Structural characterization of the dihydropyridine receptor-linked calcium channel from porcine heart.
Ca(2+)-channel was purified 230-fold from digitonin extracts of the porcine cardiac sarcolemmal membranes by means of a four-step procedure. Two antibodies, a site-directed antibody against the sequence 1691-1707 of the rabbit cardiac alpha 1 subunit (anti-CCP5) and a monoclonal antibody directed to rabbit skeletal muscle alpha 2 delta subunit-complex (MCC-1), effectively immunoprecipitated the 125I-labeled cardiac Ca(2+)-channel complex in 0.2% digitonin. SDS-PAGE analysis of the immunoprecipitates under reducing conditions revealed that the cardiac channel is mainly composed of two large polypeptides of 190 and 150 kDa, and five smaller polypeptides of 60, 55, 35, 30, and 25 kDa. An additional polypeptide of either 79 or 55 kDa is crosslinked with the 190 kDa component to form 250-270 kDa (approximately 270 kDa) to the extent of 15-20% through disulfide bond(s). The 190 kDa component (alpha 1) is responsible for photoaffinity labeling with [3H]diazepine, since minor photolabeled approximately 270 kDa was converged to the major labeled 190 kDa component when electrophoresed under reducing conditions. The 150 kDa component (alpha 2) was derived by reduction of disulfide bonds from another 190 kDa component of glycopolypeptide which was separated from the channel complex in 1% Triton X-100 and capable of binding to WGA-Sepharose. The four smaller components of 60, 35, 30, and 25 kDa were not covalently associated with the large components through disulfide bonds, whereas the 55 kDa polypeptide was suggested to be a mixture of two kinds of peptides with respect to the disulfide bond: one was crosslinked with alpha 1 through disulfide linkage and the other was not covalently associated with any other component.(ABSTRACT TRUNCATED AT 250 WORDS)
Kuniyasu
A
A
Faculty of Pharmaceutical Sciences, Hokkaido University.
Oka
K
K
Ide-Yamada
T
T
Hatanaka
Y
Y
Abe
T
T
Nakayama
H
H
Kanaoka
Y
Y
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015220:Calcium Channels / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D004095:Dihydropyridines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D009206:Myocardium / Q000737:chemistry*; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D013552:Swine
D000906:Antibodies; D015220:Calcium Channels; D004095:Dihydropyridines; C038806:1,4-dihydropyridine; D011494:Protein Kinases