Purification of dihydropyridine receptor from rabbit skeletal muscle

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6943369

Nakayama, N; Vaghy, P; Schwartz, A

Dihydropyridine (DHP)receptor was purified from T-tubules isolated from freeze-thawed rabbit skeletal muscle after French press treatment of microsomal membranes. DHP receptor was labeled with 25 nM (/sup 3/H)-(+)-PN-200-110 (PN, one of the most potent Ca-antagonists) and solubilized with 1% digitonin. The solubilized receptor was purified in the presence of protease inhibitors (0.1 mM PMSF, 1 mM iodoacetamide, 1..mu..M pepstatin A, 1 mg/l antipain and 0.2 mM o-phenanthroline) using WGA-Sepharose and DEAE-Biogel A column chromatography as well as sucrose density gradient (SDG) centrifugation. The pooled fractions of the SDG had a maximum binding of 590 pmol/mg protein even without correcting for dissociation of PN from the receptors during purification. On SDS-PAGE, a single major band (191 K dalton) was shown both in presence and absence of 20 mM N-ethyl maleimide. However, two major (145 and 103 K dalton) a few minor bands (55,46,32 and 31K dalton) were obtained if the fraction was treated with 20 mM dithiothreitol prior to electrophoresis. The authors data suggest that 191 and 145 k dalton proteins correspond to the ..cap alpha..-subunit of the DHP receptor as reported by Curtis and Catterall.

• Research Organization: Univ. of Cincinnati College of Medicine, OH
• OSTI ID: 6943369
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AZINES
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CENTRIFUGATION
CHROMATOGRAPHY
ELECTROPHORESIS
GEL PERMEATION CHROMATOGRAPHY
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANE PROTEINS
MICROSOMES
MOLECULAR WEIGHT
MUSCLES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PROTEINS
PURIFICATION
PYRIDINES
RABBITS
REACTION KINETICS
RECEPTORS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRACENTRIFUGATION
VERTEBRATES