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

C. B. Baron, S. Ozaki, Y. Watanabe, Masato Hirata, E. F. LaBelle, R. F. Coburn

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Abstract

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].

Original languageEnglish
Pages (from-to)20459-20465
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number35
DOIs
Publication statusPublished - Jan 1 1995

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All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Baron, C. B., Ozaki, S., Watanabe, Y., Hirata, M., LaBelle, E. F., & Coburn, R. F. (1995). Inositol 1,4,5-trisphosphate binding to porcine tracheal smooth muscle aldolase. Journal of Biological Chemistry, 270(35), 20459-20465. https://doi.org/10.1074/jbc.270.35.20459