Coexpression of two isoforms of calsequestrin in rabbit slow-twitch muscle

Abstract

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 electrophoretically (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 45Ca-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Mass Spectrometry of Cardiac Calsequestrin Characterizes Microheterogeneity Unique to Heart and Indicative of Complex Intracellular Transit

“…CSQ purification from canine skeletal muscle led to isolation of both CSQ isoforms, with the fast-twitch form (63 kDa) being the predominant one, as previously reported (38). Application of our phosphatase/CK2 kinase assay to the skeletal muscle preparation showed that, as in heart tissue, cardiac CSQ (55 kDa) was present mainly as the phosphoprotein (Fig.…”

Evidence that Spinach Leaves Express Calreticulin but Not Calsequestrin

“…Terminal cisternae are enriched in the content of both skeletal and cardiac isoforms of CS (Damiani et al, 1990) and were used as positive controls in immunoblot experiments with polyclonal antibodies to animal CS.…”

“…Phenyl-Sepharose (Pharmacia) column chromatography was carried out using either the procedure described by Heilmann et al (1993) for purification of CR from human liver (procedure 1) or that described by Damiani et al (1990) for purification of CS from rabbit skeletal muscle (procedure 2).…”

“…Blots were incubated as described by Damiani et al (1990) with (a) polyclonal antibodies to rabbit fast-twitch skeletal and dog cardiac CS (Damiani et al, 1990) or (b) the antiserum raised against spinach CR. In preliminary experiments, it was verified that the preimmune serum did not react with any protein of the several fractions tested.…”

“…In contrast, when an identical sample was applied to phenyl-Sepharose in the presence of 1 mM EGTA and subsequently eluted with 10 mM CaCl 2 , i.e. under experimental conditions identical to those employed for purification of CS from animal tissues (Damiani et al, 1990), neither the 56-nor the 54-kD spinach Ca 2+ -binding protein could be purified (Fig. 3).…”