Endogenous phosphoinositide precursors of inositol phosphates in rat brain cortical membranes.
The appearance of Ins1P as an index of direct PtdIns breakdown by phospholipase C was examined in rat brain cortical membranes using either exogenous [3H]PtdIns substrate or [3H]inositol-prelabeled endogenous phosphoinositide substrates. Production of [3H]Ins1P was observed using exogenous [3H]PtdIns but not with endogenous substrate over the physiological range of calcium concentrations. [3H]Ins1,4P2 and [3H]Ins4P, derived from phospholipase C breakdown of polyphosphoinositides, were formed by membranes from both exogenous [3H]PtdIns and endogenous 3H-phosphoinositides, in the presence of ATP. The contribution of endogenous PtdInsP2 and PtdInsP to the generation of inositol phosphates was examined in membranes from [3H]inositol-prelabeled brain slices by adding unlabeled Ins1,4,5P3 to trap [3H]Ins1,4,5P3 generated upon breakdown of [3H]PtdInsP2. The maximal rate of [3H]Ins1,4,5P3 appearance was attained in the presence of 150-200 microM added Ins1,4,5P3 and represented 12.5% of the combined rates of formation of [3H]Ins1,4,5P3 and [3H]Ins1,4P2, similar to the content of [3H]PtdInsP2 relative to total 3H-polyphosphoinositides. The results show that, while endogenous PtdIns is not degraded by phospholipase C, the enzyme appears to be equally effective to cleave endogenous PtdInsP2 and PtdInsP.
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Autónoma de Barcelona, Bellaterra, Spain.
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Claro
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Sarri
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Picatoste
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D066298:In Vitro Techniques; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D014316:Tritium; D007294:Inositol; D010738:Type C Phospholipases; D002118:Calcium