High-affinity inositol 1,3,4,5-tetrakisphosphate receptor from cerebellum: solubilization, partial purification and characterization.
Proteins which bind with high affinity Ins 1,3,4,5-P4 or Ins 1,4,5-P3 were solubilized from porcine cerebellar membranes. Both binding activities were separated by heparin-agarose chromatography. The Ins 1,3,4,5-P4 receptor was partially purified with an approximately 1000-fold enrichment as compared to the membrane preparation. In the receptor-enriched preparation the Ins 1,3,4,5-P413 binding protein had an affinity (Kd) for Ins 1,3,4,5-P4 of 4.6 nM. Ins 1,3,4,5,6-P5 displaced [3H]Ins 1,3,4,5-P4 binding with a comparable affinity. The Ins 1,3,4,5-P4 binding protein displayed high selectivity for Ins 1,3,4,5-P4 over other inositol-phosphates (IC50 for Ins 1,4,5,6-P4 150 nM, for Ins-P6 1 microM and for Ins 1,3,4-P3 5 microM). Most importantly, Ins 1,4,5-P3 did not displace [3H]Ins 1,3,4,5-P4 binding at concentrations up to 10 microM. Binding of Ins 1,3,4,5-P4 was maximal in the pH range between 4.5 and 6, was stable with Ca2+ concentration varied from 1 nM to 1 mM, and was suppressed by heparin (IC50 about 2 nM). The high affinity receptor for Ins 1,3,4,5-P4 reported here, which is distinct from the Ins 1,4,5-P3 receptor might allow to evaluate the possible functional role of Ins 1,3,4,5-P4 in the cellular signal transduction.
Donié
F
F
Physiologisch-Chemisches Institut, Universität Tübingen, FRG.
Hülser
E
E
Reiser
G
G
D000818:Animals; D002118:Calcium / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000502:physiology*; D006493:Heparin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility; D013552:Swine
D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C061107:inositol-1,3,4,5-tetrakisphosphate receptor; C081655:inositol-1,3,4,5-tetrakisphosphate; C081651:inositol-1,4,5,6-tetrakisphosphate; D006493:Heparin; D002118:Calcium