Purification and characterization of spermidine N¹‐acetyltransferase from chick duodenum

Abstract

We have reported that spermidine N' -acetyltransferase has a larger role than ornithine decarboxylase in putrescine synthesis in chick duodenum induced by 1 a,25-dihydroxycholecalciferol (calcitriol) [Shinki, T., Kadofuku, T., Sato, T. and Suda, T. (1986) J. Biol. Chem. 261, In the present study, spermidine N'-acetyltransferase was purified from the duodenal cytosol of calcitriol-treated chicks to homogeneity judged by SDS/polyacrylamide gel electrophoresis. The purified enzyme converted spermidine only to N'-acetylspermidine. The apparent molecular mass of the purified spermidine N'-acetyltransferase was found to be 36 kDa by gel filtration on Sephacryl S-200 and 18 kDa by SDS/polyacrylamide gel electrophoresis. When duodenal crude 105000 x g extracts were directly applied to a Sephacryl S-200 column without prior purification, three peaks with spermidine N'-acetyltransferase activity appeared. The first peak was in the void volume, the second peak was in the fraction corresponding to an apparent molecular mass of 70 kDa, and the third peak was in the fraction corresponding to 36 kDa. These results suggest that spermidine N'-acetyltransferase exists as a dimer of the 38 kDa subunits and is stabilized in (a) form(s) bound to other components or proteins in intact cells.Recently, a number of reports have indicated the presence of enzymes acetylating polyamines and histones [l -101. Some of them catalyze the acetylation reaction of either polyamines or histones, but some can catalyse both. Thus, intact cells and crude cell preparations contain a mixture of enzymes capable of catalyzing the formation of acetylated spermidine and spermine using acetylcoenzyme A as a substrate. Of these, enzymes acetylating both polyamines and histones form N8-acetylspermidine, which are not native metabolites in the interconversion of polyamines. Only spermidine/spermine N'-acetyltransferase is considered to play an important role in the regulation of intracellular concentrations of polyamines [4,5,8, 111. This enzyme catalyzes the rate-limiting step of the interconversion of polyamines to form N'-acetylspermidine and N'-acetylspermine, which are further converted to putrescine and spermidine, respectively, by polyamine oxidase [12, 131. Calcitriol, the most potent metabolite of calciol, is now considered to be a hormone which mediates calcium transport in target tissues such as intestine and bone [14-161.

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma

“…The inability to precipitate as much of the SSAT activity as reported for the rat protein (Persson & Pegg, 1984) may be due to comparatively poorer recognition of the human protein by the anti-(rat SSAT) antiserum. However, it is clear from Western-blot and silver-stain analysis that the human SSAT has an Mr of -20000, similar to the Mr of -18000 previously observed for the purified rat and chicken proteins (Persson & Pegg, 1984;Shinki & Suda, 1989).…”

Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli

“…It has been suggested that the very high level of induction of SSAT in mammalian cells by BESM and related compounds is related to the inhibition of growth by these polyamine analogs. This is supported by a correlation between the extent of SSAT induction and growth inhibition in a series of tumor cell lines (Casero & Pegg, 1993;Casero et al, 1989;Saab et al, 1993;Shappell et al, 1993;Libby et al, 1989; Della Ragione & Pegg, 1982; Shinki & Suda, 1989). However, other experiments suggest that the analogs are themselves antiproliferative, possibly by virtue of their abilities to interact with DNA (Basu et al, 1989(Basu et al, , 1993Albanese et al, 1993;Fukuchi et al, 1992).…”

“…SSAT has been purified to homogeneity from rat liver (Della Ragione & , chicken duodenum (Shinki & Suda, 1989), human lung H157 cells (Casero et al, 1990), and human melanoma cells (Libby et al, 1991) following induction with calcitrol, carbon tetrachloride, BESM, and BENSM, respectively. Only minute amounts of protein were obtained from these sources which were limited by either the low specific activity of the starting material or the difficulty of growing large numbers of these mammalian cells.…”

Biogenic-amine acetylation: an additional function of the N-acetyltransferase from Fasciola hepatica

“…The trematode acetylase is clearly different from the mammalian liver polyamine N-acetyltransferase and the isofunctional enzyme from the chicken duodenum (Della Ragione and Pegg, 1983;Shinki and Suda, 1989). Whereas these other acetylases catalysed only polyamine acetylation, the N-acetyltransferase from F. hepatica accepts a rather broad range of substrates.…”