Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P₃ affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P₃ affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P₃-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P₃- binding proteins but with no Ins(1,4,5)P₃ metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the δ-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P₃-recognizing proteins hitherto examined. Ins(1,4,5)P₃ at concentrations greater than 1 μM strongly inhibited 85-kDa phospholipase Cδ activity, without changing its dependence on the concentrations of free Ca²⁺ and H⁺. Among inositol phosphates examined, Ins(3,4,5,6)P₄ inhibited the binding of [³H]Ins(1,4,5)P₃ to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P₃. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P₃-binding proteins in the rat brain, one of which is the δ isozyme of phospholipase C.