Abstract
We established a novel method to isolate a single type of inositol 1,4,5-trisphosphate receptor (IP3R) among the heterogeneous population of receptors to study the regulatory mechanism of Ca2+ release. We raised in the rabbit a polyclonal antibody against synthetic peptide corresponding to amino acids 2736-2747 (pep 6) of type I IP3R (IP3-R-I) that is most abundant in cerebellum. We purified IP3R-I from a 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid solubilized mouse cerebellar microsomal fraction by immunoaffinity chromatography on an anti-pep 6 antibody-Sepharose 4B column with specific elution by the pep 6 peptide (GHPPHMNVNPQQ) of the IP3R-I C terminus. Immunoaffinity-purified IP3R reconstituted into lipid vesicles formed a homotetramer structure. Monoclonal antibody 18A10, which partially blocks the Ca2+ release from cerebellar microsome, almost completely inhibited IP3-induced 45Ca2+ influx into proteoliposomes, whereas monoclonal antibody that recognizes other regions did not inhibit Ca2+ influx. Both the rate and extent of 45Ca2+ influx into proteoliposomes increased 20% after incubation with the catalytic subunit of cyclic AMP-dependent protein kinase, accompanied by stoichiometric phosphorylation of IP3R protein.
Highlights
Shimamoto-ch, Mishima-gun, Osaka 618, the $Departmentof Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai,Minato-ku, Tokyo 108, and the TMolecular Neurobiology Laboratory, the Institute of Physical and Chemical Research (RIKEN), Tsukuba LifeScience Center, Koyodai 3-1-1, Tsukuba, Ibaragi305,Japan
IP3R contains two consensus the Cal+ release fromcerebellarmicrosome,almost amino acid sequences that fulfill the criteria for protein kinase (PKA) action completelyinhibitedIPS-induced“Ca2+ influx into [5], andthese were shown to be phosphorylated by PKA both proteoliposomes,whereasmonoclonalantibodythat in vitro [17] and in intact cells [18].Supattapone et al [17]
In order to purify the functional IP3R-I alone, we have developed a novel method for the rapid, gentle purification of the receptor using immunoaffinity chromatography
Summary
EXPERIMENTALPROCEDURES at 95 "C and dialyzed against PBS containing 1%CHAPS, 0.1 mM. Materiuls--IP3 and CHAPS were obtained from Dojindo Laboratories (Kumamoto, Japan). [3H]IP3and "Ca2+were obtained from DuPont NEN. [y3'P]ATP (6000 Ci/mmol), ECL Western blotting system, and Hyper film-ECL were obtained from Amersham Corp. Tissues were mixed with 9 volumes of the solution containing 0.32 M SDS, 10% 2-mercaptoethanol, 20%glycerol,0.125 M Tris-HC1, pH sucrose, 1mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 6.8, and were heated in a boiling water bath for 3 min. Proteolipophosphatidylcholine/ml, and IP3R was eluted by pep 6 at a somes formed were collected by centrifugation at 100,000 X g concentration of 5 p~ in buffer A containing 1mg of phosphatidyl- for 20 min, and the resulting pellets were suspended with buffer C choline/ml. 10 pl of sample was solubilized in agarose-PAGE sample buffer a t a final concentration of 1%SDS, 1mM EDTA, 5% 2-mercaptoethanol, 10 mM Tris-HC1, pH 8.0, 10% glycerol and heated in a boiling water bath for 3 min. Purification of IPa with Conventional Method and Measurement of I3H]IP3Binding-The methods are described elsewhere [4]
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