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(Received for publication, December 1, 1994; and in revised form, May 30,
1995) Long-chain (sphingoid) bases are highly bioactive intermediates
of sphingolipid metabolism, yet relatively little is known about how
the amounts of these compounds are regulated. This study used J774A.1
cells to characterize the ``burst'' of sphinganine and
sphingosine, or the transient increase of up to 10-fold in long-chain
base mass, that occurs when cells in culture are changed to fresh
medium. The increase in sphinganine was attributable to de novo sphingolipid biosynthesis because: 1) there is increased
incorporation of [
Volume 270,
Number 32,
Issue of August 11, pp. 18749-18758, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Burst
of Free Sphingosine and Sphinganine, and Their
1-Phosphates and N-Acyl-Derivatives, That Occurs upon Changing
the Medium of Cells in Culture
H]serine and
[H]palmitate into sphinganine; 2) the
incorporation of [H]serine was equivalent to the
increase in sphinganine mass; 3) 
-F-alanine, an inhibitor of
serine palmitoyltransferase, blocked the sphinganine burst; 4) the
magnitude of the burst depended on the concentration of serine in the
medium, which is known to affect long-chain base biosynthesis; and 5)
the appearance of sphinganine was relatively unaffected by
lyso-osmotrophic agents (NH
Cl and chloroquine) that blocked
sphingolipid hydrolysis in these cells. In contrast, the sphingosine
burst arose mainly from turnover of complex sphingolipids because no
incorporation of [H]serine or
[H]palmitate into sphingosine was detected;
sphingosine mass was not affected by -F-alanine or the serine
concentration; and, the burst could be followed by the release of
sphingosine and ceramide from complex sphingolipids (especially
sphingomyelin) in a process that was inhibited by NHCl and
chloroquine. Additionally, the fate of these long-chain bases differed:
sphinganine was mostly (80-85%) acylated and incorporated into
dihydroceramide and complex sphingolipids, whereas most of the
sphingosine (70%) was phosphorylated and degraded, with incorporation
of the resulting ethanolamine phosphate into phosphatidylethanolamine.
Sphinganine, however, could be diverted toward degradation by adding an
inhibitor of N-acylation (fumonisin B
). In
accounting for the elevation in sphingosine and sphinganine after cells
are changed to new medium, these studies have provided fundamental
information about long-chain base metabolism. The existence of
differential changes in sphinganine and sphingosine, as well as their
1-phosphates and N-acyl-derivatives, should be considered when
evaluating the roles of sphingolipid metabolites in cell regulation.
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