ALBERT

Notes: Summary A calsequentrin (CS)-like glycoprotein is present in the sarcoplasmic reticulum (SR) of chicken pectoralis muscle, which displays unusual properties: it binds relatively low amounts of Ca2+, compared to CS in mammalian skeletal muscle (Yap & MacLennan, 1976), it does not exhibit a marked pH-dependent shift in mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and its metachromatic staining properties with Stains All are likewise peculiar (Damianiet al., 1986). We have now definitively localized the same protein to the junctional terminal cisternae (TC) fraction of the SR of chicken pectoralis muscle and have further characterized it, following purification by crystallization with Ca2+ and by Ca2+-dependent elution from phenyl-Sepharose columns. The purified protein (apparent Mr: 51 kDa), isoelectrofocuses at pH 4.5, and is readily identified on blots by a45Ca overlay technique, similar to CS of rabbit skeletal muscle, but it binds half as much Ca2+ (about 20 moles of Ca2+ per mole of protein), as estimated by equilibrium dialysis. However, the chicken protein shares extensive similarities with mammalian CSs, concerning Ca2+-induced changes in maximum intrinsic fluorescence and the Ca2+-modulated interaction with phenyl-Sepharose, as well as in being protected by Ca2+ from proteolysis by either trypsin or chymotrypsin. We discuss how the presence of a Ca2+-regulated hydrophobic site in the CS molecule appears to be the most invariant property of the CS-family of Ca2+-binding proteins.

Notes: Summary We have investigated high-affinity ryanodine-binding sites in membrane preparations from representative fast-twitch and slow-twitch muscles of the rabbit and rat, as well as from human mixed muscle. Our results, obtained in high-ionic strength binding buffer, demonstrate extensive similarities in binding affinity for [3H]ryanodine (Kd: about 10 nM) and a two-fold to four-fold difference in membrane density of the ryanodine receptor between fast-twitch and slow-twitch muscle of the rat and rabbit, respectively. The [3H]ryanodine-pCa relationship for the Ca2+-activation curve of ryanodine binding was found to be similar for all mammalian muscles, as tested at 20 nM ryanodine. With 10 mM caffeine or 50 μM doxorubicin the pCa for half-maximal activation of [3H]ryanodine binding invariably shifted from an average pCa value of 6.5 to pCa 7.1–7.3. IC50 values for the inhibition of [3H]ryanodine binding by Ruthenium Red, a Ca2+-release channel blocker, did not differ significantly (range 0.3–1.0 μM). The Ca2+-dependence curve (range 1 nM–10 mM free Ca2+) that we have observed at 5 nM ryanodine, for [3H]ryanodine binding to terminal cisternae from rabbit fast-twitch, as well as slow-twitch muscle, is bell-shaped and differs from that obtained with cardiac terminal cisternae from the same species. Cardiac ryanodine receptor is also clearly distinguishable for electrophoretic mobility, Cleveland's peptide maps, and, most strikingly, for total lack of cross-reactivity with polyclonal antibody to fast skeletal RyR. By the same properties, the ryanodine receptor of fast- and slow-twitch muscle appear to be the same or a similar protein. On investigating the composition of calsequestrin in rat and human skeletal muscles, both in membrane-bound form and after purification by phenyl-Sepharose chromatography, we have been able to show that, independent of the animal species, the cardiac isoform, as characterized by the identical amino-terminal amino-acid sequence, pattern of immunoreactivity, and lack of Ca2+-dependent shift in mobility on SDS-PAGE, is exclusively expressed in slow-twitch fibres, together with the main fast-skeletal calsequestrin isoform. While our experimental findings strongly argue for the presence of only one population of skeletal-specific Ca2+-release channels in junctional terminal cisternae of mammalian fast-twitch and slow-twitch muscle, they at the same time suggest the existence of differences in calsequestrin modulation of Ca2+-release, depending on its isoform composition.