Biochemical characterization of ultrastructural localization of a major junctional sarcoplasmic reticulum glycoprotein (triadin).
Monoclonal antibodies were used to identify a 94-kDa protein that was greatly enriched in traids and junctional face membranes (9.3 +/- 0.2%) but not detected in the transverse tubular and nonjunctional sarcoplasmic reticulum membranes. The 94-kDa protein is a hydrophobic glycoprotein based on endoglycosidase H sensitivity, concanavalin A binding, and labelling with a hydrophobic probe. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of reducing agents suggests that this protein is present as a population of multimeric structures containing a variable number of the 94-kDa subunits. Immunofluorescent staining of serial transverse sections of skeletal muscle shows staining of all fiber types with preferential staining of type II fast fibers. Specific immunofluorescence staining in longitudinal sections of skeletal muscle is confined to the interface between the A- and I-bands where the triad structures are localized. Immunocolloidal gold labeling revealed the 94-kDa glycoprotein to be localized over a region of the junctional sarcoplasmic reticulum where the ryanodine receptor/Ca2+ release channel is localized. The distribution and high abundance of the 94-kDa glycoprotein in the junctional membrane suggest that it performs a structural or functional role in the storage or release of calcium from the junctional sarcoplasmic reticulum in skeletal muscle.
Howard Hughes Medical Institute, Iowa City, Iowa.
C M
CM
Knudson
K K
KK
Stang
A O
AO
Jorgensen
K P
KP
Campbell
D000818:Animals; D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D002352:Carrier Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis*; D008854:Microscopy, Electron; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis*; D009132:Muscles / Q000737:chemistry / Q000648:ultrastructure*; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D002352:Carrier Proteins; D006023:Glycoproteins; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin; D012433:Ryanodine