Immunochemical quantification of sarcoplasmic reticulum Ca-ATPase, of calsequestrin and of parvalbumin in rabbit skeletal muscles of defined fiber composition

Abstract

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 CaATPase 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 CaATPase, 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 2 IIa & I. Calsequestrin was uniformly distributed in fast-twitch muscles independently of their IIa/Ilb fiber ratio and displayed 50% lower concentrations in slow than in fast-twitch muscles (IIb = IIa > 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 > IIa % I).The major components of the Ca-sequestering system in mammalian skeletal muscle are the Ca-pumping ATPase and the Ca-binding calsequestrin of the sarcoplasmic reticulum (SR). The functional properties and the amount of these components determine the Ca-sequestering capacity of a given muscle. It is now generally accepted that differences exist with regard to this system between fast and slow-twitch muscles. Studies on isolated microsomal preparations have shown higher Ca-uptake capacities and Ca-ATPase activities in SR from fast-twitch than in SR from slow-twitch mammalian muscles [l -121. In addition, electron microscopy has revealed a higher fractional volume of the SR in fast than in slowtwitch muscle fibers [13-161. These data are in accordance with the higher Ca-uptakc capacities measured in cryostat sections [I 71 and in homogenates of fast-twitch muscles [5, 91. More recently, evidence indicates that mammalian muscles contain fiber-type-specific Ca-ATPase isozymes [18, 191. It has been suggested that the acidic, cytosolic Ca-binding protein parvalbumin may also play a major role in Ca regulation (for reviews see [20 -221) parvalbumin [23 -251. Therefore, the suggested role of parvalbumin may be restricted to fast-twitch fibers.The aim of the present study was to investigate the concentrations of these major Ca-seq...

Thyroid hormone differentially affects mRNA levels of Ca‐ATPase isozymes of sarcoplasmic reticulum in fast and slow skeletal muscle

“…In agreement with the published difference in Ca-ATPase content between EDL and SOL [5,14,28], we found for the euthyroid condition a 7-fold higher Ca-ATPase activity in EDL as compared to SOL, and the maximal T3-dependent stimulation of this activity (hyper-versus hypothyroid) was 6-fold for SOL and 1.5-fold for EDL (see legend to Fig. 3 for data).…”

“…Measurement of SR Ca-ATPase activity, or coupled Ca 2 + -transport, in muscle homogenates or isolated SR [1][2][3][4]17], as well direct determination of enzyme levels [5] and electron microscopy [6] has shown that Ca-ATPase levels are increased by T3 up to 6-fold in slow muscle and 1.5-fold in fast muscle. Slow muscle fibers (type I) express the cardiac isoform of the CaATPase (designated type I in this paper), and fast muscle fibers (type II) express 5 to 7 times higher levels of the somewhat more active isoform of the Ca 2 + pump (designated type II) [5,14,28]. Both isoforms are encoded by separate genes [7,8], and it is not known whether T3 merely stimulates the expression of the isoform already present in a muscle fiber, or that the two isoforms are differently affected.…”

Postnatal development of Ca2+‐sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice

“…Tissue contents of CaZ+-ATPase and calsequestrin were immunochemically determined by the use of a sandwich enzyme-linked immunoadsorbent assay (ELISA) [20]. The competitive ELISA used for distinguishing isoforms of Ca2…”

“…ATPase was the same as in [20]. The conditions for measurement of phosphoprotein formation and for the spectrophotometric assay of Ca2+,Mg2+-ATPase activity in total SR 7-8 (S), 12-16 (14), 16-20 (lS), 20-24 (22), 24-28 (26 …”

“…A preparation enriched in sarcolemmal membranes was obtained by sucrose density centrifugation of the microsomal preparation after calcium oxalate loading. This fraction was on top of the 20 -51 YO sucrose density gradient.Tissue contents of CaZ+-ATPase and calsequestrin were immunochemically determined by the use of a sandwich enzyme-linked immunoadsorbent assay (ELISA) [20]. The competitive ELISA used for distinguishing isoforms of Ca2…”