The effects of a potent spermidine synthase inhibitor, trans-4-methylcyclohexylamine (4MCHA), and a spermine synthase inhibitor, N-(3-aminopropyl)cyclohexylamine (APCHA), on polyamine biosynthesis and cell growth have been studied in rat hepatoma cells (HTC cells) in culture. Treatment of HTC cells with 4MCHA or APCHA caused a marked decrease of spermidine or spermine with a compensatory increase of putrescine and spermine or spermidine, respectively, in a dose-dependent manner, suggesting specific and potent inhibition of each target enzyme. When 250 microM 4MCHA or APCHA was administered to the cells for 8 days, spermidine was decreased to 2% of control culture or spermine below 1%, respectively, while total polyamine (sum of putrescine, spermidine, and spermine) remained almost unchanged during the culture. There were no significant changes in the growth rate during treatment with the inhibitors at 250 microM concentration. The results suggest that in the growth of HTC cells, putrescine and spermine can be substituted for most of the fraction of cellular spermidine, and spermidine for most of the fraction of cellular spermine. Of five enzymatic activities involved in polyamine biosynthesis and interconversion, S-adenosylmethionine decarboxylase activity increased 8-fold with 250 microM 4MCHA, and 3-fold with 250 microM APCHA during the treatment. This increase was partially due to the increase of half-life of the enzyme. Separate roles for spermidine and spermine in the biosynthesis of the enzyme protein were also suggested.
Spermidine synthase (spd syn, putrescine aminopropyltransferase) has been shown to catalyze the transfer reaction of the aminopropyl moiety of decarboxylated S-adenosyl-Lmethionine (dcAdoMet) into putrescine in the formation of spermidine, which is widely distributed among living organisms with increasing levels related to cell growth. [1][2][3] In addition to the reported amino acid sequence of spd syn from various sources, 4,5) the crystal structures of some of these bacterial enzymes have been recently reported 6) (PDB accession codes 1INL, 1JQ3, 1MJF, 1IY9, and 1UIR). One of the significant differences between the amino acid sequence of mammalian and bacterial enzymes is the content of cysteine residue, i.e. ten residues for mammalian enzymes, three for Thermotoga maritima, and none for Bacillus subtilis and Pyrococcus furiosus. Accordingly, the determination of each cysteine residue as either sulfhydryl or disulfide is essential for the accurate homology modeling of mammalian spd syn. The primary sequence of rat spd syn is shown in Fig. 1. 7)The use of specific inhibitors is another approach in the study of the substrate-binding site. Using a number of monoamine and diamine compounds, we have proposed a model of the putrescine-binding site of pig spd syn, 8) which features a relatively large hydrophobic cavity adjacent to a negatively charged site. Presumably, one of the amino group of putrescine is protonated and binds to this charged site, and the other amino group is not protonated and binds to the hydrophobic cavity, to be aminopropylated by dcAdoMet. The substrate-binding site of our model shows good agreement with that of the crystal structure of the T. maritima enzyme.6)The present study was undertaken to identify the ten cysteine residues of rat spd syn exist as sulfhydryl or disulfide, and to investigate the putrescine-binding site using several compounds that are based on two known potent inhibitors for mammalian spd syn, trans-4-methylcyclohexylamine (4MCHA) and n-butylamine (BA). Based on the results, a three-dimensional model for mammalian spd syn was proposed using the homology modeling approach based on the crystal structure of the T. maritima enzyme. MATERIALS AND METHODSChemicals Dithiothreitol (DTT), guanidine hydrochloride, monoiodoacetic acid (MIA), trans-4-hydroxycyclohexyl amine (4HCHA), 5-[N-(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (IAEDANS), 2-nitro-5-thiocyanobenzoic acid (NTCB), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma. Decarboxylated S-adenosylmethionine (dcAdoMet) was prepared in our laboratories. 9) N-Hydroxysuccinimidyl bromoacetate (HSBA) was also prepared in our laboratories using bromoacetic acid, N-hydroxysuccinimide, and dicyclohexylcarbodiimide. n-Propylamine, n-butylamine (BA), n-pentyl amine (PA), n-hexylamine (HA), and cyclohexylamine (CHA) were purchased from Tokyo Kasei Kogyo Co. Ltd., and were recrystallized as their hydrochlorides. 4-Methylcyclohexylamine (as a mixture of cis-and trans-isomers) was purchased fro...
Two unusual aminopropyl acceptors found in a survey of putrescine binding sites of mammalian spermidine synthase, N-methylputrescine (I) and 4-aminomethylpiperidine (II), were examined for their aminopropyl derivatives. Studies under in vitro incubation conditions suggested that the aminopropyl derivatives of the secondary amine of I and II, N4-methylspermidine (Is) and 1-N-(3-aminopropyl)-4-aminomethylpiperidine (IIs), and of the primary amine of I and II, N8-methylspermidine (Ip) and 4-[N-(3-aminopropyl)aminomethyl]piperidine (IIp), respectively, were biosynthesized by rat spermidine synthase. Studies on the cell culture system of cultured rat hepatoma (HTC) cells treated with alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor, clearly showed the presence of Is and Ip when I was administered, and IIs and IIp when II was administered, with no detection of putrescine or spermidine. These results suggested that mammalian spermidine synthase can transfer the aminopropyl moiety of decarboxylated S-adenosylmethionine to certain secondary amines in living cells.
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