Characterization of human spermidine/spermine N1-acetyltransferase purified from cultured melanoma cells.
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 microM N1,N11-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 sulfate-polyacrylamide 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 mumol/min/mg for spermidine and Km values of 5.9 microM for acetylcoenzyme A, 55 microM for spermidine, 5 microM for spermine, 36 microM for N1-acetylspermine, 1.6 microM for norspermidine, and 4 microM for norspermine. Homologs of BENSPM were found to be competitive inhibitors of spermidine acetylation, with Ki values of 0.8 microM for BENSPM, 1.9 microM for N1,N12-bis-(ethyl)spermine and 17 microM for N1,N14-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.
Libby
P R
PR
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263.
Ganis
B
B
Bergeron
R J
RJ
Porter
C W
CW
D000123:Acetyltransferases / Q000032:analysis*; D003065:Coenzyme A / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008545:Melanoma / Q000188:drug therapy / Q000201:enzymology*; D008970:Molecular Weight; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D003065:Coenzyme A; D013095:Spermidine; D013096:Spermine; D000099228:Diamine N-Acetyltransferase