Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

Peter S Mcpherson; Kevin P Campbell

doi:10.1016/S0021-9258(17)44774-0

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Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

Journal article

Open access

Peer reviewed

Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

Peter S Mcpherson and Kevin P Campbell

The Journal of biological chemistry, Vol.265(30), pp.18454-18460

10/25/1990

DOI: 10.1016/S0021-9258(17)44774-0

PMID: 2211713

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Abstract

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.

Rabbits

Centrifugation, Density Gradient

Molecular Weight

Ryanodine - metabolism

Receptors, Cholinergic - metabolism

Ryanodine Receptor Calcium Release Channel

Solubility

Precipitin Tests

Cell Membrane - chemistry

Animals

Chromatography, Affinity

Receptors, Cholinergic - immunology

Brain Chemistry

Sarcoplasmic Reticulum - metabolism

Heparin

Receptors, Cholinergic - isolation & purification

Details

Title: Subtitle: Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes
Creators: Peter S Mcpherson - Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242
Kevin P Campbell
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.265(30), pp.18454-18460
DOI: 10.1016/S0021-9258(17)44774-0
PMID: 2211713
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: HL 39265 / NHLBI NIH HHS
Language: English
Date published: 10/25/1990
Academic Unit: Neurology; Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984068370002771

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