RESUMO
BACKGROUND: Altered levels of inositol phosphate in the central nervous system (CNS) are hypothesized to produce distorted brain signaling and lead to numerous neurologic maladies. Little is known of mechanisms controlling the complex metabolic flux of inositol phosphate. Less is known of controls that regulate inositol-phosphate biosynthesis in the mammalian brain. The expression of 1L-myo-inositol-1 phosphate synthase (MIP), the only enzyme known to synthesize inositol phosphate, was studied in the brain of normal (CBA) and curly tail (CT) mutant mice. The CT strain exhibits a neural tube defect, spina bifida, responsive to inositol supplementation, but not to folic acid treatment. METHODS: Utilizing enzyme assays to determine the specific activity of MIP, Western blotting to detect expression, gas chromatography/mass spectrometry to measure inositol concentration, and statistical analyses to evaluate quantitative data, MIP expression was analyzed in newborn, young, and adult brains of CBA and CT (curly tail [ct-CT] and straight tail [st-CT]) mutant mice. RESULTS: Data analyses suggest there is a significant difference in MIP activity in the brain of CBA mice as compared to that of CT mutant mice and that temporal and spatial control of MIP expression and inositol concentrations are altered in the brain of both the ct-CT and phenotypically normal st-CT mutant. Moreover, two differentially expressed forms of MIP were identified in the adult mouse brain. CONCLUSIONS: These findings implicate a role for MIP in the maturation of the CNS and evoke a hypothesis regarding the regulation of inositol phosphate biosynthesis in brain development.
Assuntos
Encéfalo/crescimento & desenvolvimento , Fosfatos de Inositol/biossíntese , Animais , Animais Recém-Nascidos , Western Blotting , Encéfalo/enzimologia , Encéfalo/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Inositol/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Camundongos Mutantes , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fenótipo , Especificidade da EspécieRESUMO
We have studied the expression of 1L-myoinositol-1-phosphate synthase (MIPS; EC 5.5.1.4) in developing organs of Phaseolus vulgaris to define genetic controls that spatially regulate inositol phosphate biosynthesis. MIPS, the pivotal biosynthetic enzyme in inositol metabolism, is the only enzyme known to catalyze the conversion of glucose 6-phosphate to inositol phosphate. It is found in unicellular and multicellular eukaryotes and has been isolated as a soluble enzyme from both. Thus, it is widely accepted that inositol phosphate biosynthesis is largely restricted to the cytosol. Here, we report findings that suggest the enzyme is also expressed in membrane-bound organelles. Microscopic and biochemical analyses detected MIPS expression in plasma membranes, plastids, mitochondria, endoplasmic reticula, nuclei, and cell walls of bean. To address mechanisms by which the enzyme could be targeted to or through membranes, MIPS genes were analyzed for sorting signals within primary structures and upstream open reading frames that we discovered through our sequence analyses. Comprehensive computer analyses revealed putative transit peptides that are predicted to target the enzyme to different cellular compartments. Reverse transcriptase PCR experiments suggest that these putative targeting peptides are expressed in bean roots and leaves.