Immunochemical quantification of sarcoplasmic reticulum Ca-ATPase, of calsequestrin and of parvalbumin in rabbit skeletal muscles of defined fiber composition.
Antibodies directed against purified Ca-ATPase from sarcoplasmic reticulum, calsequestrin and parvalbumin from rabbit fast-twitch muscle were raised in sheep. The specificity of the antibodies was shown by immunoblot analysis and by enzyme-linked immunoadsorbent assays (ELISAs). IgG against the sarcoplasmic reticulum Ca-ATPase inhibited the catalytic activities of Ca-ATPase from fast-twitch (psoas, tibialis anterior) and slow-twitch (soleus) muscles to the same degree. In non-equilibrium competitive ELISAs the anti(Ca-ATPase) IgG displayed a slightly higher affinity for the Ca-ATPase from fast-twitch muscle than for that from slow-twitch muscle. This suggests a fiber-type-specific polymorphism of the sarcoplasmic reticulum Ca-ATPase. Quantification of Ca-ATPase, calsequestrin and parvalbumin in various rabbit skeletal muscles of histochemically determined fiber composition was achieved by sandwich ELISA. Ca-ATPase was found to be 6-7 times higher in fast than in slow-twitch muscles. A slightly higher concentration was found in fast-twitch muscles with a higher percentage of IIb fibers when compared with fast-twitch muscles with a higher percentage of IIa fibers. Thus Ca-ATPase is distributed as follows, IIb greater than or equal to IIa much greater than I. Calsequestrin was uniformly distributed in fast-twitch muscles independently of their IIa/IIb fiber ratio and displayed 50% lower concentrations in slow than in fast-twitch muscles (IIb = IIa greater than I). Parvalbumin contents were 200-300-fold higher in fast than in slow-twitch muscles. Significantly lower parvalbumin concentrations were found in fast-twitch muscles with a higher percentage of IIa fibers than in fast-twitch muscles with a higher percentage of IIb fibers (IIb greater than IIa much greater than I).
D000818:Animals; D000918:Antibody Specificity; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002155:Calsequestrin / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007120:Immunochemistry; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000032:analysis* / Q000033:anatomy & histology / Q000201:enzymology; D010320:Parvalbumins / Q000302:isolation & purification*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000032:analysis / Q000201:enzymology
D002155:Calsequestrin; D009124:Muscle Proteins; D010320:Parvalbumins; D000252:Calcium-Transporting ATPases