Physiological functions of polyamines and its transport

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1.Spermidine regulation of protein synthesis (stimulation at low concentration and inhibition at high concentration) was studied in a rabbit reticulocyte cell-free system. When the 5'-untranslated region (5'-UTR) of mRNA contained a continuous GC stem region, the synthesis of ornithine decarboxylase, S-adenosylmethionine decarboxylase and beta-actin was strongly stimulated by low spermidine concentrations and greatly inhibited by high spermidine concentrations. Spermidine regulation of protein synthesis reflected the initiation complex formation of Met-tRNA_i, mRNA and ribosomes. However, the binding of mRNA to ribosomes in the presence and absence of Met-tRNAi or the binding of Met-tRNA_i to ribosomes in the absence of mRNA was not significantly influenced by spermidine. The results suggest that the binding sites of mRNA and Met-tRNA_i to ribosomes partially overlap, and spermidine regulates the simultaneous binding of mRNA and Met-tRNA_i to ribosomes through the configurational chang … More e of RNA by spermidine.2.We recently obtained and characterized three clones of polyamine transport genes in Escherichia coli. Two of them were spermidine-preferential and putrescine-specific uptake systems, respectively. Spermidine-preferential uptake system consisted of four kinds of proteins : periplasmic substrate-binding protein (potD) , membrane-associated ATP binding protein (potA) and two transmembrane proteins (potB and C). From activity measurement and cellular localization of mutated proteins obtained by site-directed mutagenesis, it was found that ATP binding domain and active center of ATPase activity were located in the NH_2-terminal of potA protein and COOH-terminal of the protein stimulated association between potA and potB,C transmembrane proteins. Spermidine binding site of potD protein was also determined by X-ray crystal analysis of the purified protein and activity measurement of mutated proteins. It was found that Glu36 and Glu171 were important for the recognition of primary amines and Asp257 was important for the recognition of secondary amine of spermidine. Less