A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex.
P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/microgram and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides approximately 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.
Maeda
N
N
Division of Regulation of Macromolecular Function, Osaka University, Japan.
Niinobe
M
M
Mikoshiba
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D002531:Cerebellum / Q000032:analysis*; D002845:Chromatography; D015151:Immunoblotting; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D008861:Microsomes / Q000032:analysis; D008928:Mitochondria / Q000032:analysis; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018160:Receptors, Cytoplasmic and Nuclear; D012697:Serine Endopeptidases / Q000378:metabolism; D012995:Solubility
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; C025389:Itpr1 protein, mouse; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase