Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes.
The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.
Kamata
H
H
Department of Biochemistry, Jichi Medical School, Tochigi.
Hirata
M
M
Ozaki
S
S
Kusaka
I
I
Kagawa
Y
Y
Hirata
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002417:Cattle; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007477:Ions; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000737:chemistry* / Q000378:metabolism; D009994:Osmolar Concentration; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000494:pharmacology; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D013268:Stimulation, Chemical; D014316:Tritium
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007477:Ions; D008081:Liposomes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D011510:Proteolipids; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D014316:Tritium; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D002118:Calcium