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)P-3) 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 (aldolase(4)) content was 34.5 pmol/100 nmol lipid P-i. Ins(1,4,5)P-3) binding activity coeluted with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P-3 affinity chromatography. Ins(1,4,5)P-3 bound to purified aldolase (at 0 degrees C) in a dose-dependent manner over the range [Ins(1,4,5)P-3] 20 nM to 20 mu M, with maximal binding of 1 mol of Ins(1,4,5)P-3/mol aldolase, and a K-d of 12-14 mu M. Fru(1,6)P-2 and Fru(2,6)P-2 displaced bound Ins(1,4,5)P-3) with a 50% inhibition at 30 and 170 mu M, respectively. Ins(1,3,4)P-3 (20 mu M) and glyceraldehyde 3-phosphate (2 mM) were also potent inhibitors of Ins(1,4,5)P-3 binding, but not inositol 4-phosphate or inositol 1,4-bisphosphate (20 mu M each). Aldolase-bound Ins(1,4,5)P-3 may play a role in phospholipase C-independent increases in free [Ins(1,4,5)P-3]