Purification and characterization of the 1,200-kDa subfragment of connectin filaments produced by 0.1 mM calcium ions.
When myofibrils prepared from chicken leg muscle were treated with a solution containing 0.1 mM CaCl2 and 30 micrograms/ml of leupeptin, alpha-connectin, which exists as a longitudinal thin filament in a sarcomere, was split into beta-connectin and a 1,200-kDa subfragment. The native subfragment was successfully purified without using any denaturant: It was extracted with 1 M KI solution from the Ca-treated myofibrils and purified by TSKgel G6000PW column chromatography. About 10 mg of the subfragment was yielded from 100 g of starting muscle. Using immunofluorescence microscopy and immunoelectron microscopy, we show here that polyclonal antibodies against the 1,200-kDa subfragment bind to the Z-disk and the epitope, which is about 0.34 micron apart from the Z-disk at a sarcomere length of 2.6 microns; the 1,200-kDa subfragment constitutes the proximal region of connectin filaments. Purified alpha-actinin decorated alpha-connectin and the 1,200-kDa subfragment on nitrocellulose blots of myofibrillar proteins separated by SDS-PAGE. Therefore, we conclude that connectin filaments are anchored to the Z-disk by the binding of the 1,200-kDa subfragment to alpha-actinin.
Tanabe
R
R
Department of Animal Science, Faculty of Agriculture, Hokkaido University.
Tatsumi
R
R
Takahashi
K
K
D000185:Actinin / Q000032:analysis; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D002645:Chickens; D002850:Chromatography, Gel; D064211:Connectin; D004591:Electrophoresis, Polyacrylamide Gel; D007976:Leupeptins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D009210:Myofibrils / Q000737:chemistry* / Q000187:drug effects; D010446:Peptide Fragments; D011485:Protein Binding; D011494:Protein Kinases
D002352:Carrier Proteins; D064211:Connectin; D007976:Leupeptins; D009124:Muscle Proteins; D010446:Peptide Fragments; D000185:Actinin; D011494:Protein Kinases; C032854:leupeptin; D002122:Calcium Chloride