Subunit composition of the purified dihydropyridine binding protein from skeletal muscle

Abstract

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)

Structure of the voltage-gated L-type Ca 2+ channel by electron cryomicroscopy

“…Purified skeletal muscle L-type Ca 2ϩ channel was prepared as described (18). The identity of the bands seen in SDS polyacrylamide gels of the purified Ca 2ϩ channel was confirmed by Western blotting with monoclonal subunit-specific antibodies (Fig.…”

“…Skeletal muscle membranes were prepared from rabbit fast-twitch skeletal muscle, as described in Hawkes et al (17). Homogeneous preparations of the skeletal muscle Ca 2ϩ channel suitable for studies by electron microscopy were purified by using WGA-Affi-Gel column and DEAE-Trisacryl M ion exchange as described (18) with some modifications to adapt this procedure for structural studies. Protease inhibitors (100 M PMSF͞1 M aminobenzamidine͞1 M pepstatin A͞1 g/ml leupeptin͞1 g/ml aprotinin) were included in all buffers used throughout the preparations of microsomal membranes and Ca 2ϩ channel.…”

Full-length myotonin protein kinase (72 kDa) displays serine kinase activity.

“…6A). Biochemically purified DHPR (17), kindly provided by S. Hamilton, was used in the reaction mixture with Mt-PK fusion protein. DHPR contains an endogenous kinase that phosphorylates the major proteins of DHPR (18) requiring use of a low concentration of DHPR (60 ,g/ml).…”

Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca 2+ ·calmodulins

“…Splice variants 9 and 26 have the following substitution: 1618 LRIKTEGNLEQANE-ELR A IIKKIWKRTSMKLLDQVVPPAGDDEVTVGKF-YATFLIQEYFRKFKKRKEQGLVGKPSQRNALSL 1699 3 LRETELSSQVQYQAKEASLLERRRKSSHPKSSTKPNK-LLSSGGSTGWVEDARALEGQVLARGCGWLGSLEER-ERGPHHPPLGF. In isoforms 10,13,14,15,24,25,27 and 29, the glutamic residue 1623 is replaced with the sequence EEG-PSPSEAHQGAEDPFRPA. In all of these splice variants, the coiled-coil interaction is expected to be totally disrupted (Fig.…”

“…The first peak elutes at 43.7 mL and correlates to a dimer of Ca V 1.2 and the second peak is the monomer Ca V 1.2. A third peak is also detected and appears to correspond to a partially dissociated form of Ca V 1.2 because of the subunit dissociation in digitonin (10). Specificity of the [ 3 H]PN200-110 binding was shown by incubating the cardiac microsomes with 1 M isradipine, a nonradiolabeled dihydropyridine (Fig.…”