TY - JOUR
T1 - Inositol 1,4,5-trisphosphate binding to porcine tracheal smooth muscle aldolase
AU - Baron, C. B.
AU - Ozaki, S.
AU - Watanabe, Y.
AU - Hirata, M.
AU - LaBelle, E. F.
AU - Coburn, R. F.
PY - 1995
Y1 - 1995
N2 - 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].
AB - 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].
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U2 - 10.1074/jbc.270.35.20459
DO - 10.1074/jbc.270.35.20459
M3 - Article
C2 - 7657622
AN - SCOPUS:0029156476
VL - 270
SP - 20459
EP - 20465
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 35
ER -