title: Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase.
abstract: In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N(1) -acetyl-transferase (SSAT-1) and then oxidized by polyamine oxidase to produce spermidine and putrescine respectively. Herein we apply homology-search methods to identify novel sequences belonging to a second SSAT, SSAT-2, with a chromosomal location at 17p13.1, which is distinct from SSAT-1 at Xp22. Human SSAT-2 cDNA derived from small-cell lung carcinoma was deduced to encode a 170-amino-acid protein having 46% sequence identity and 64% sequence similarity with SSAT-1. When transiently transfected into HEK-293 cells, SSAT-1 decreased spermidine and spermine pools by approximately 30%, while, at the same time, significantly increasing putrescine, N (1)-acetylspermidine, N (1)-acetylspermine and N (1), N (12)-diacetylspermine pools. By contrast, transfected SSAT-2 had no effect on intracellular polyamine or acetylated polyamine pools. When enzyme activity was assayed on enzyme extracts from transfected cells, both SSAT-1 and SSAT-2 demonstrated much higher acetylating activity than vector-transfected cells. The data suggest that, in intact cells, SSAT-2 may be compartmentalized or it may be inefficient at low intracellular polyamine concentrations. By substituting candidate substrates in the enzyme assay, we determined that SSAT-1 shows the substrate preference norspermidine=spermidine>>spermine> N (1)-acetylspermine>putrescine, whereas SSAT-2 shows the preference norspermidine>spermidine=spermine>> N (1)-acetylspermine=putrescine. Analysis of mRNA levels in cell lines and ESTs (expressed sequence tags) from various tissues by digiNorthern (a web-based tool for virtually displaying expression profiles of query genes based on EST sequences) indicated that SSAT-1 tends to be more widely and highly expressed than SSAT-2. While SSAT-1 mRNA was inducible by polyamine analogues in a variety of cell lines, SSAT-2 was not. The existence of an active, but possibly sequestered, SSAT-2 enzyme suggests that, under certain conditions, it may be recruited into basal or perturbed polyamine metabolism.
authors.affiliation: Grace Cancer Drug Center and Department of Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Slavoljub
authors.initials: S
authors.lastname: Vujcic
authors.forename: Ping
authors.initials: P
authors.lastname: Liang
authors.forename: Paula
authors.initials: P
authors.lastname: Diegelman
authors.forename: Debora L
authors.initials: DL
authors.lastname: Kramer
authors.forename: Carl W
authors.initials: CW
authors.lastname: Porter
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D041321:Chromosomes, Human, X; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000099228:Diamine N-Acetyltransferase
chemical_list: D000123:Acetyltransferases; D018076:DNA, Complementary; D000099228:Diamine N-Acetyltransferase