High-affinity inositol 1,3,4,5-tetrakisphosphate receptor from cerebellum: solubilization, partial purification and characterization

• Published in: FEBS letters 1990-07, Vol.268 (1), p.194-198
• Main Authors: Donié, Frédéric, Hülser, Eckehard, Reiser, Georg
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Calcium - physiology; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; cerebellum; Cerebellum - physiology; Fundamental and applied biological sciences. Psychology; Heparin; Heparin - metabolism; Hydrogen-Ion Concentration; Inositol 1,3,4,5-tetrakisphosphate; Inositol 1,4,5-trisphosphate; Inositol Phosphates - metabolism; Inositolphosphate receptor; Ins-P3 (Ins 1,4,5-P3), D-myo-inositol 1,4,5-P3; Ins-P4 (Ins 1,3,4,5-P4), D-myo-inositol 1,3,4,5-P4; Miscellaneous; Molecular and cellular biology; Receptors, Cell Surface - isolation & purification; Receptors, Cell Surface - metabolism; Receptors, Cytoplasmic and Nuclear; Solubility; Swine
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(90)81006-A

Proteins which bind with high affinity Ins 1,3,4,5-P 4 or Ins 1,4,5-P 3 were solubilized from porcine cerebellar membranes. Both binding activities were separated by heparin-agarose chromatography. The Ins 1,3,4,5-P 4 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-P 413 binding protein had an affinity ( K d) for Ins 1,3,4,5-P 4 of 4.6 nM. Ins 1,3,4,5,6-P 5 displaced [ 3H]Ins 1,3,4,5-P 4binding with a comparable affinity. The Ins 1,3,4,5-P 4binding protein displayed high selectivity for Ins 1,3,4,5-P 4 over other inositolphosphates (IC 50 for Ins 1,4,5,6-P 4 150 nM, for Ins-P 6 1 μM and for Ins 1,3,4-P 3 5 μM). Most importantly. Ins 1,4,5-P 3 did not displace [ 3H]Ins 1,3,4,5-P 4binding at concentrations up to 10μM. Binding of Ins 1,3,4,5-P 4 was maximal in the pH range between 4.5 and 6, was stable with Ca 2+ concentration varied from 1 nM to 1 mM, and was suppressed by heparin (IC 50 about 2 nM). The high affinity receptor for Ins 1,3,4,5-P 4 reported here, which is distinct from the Ins 1,4,5-P 3 receptor might allow to evaluate the possible functional role of Ins 1,3,4,5-P 4 in the cellular signal transduction.