COEXPRESSION OF 2 ISOFORMS OF CALSEQUESTRIN IN RABBIT SLOW-TWITCH MUSCLE

The cardiac and fast-twitch skeletal muscle forms of the Ca2+-binding protein calsequestrin (CS) are the products of two different genes, both of which are transcribed in slow-twitch skeletal muscle, though at much different rates (Scott et al., 1988., Fliegel et al., 1989). We have investigated this problem more closely at the protein level, on isolated terminal cisternae (TC) of the sarcoplasmic reticulum (SR) of rabbit slow-twitch muscle, and following purification of two distinct forms of CS from whole tissue by DEAE-Cellulose chromatography and Ca2+-dependent elution from phenyl-Sepharose. Two eletrophoretically (apparent molecular mass of 64 kDa and 54 kDa, respectively), and antigenically distinct forms of CS, here shown to be related to the fast-twitch skeletal muscle and to cardiac-type isoform of CS, respectively, colocalize to junctional TC of slow-twitch muscle. The cardiac-type isoform that is expressed in slow-twitch muscle accounts for about 25% of total CS present in isolated TC, it binds Ca2+ as effectively as the major CS form, using a Ca-45-overlay technique, and it shares extensive similarities with dog cardiac CS, not only in size and antigenically, but also in pl, as well as in the DEAE-elution characteristics. No difference in behaviour with phenyl-Sepharose resin were observed between the two CS isoforms from slow-twitch muscle. Purified CS from fast-twitch muscle and the major CS isoform of slow-twitch muscle were found to be identical proteins, when compared between each other for peptide composition, after partial digestion with S. aureus V8 protease and for amino-terminal amino acid sequences, as well as concerning Ca2+-induced structural changes, as determined by intrinsic fluorescense spectroscopy. On the other hand, by all these properties the minor CS form from slow-twitch muscle appears to be closely related or identical to cardiac CS.