Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes.
A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242.
P S
PS
McPherson
K P
KP
Campbell
D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D006493:Heparin; D008970:Molecular Weight; D011233:Precipitin Tests; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012995:Solubility
D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D006493:Heparin