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Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal muscle and is characterized by the presence of an amplified trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene. In this study, the subcellular localization of the myotonic dystrophy protein kinase in muscle tissues has been investigated at both morphological and biochemical level, by using antibodies against the myotonic dystrophy protein kinase. Immunofluorescence studies and Western-blot analysis were carried out with antibodies raised against both a synthetic peptide and a recombinant fusion protein fragment specific for the myotonic dystrophy protein kinase. The kinase is localized both to the surface membranes, and within the skeletal fibres in the region of the A-I band boundary. Consistent with the A-I location of the kinase is that Western-blot analysis of purified fractions from sarcoplasmic reticulum show that triads and sarcoplasmic reticulum terminal cisternae are immunoreactive for two myotonic dystrophy protein kinase proteins of different molecular weight (85 and 54 kDa). The relative amount of these two proteins is different in relation to the muscle type, the 85 kDa protein being more evident in skeletal than in cardiac fibres. In addition, immunofluorescence studies of cardiac muscle reveal a heavy concentration of DM-PK localized to the intercalated discs, as well as a weaker reaction in the sarcoplasm. These results taken together suggest that multiple isoforms of the DM-PK may exist and that they may be differentially located in muscle tissues.

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 (Damiani et 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 a 45Ca 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.

Calsequestrin was identified in the isolated sarcoplasmic reticulum from skeletal muscle of three mammalian species (man, rat and rabbit) and from frog and chicken muscle, using electrophoretic and immunoblot techniques. It was further characterized in sarcoplasmic reticulum protein mixtures and at several stages of purification, following extraction with EDTA. We found extensive similarities in apparent molecular weight values, Stains All staining properties and in Cleveland's peptide maps, between mammalian calsequestrins, and no detectable difference within a species between fast and slow muscle. Human calsequestrin, with an apparent molecular weight of 60,000 when measured at alkaline pH and of 41,000 when measured at neutral pH, appears to be the smallest in size. Frog calsequestrin, although weakly cross-reactive with rabbit calsequestrin and having a relatively higher apparent molecular weight at alkaline pH (72,000), shares several significant properties with mammalian calsequestrins. It bound calcium with a high capacity (1300 nmol per mg protein), it contained about 32% acidic amino acid residues and focused at closely similar pI values. We observed the formation of a complex with Stains All absorbing maximally at 535 nm, rather than at 600 nm, and an even more marked shift in apparent molecular weight at neutral pH. We found distinct differences in the case of chicken calsequestrin, in addition to those previously reported. It is a highly acidic, calcium-precipitable protein, but its amino acid composition is contradistinguished by a higher ratio of glutamate to aspartate and its rate of electrophoretic mobility is minimally affected by changes in pH. It stained deep bluish with Stains All after gel electrophoresis and yielded a protein-dye complex in aqueous solution, absorbing maximally at 560 nm, and finally, it bound fluorescent Concanavalin A.