Inositol 1,4,5-trisphosphate binding to porcine tracheal smooth muscle aldolase

A cytoskeletal fraction of porcine tracheal smooth muscle (PTSM) was found to contain >90% of total cellular aldolase (fructose 1,6-bisphosphate aldolase, EC 4.1.2.13) activity. PTSM aldolase was purified by DEAE and inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) affinity chromatography and found to react with an antibody directed against human aldolase C, but not anti-aldolase A and B. The molecular mass of native aldolase was about 138 kDa (on Sephacryl S-300); SDS-denatured enzyme was 35 kDa (comigrated with rabbit skeletal muscle aldolase). Total cellular aldolase tetramer (aldolase₄) content was 34.5 pmol/100 nmol lipid P(i). Ins(1,4,5)P₃) binding activity coeluted with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P₃ affinity chromatography. Ins(1,4,5)P₃ bound to purified aldolase (at 0 °C) in a dose-dependent manner over the range [Ins(1,4,5)P₃] 20 nm to 20 μM, with maximal binding of 1 mol of Ins(1,4,5)P₃/mol aldolase₄ and a K(d) of 12-14 μM. Fru(1,6)P₂ and Fru(2,6)P₂ displaced bound Ins(1,4,5)P₃) with a 50% inhibition at 30 and 170 μM, respectively. Ins(1,3,4)P₃ (20 μM) and glyceraldehyde 3-phosphate (2 mM) were also potent inhibitors of Ins(1,4,5)P₃ binding, but not inositol 4-phosphate or inositol 1,4-bisphosphate (20 μM each). Aldolase-bound Ins(1,4,5)P₃ may play a role in phospholipase C-independent increases in free [Ins(1,4,5)P₃].