Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol.
In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.
Kanematsu
T
T
Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Takeya
H
H
Watanabe
Y
Y
Ozaki
S
S
Yoshida
M
M
Koga
T
T
Iwanaga
S
S
Hirata
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism
D015220:Calcium Channels; D002413:Cations, Divalent; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D007527:Isoenzymes; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D010738:Type C Phospholipases; D002118:Calcium