Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1986

OSTI ID:6713230

Curtis, B M

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
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BODY
BRAIN
CALCIUM COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
CHROMATOGRAPHY
ELECTROPHORESIS
ION EXCHANGE CHROMATOGRAPHY
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MEMBRANES
MOLECULAR STRUCTURE
NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANS
PEPTIDES
PHOSPHORYLATION
POLYPEPTIDES
PROTEINS
PURIFICATION
RATS
RECEPTORS
RODENTS
SEDIMENTATION
SEPARATION PROCESSES
TRITIUM COMPOUNDS
VERTEBRATES