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Volume 270,
Number 35,
Issue of September 01, pp. 20459-20465, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inositol
1,4,5-Trisphosphate Binding to Porcine Tracheal Smooth Muscle Aldolase
(Received for publication, March 9, 1995; and in revised form, May 23, 1995 )
Carl B.
Baron
, <WBR>
Shoichiro
Ozaki
, <WBR>
Yutaka
Watanabe
, <WBR>
Masato
Hirata
, <WBR>
Edward
F.
LaBelle
, <WBR>, <WBR>
Ronald F.
Coburn
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 ) 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 ) content was 34.5 pmol/100 nmol
lipid P . Ins(1,4,5)P ) binding activity coeluted
with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P affinity chromatography. Ins(1,4,5)P bound to
purified aldolase (at 0 °C) in a dose-dependent manner over the
range [Ins(1,4,5)P ] 20 nM to 20
µM, with maximal binding of 1 mol of
Ins(1,4,5)P /mol aldolase and a K of 12-14 µM.
Fru(1,6)P and Fru(2,6)P displaced bound
Ins(1,4,5)P ) with a 50% inhibition at 30 and 170
µM, respectively. Ins(1,3,4)P (20
µM) and glyceraldehyde 3-phosphate (2 mM) were
also potent inhibitors of Ins(1,4,5)P binding, but not
inositol 4-phosphate or inositol 1,4-bisphosphate (20 µM each). Aldolase-bound Ins(1,4,5)P may play a role in
phospholipase C-independent increases in free
[Ins(1,4,5)P ].

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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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