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)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 degree C) in a dose-dependent manner over the range [Ins(1,4,5)P3] 20 nM to 20 microM, with maximal binding of 1 mol of Ins(1,4,5)P3/mol aldolase4 and a Kd of 12-14 microM. Fru(1,6)P2 and Fru(2,6)P2 displaced bound Ins(1,4,5)P3) with a 50% inhibition at 30 and 170 microM, respectively. Ins(1,3,4)P3 (20 microM) 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 microM each). Aldolase-bound Ins(1,4,5)P3 may play a role in phospholipase C-independent increases in free [Ins(1,4,5)P3].
Baron
C B
CB
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Ozaki
S
S
Watanabe
Y
Y
Hirata
M
M
LaBelle
E F
EF
Coburn
R F
RF
D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005636:Fructosephosphates / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D009130:Muscle, Smooth / Q000201:enzymology*; D011817:Rabbits; D013552:Swine; D014132:Trachea / Q000201:enzymology*
D000906:Antibodies; D005636:Fructosephosphates; D007295:Inositol Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D005634:Fructose-Bisphosphate Aldolase; D002118:Calcium