspermine (BENSPM) for 48–72 h increased SSAT activity by some 1000- to 4000-fold and enabled purification of the enzyme by established procedures—binding on immobilized spermine and elution with spermine followed by binding on Matrex Blue A and elution with coenzyme A. The enzyme showed a single band by sodium dodecyl sulfatepolyacrylamide gel electrophoresis with a single subunit species and molecular weight of approximately 20,300 Da. By gel permeation chromatography, the holoenzyme was found to have a molecular weight of 80,000 Da, suggesting a total of four identical subunits. Purified SSAT had a specific activity of 285 μmol/min/mg for spermidine and K m values of 5.9 μ m for acetylcoenzyme A, 55 μ m for spermidine, 5 μ m for spermine, 36 μ m for N 1-acetylspermine, 1.6 μ m for norspermidine, and 4 μ m for norspermine. Homologs of BENSPM were found to be competitive inhibitors of spermidine acetylation, with K i values of 0.8 μ m for BENSPM, 1.9 μ m for N 1,N 12 -bis-(ethyl)spermine and 17 μ m for N 1,N 14 -bis-(ethyl)-homospermine. Correlation of these values with the relative abilities of the homologs to increase SSAT in intact cells suggests that formation of an enzyme inhibitor complex may play a contributing role in enzyme induction.