Abstract
We report evidence for two foot protein isoforms in chicken pectoral muscle. (i) Two polypeptides with molecular masses of approximately 500 kDa copurify with [3H]ryanodine binding. (ii) Both polypeptides are associated with oligomeric proteins similar in size to the mammalian skeletal muscle foot protein. (iii) The polypeptides are shown to be unique by limited proteolysis. (iv) By using isoform-specific antibodies, the polypeptides are shown to be subunits of different [3H]ryanodine-binding proteins. Using immunolabeling techniques, we have localized these proteins in chicken breast muscle by both light and electron microscopy. (v) From immunofluorescent light microscopy of longitudinal sections, it was determined that both ryanodine-binding protein isoforms exhibit identical repetitive punctate distributions near the Z-lines. (vi) In serial cross-sections both proteins have similar distributions in the same fibers. (vii) Both proteins were found to be associated with the terminal cisternae of the sarcoplasmic reticulum by immunoelectron microscopy. Based on their localization to the triadic junction, their large size and their ability to bind [3H]ryanodine, these proteins are identified as foot proteins. In conclusion, two distinct homo-oligomeric foot proteins coexist in avian fast twitch skeletal muscle. We have termed these proteins, alpha and beta foot proteins.
Highlights
From the SDepartment of Pharmacology and the Cell and Molecular Biology Program, University of Nevada, Rena, Nevada
We report evidence for two foot protein isoforms in chicken pectoral muscle. (i) Two polypeptides with molecular masses of -500 kDa copurify with [‘Hlryanodine binding. (ii) Both polypeptides are associated with oligomeric proteins similar in size to the mammalian skeletal muscle foot protein. (iii) The polypeptides are shown to be unique by limited proteolysis. (iv)
By using isoform-specific antibodies, the polypeptides are shown to be subunits of different [‘Hlryanodinebinding proteins. We have localized these proteins in chicken breast muscle by both light and electron microscopy. (v) From immunofluorescent light microscopy of longitudinal sections, it was determined that both ryanodine-binding protein isoforms exhibit identical repetitive punctate distributions near the Z-lines. (vi) In serial cross-sections both proteins have similar distributions in the same fibers. (vii) Both proteins were found to be associated with the terminal cisternae of the sarcoplasmic reticulum by immunoelectron microscopy
Summary
Materials-T-61 euthanasia solution was purchased from American Hoechst Corp. (Somerville, NJ); leupeptin, PMSF,’ aprotinin, benzamide, iodoacetamide, pepstatin A, diisopropryl fluorophosphate, CHAPS, L-a-phosphatidylcholine, DEAE-Sepharose, polyethylene glycol, agarose-linked goat anti-mouse IgG (whole molecule) antibodies, trypsin, Staphylococcus aureus V8 protease, and calcium-activated neutral protease (calpain) were from Sigma; and alkaline phosphatase conjugated goat anti-mouse IgG and goat anti-rabbit IgG an&bodies were from Tago Aliquots of 0.1 ml of the beads were incubated with an equal volume of anti-ryanodine-binding protein monoclonal antibody (l-10 mg protein/ml) for either 3 h at room temperature or overnight at O-4 “C. The concentrations of primary antibody and solubilized protein resulting in precipitation of equivalent levels of each of the ryanodine-binding protein isoforms were determined in preliminary experiments to insure that approximately equal amounts of the isoforms were loaded on each gel. This was necessary because the isoforms represented a relatively minor. Electron micrographs were obtained with a JEOL 1OOCX electron microscope at 100 Kev
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