Subunit composition of the purified dihydropyridine binding protein from skeletal muscle.
The dihydropyridine (DHP) receptor from rabbit skeletal muscle has been characterized by affinity labeling and purification. Two procedures were used for purification: one that was a procedure modified from that of Curtis and Catterall (1984) and one that employed an anti alpha 1 monoclonal antibody (Mab) affinity column. In addition, both digitonin and CHAPS solubilizations were utilized with each purification technique. The major findings are as follows: (1) In contrast to the behavior in digitonin, neither the 52K (beta) nor the 140K (alpha 2) polypeptide quantitatively copurifies with the 170K (alpha 1) polypeptide when the purification is carried out in CHAPS. This has been shown by use of both wheat germ and monoclonal antibody columns. The digitonin-extracted receptor complex bound to the Mab affinity column loses alpha 2 and beta when the digitonin is replaced by CHAPS, and when the complex is bound to a WGA column, a CHAPS wash causes dissociation of alpha 1, beta, and gamma from alpha 2. Loss of binding of dihydropyridines occurs with the CHAPS wash but can be partially restored by the addition of the CHAPS wash to the material eluted from the column with N-acetylglucosamine. (2) Although both detergents solubilized greater than 80% of the polypeptides associated with the DHP binding site, the ability of these proteins to bind dihydropyridines is reduced more by CHAPS treatment than by digitonin treatment, raising the possibility that subunit interactions contribute to high-affinity binding. Alternatively, CHAPS may remove tightly bound lipids necessary for binding or cause irreversible denaturation of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Hamilton
S L
SL
Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Hawkes
M J
MJ
Brush
K
K
Cook
R
R
Chang
R J
RJ
Smilowitz
H M
HM
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004095:Dihydropyridines / Q000032:analysis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009132:Muscles / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000302:isolation & purification; D012995:Solubility
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D004095:Dihydropyridines; D011956:Receptors, Cell Surface; C038806:1,4-dihydropyridine