Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes

Journal Article · Sun May 01 00:00:00 EDT 1988 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.85.9.2969· OSTI ID:6245043

Kanngiesser, U; Nalik, P; Pongs, O

Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca/sup 2 +/ channel) has been purified as a 340-kDa protein complex to approx.80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two step-chromatographic purification procedure. The equilibrium dissociation constant of (/sup 3/H) (+) -PN200-110 binding (K/sub d/; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approx.300 kDa. The 195-kDa subunit was affinity-labeled with (/sup 3/H)azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).

• Research Organization: Ruhr-Universitaet Bochum (Germany, F.R.)
• OSTI ID: 6245043
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 85:9
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
MEMBRANES
MOLECULAR STRUCTURE
RECEPTORS
PURIFICATION
CALCIUM COMPOUNDS
CATIONS
ELECTROPHORESIS
FAR ULTRAVIOLET RADIATION
LABELLING
MUSCLES
PYRIDINES
RABBITS
TRITIUM COMPOUNDS
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
AZINES
CHARGED PARTICLES
ELECTROMAGNETIC RADIATION
HETEROCYCLIC COMPOUNDS
IONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
RADIATIONS
ULTRAVIOLET RADIATION
VERTEBRATES
550201* - Biochemistry- Tracer Techniques