Ryanodine-affinity chromatography purifies 106 kD Ca2+ release channels from skeletal and cardiac sarcoplasmic reticulum.
A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.
Salama
G
G
Department of Physiology, School of Medicine, University of Pittsburgh, Pennsylvania.
Nigam
M
M
Shome
K
K
Finkel
M S
MS
Lagenaur
C
C
Zaidi
N F
NF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D015220:Calcium Channels / Q000276:immunology / Q000378:metabolism; D002846:Chromatography, Affinity; D004285:Dogs; D015640:Ion Channel Gating / Q000187:drug effects; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009124:Muscle Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D009132:Muscles / Q000737:chemistry* / Q000648:ultrastructure; D009206:Myocardium / Q000737:chemistry* / Q000648:ultrastructure; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification* / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D008565:Membrane Proteins; D009124:Muscle Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D013439:Sulfhydryl Reagents; D012433:Ryanodine