Inositol 1,4,5-Trisphosphate Binding to Porcine Tracheal Smooth Muscle Aldolase (∗)

• Published in: The Journal of biological chemistry 1995-09, Vol.270 (35), p.20459-20465
• Main Authors: Baron, Carl B., Ozaki, Shoichiro, Watanabe, Yutaka, Hirata, Masato, LaBelle, Edward F., Coburn, Ronald F.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; AZUCARES FOSFATOS; Binding Sites; Binding, Competitive; Calcium - metabolism; Cell Fractionation; CELL STRUCTURE; CERDO; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Chromatography, Gel; CYTOSKELETON; Electrophoresis, Polyacrylamide Gel; ESTRUCTURA CELULAR; FRUCTOSE 1,6-BISPHOSPHATE; FRUCTOSE 2,6-BISPHOSPHATE; FRUCTOSE-BISPHOSPHATE ALDOLASE; Fructose-Bisphosphate Aldolase - chemistry; Fructose-Bisphosphate Aldolase - isolation & purification; Fructose-Bisphosphate Aldolase - metabolism; Fructosephosphates - pharmacology; Humans; Hydrogen-Ion Concentration; INHIBICION; INHIBITION; INOSITOL; Inositol 1,4,5-Trisphosphate - metabolism; Inositol Phosphates - metabolism; Kinetics; LIASAS; LYASE; LYASES; MOLECULAR WEIGHT; MUSCLE; Muscle, Skeletal - enzymology; Muscle, Smooth - enzymology; MUSCLES; MUSCULOS; PESO MOLECULAR; POIDS MOLECULAIRE; PORCIN; PURIFICACION; PURIFICATION; Rabbits; STRUCTURE CELLULAIRE; SUCRE PHOSPHATE; SUGAR PHOSPHATES; SWINE; TRACHEA; Trachea - enzymology; TRACHEE; TRAQUEA
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.35.20459

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 Pi. 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 Kd 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].