Abstract
A transport system for polyamines was studied with both intact cells and membrane vesicles of an Escherichia coli polyamine-deficient mutant. Polyamine uptake by intact cells and membrane vesicles was inhibited by various protonophores, and polyamines accumulated in membrane vesicles when D-lactate was added as an energy source or when a membrane potential was imposed artificially by the addition of valinomycin to K+-loaded vesicles. These results show that the uptake was dependent on proton motive force. Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m-chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. However, they were excreted by membrane vesicles, although the degree of spermidine efflux was much lower than that of putrescine efflux. These results suggest that the apparent unidirectionality in intact cells has arisen from polyamine binding to nucleic acids, thus giving rise to a negligible free intracellular concentration of polyamines. Polyamine uptake, especially putrescine uptake, was inhibited strongly by monovalent cations. The Mg2+ ion inhibited spermidine and spermine uptake but not putrescine uptake.
Highlights
Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m-chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. They were excreted by membrane vesicles, the degree of spermidine efflux was much lower than that of putrescine efflux. These results suggest that the apparent unidirectionality in intact cells has arisen from polyamine binding to nucleic acids, giving rise to a negligible free intracellular concentration of polyamines
An energydependent polyamine uptake system has been shown in Escherichia coli B [20], and two transport systems for putrescine have been demonstrated in E. coli K-12 grown in a low-osmolarity medium [16]
Polyamine uptake in intact cells and membrane vesicles was inhibited by various protonophores, and polyamines accumulated in membrane vesicles when D-lactate was added as an energy source or when a membrane potential was imposed artifically by the addition of valinomycin to K+-loaded vesicles
Summary
Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m-chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. They were excreted by membrane vesicles, the degree of spermidine efflux was much lower than that of putrescine efflux. Since the E. coli cells used in the studies described above can synthesize polyamines, there is a possibility that such polyamines, especially putrescine, may be excreted from the cells during the uptake of polyamines added to the medium [16, 20] If this occurs, an exact analysis of polyamine transport would be difficult. Polyamine transport was apparently unidirectional, and the uptake was dependent on proton motive force
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