Abstract
Myo-inositol is an important constituent of membrane phospholipids and is a precursor for the phosphoinositide signaling pathway. It is synthesized from glucose 6-phosphate by myo-inositol-3-phosphate synthase (IP synthase), a homotrimer composed of a 68-kDa polypeptide in most mammalian tissues. It is a putative target for mood-stabilizing drugs such as lithium and valproate. Here, we show that the rat gene (Isyna1) encoding this enzyme generates a number of alternatively spliced transcripts in addition to the fully spliced form that encodes the 68-kDa subunit (the alpha isoform). Specifically, we identify a small 16-kDa subunit (the gamma(c) isoform) derived by an intron retention mechanism and provide evidence for its existence in rat tissues. The gamma(c) isoform is highly conserved in mammals, but it lacks the catalytic domain while retaining the NAD(+) binding domain. Both alpha and gamma(c) isoforms are predominantly expressed in many rat tissues and display apparent stoichiometry in purified enzyme preparations. An IP synthase polyclonal antibody not only detects the alpha and gamma(c) isoforms but also several other isoforms in pancreas, intestine, and testis suggesting that the holoenzyme is composed of unique subunits in various tissues. Interestingly, the alpha isoform is not expressed in the intestine. IP synthase activity assays using purified alpha and gamma(c) isoforms indicate that the latter negatively modulates alpha isoform activity, possibly by competing for NAD(+) molecules. Our findings have important ramifications for understanding the mood stabilization process and suggest that inositol biosynthesis is a highly regulated and dynamic process.
Highlights
Inositol, a ubiquitous six-carbon cyclic sugar, exists in several stereoisomeric forms, of which the most active physiological form is myo-inositol [1]
It is synthesized from glucose 6-phosphate by myoinositol-3-phosphate synthase (IP synthase), a homotrimer composed of a 68-kDa polypeptide in most mammalian tissues
A, translation for ␥n isoform is initiated in intron 4 at the first methionine shown, generating a 22-amino acid novel N-terminal peptide for rat and mouse that is in-frame with the inositol phosphates (IPs) synthase polypeptide encoded by exons 5–11
Summary
Materials—Unless specified, reagents were of analytical grade procured from established manufacturers. The pellet obtained after centrifugation was dissolved in 50 mM Tris-HCl, pH 7.0 buffer and loaded onto a 16/10 HiTrap phenyl-Sepharose column (Amersham Biosciences) equilibrated with 25% ammonium sulfate in 50 mM Tris-HCl, pH 7.0, containing 0.2 mM dithiothreitol. 5-ml fractions were collected, and IP synthase-containing fractions were identified by Western blot analysis using the R-10 antibody (see below) These fractions were pooled, dialyzed against 50 mM Tris acetate, pH 7.4, buffer and passed over a Q-Sepharose column equilibrated with Tris acetate buffer. Peak II fractions were pooled and concentrated by ammonium sulfate precipitation and dialyzed in Tris acetate buffer These fractions were used for IP synthase activity determinations.
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