Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel
Treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with (/sup 3/H)nitrendipine from rat brain membranes. The solubilized complex retains allosteric coupling to binding sites for diltiazem, verapamil, and inorganic calcium antagonist sites. The calcium antagonist receptor from cardiac sarcolemma and the transverse-tubule membrane of skeletal muscle is also efficiently solubilized with digitonin and the receptor in all three tissues is a large glycoprotein with a sedimentation coefficient of 20 S. The T-tubule calcium antagonist receptor complex was extensively purified by a combination of chromatography on WGA-Sepharose, ion exchange chromatography, and sedimentation on sucrose gradients to yield preparations estimated to be 41% homogeneous by specific activity and 63% homogeneous by SDS gel electrophoresis. Analysis of SDS gels detect three polypeptides termed ..cap alpha..(Mr 135,000), ..beta..(Mr 50,000), and ..gamma..(Mr 32,000) as noncovalently associated subunits of the calcium antagonist receptor. The ..cap alpha.. and ..gamma.. subunits are glycosylated polypeptides, and the molecular weight of the core polypeptides are 108,000 and 24,000 respectively. The calcium antagonist receptor was reconstituted into a phospholipid bilayer by adding CHAPS and exogeneous lipid to the purified receptor followed by rapid detergent removal. This procedure resulted in the incorporation of 45% of the calcium antagonist receptor into closed phospholipid vesicles. Data suggests that the ..cap alpha.., ..beta.., and ..gamma.. subunits of the T-tubule calcium antagonist receptor are sufficient to form a functional calcium channel.
- Research Organization:
- Washington Univ., Seattle (USA)
- OSTI ID:
- 6713230
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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MEMBRANE TRANSPORT
RECEPTORS
MOLECULAR STRUCTURE
PURIFICATION
BRAIN
CELL MEMBRANES
ELECTROPHORESIS
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PHOSPHORYLATION
POLYPEPTIDES
PROTEINS
RATS
SEDIMENTATION
TRITIUM COMPOUNDS
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CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
CHROMATOGRAPHY
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
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NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANS
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550201* - Biochemistry- Tracer Techniques