| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Long chain fatty acyl-CoA is a metabolically active form of fatty acid, and various modes of enzymatic, conversion of fatty acids, including desaturation, chain elongation and oxidation, occur mainly in the form of fatty acyl-CoA in mammalian tissues. Another important role, of acyl-CoA is as an acyl donor in the formation of various types of simple aiid complex lipid molecules. In addition to its role as a metabolic intermediate, acyl-CoA is known to modulate various enzyme and cell functions. As for the biosynthesis of acyl-CoA, long chain acyl-CoA synthetases are known to catalyze the ligation reaction of free fatty acid and CoA with the consumption of ATP. The synthetase enzymes possess a conserved ATP-binding site and comprise a gene family.
Previously we found another acyl-CoA synthetic pathway, involving ATP-independent and coA-dependent synthesis of acyl-CoA in microsomal fractions of mammalian tissues. Substantial acyl-CoA was produced when microsomes were incubated with CoA in
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the absence of ATP-Mg^<2+>. We considered that the novel acyl-CoA synthetic activity may be due to the reverse reaction of acyl-CoA : lysophospholipid acyltransferases, because the generation of lysophospholipids, such as lysd phosphatidyicholine (LPC) and lysophosphatidylinositol (LPI), occurred. To clarify the hypothesis, we investigate d whether or not ATP-independent acyl-CoA formation catalyzed by recombinant lysophosphatidic acid (LPA) acyltransferase (LPAAf). The present results for LPAAT gene-transfected cells revealed a mechanism for ATP-independent acyl-CoA formation from phosphatidic acid (PA). Several lines of evidence indicate that this acyl-CoA synthetic route is due to the reverse reaction of LPAAT : This is the first direct and conclusive evidence for an acyl-CoA synthesizing enzyme other than acyl-CoA synthelase, and it also reveals a novel function for LPAAT.
The question of whether or not the hypothesis is applies to other acyl-CoA : lysophospholipid acyltransferases needs to be considered. We tried to purify LPI acyltranferase (LPIAT), one of the acyl-CoA : lysophospholipid acyltransferases, in order to examine whether LPIAT could operate in reverse to form acyl-CoA and LPI. During the purification, LPIAT was separated from other acyl-CoA acyltransferases. The purified LPIAT fraction exhibited ATP.-independent acyl-CoA synthetic activity and CoA-dcpendent LPI generation from PI, suggesting that LPIAT could operate in reverse to form acyl-CoA and LPI.
In conclusion, we have found an ATP-independent biosynthetic pathway for acyl-CoA, which is based on the reverse reaction of LPAAT and LPIAT which may be a prototype for other microsomal ATP-independent acyl-CoA synthetic activity from other phospholipids. Less
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