1,720,994 research outputs found
Amino acid sequence of chicken calsequestrin deduced from cDNA: comparison of calsequestrin and aspartactin
No full textWe have previously reported the amino terminal sequence of adult chicken calsequestrin, an intraluminal Ca2(+)-binding protein isolated from fast-twitch skeletal muscle. The partial sequence showed homology with mammalian calsequestrins contained in the PIR data bank and complete identity with the amino terminus of a putative laminin-binding protein of the extracellular matrix, aspartactin. Based on these data, oligonucleotide primers were synthesized for PCR amplification and direct DNA sequencing. We report herein the primary sequence of chicken calsequestrin, deduced from cDNA. The sequence has been verified by amino acid sequencing of internal tryptic peptides. Importantly, the data show the primary structure of calsequestrin to be identical to the amino acid sequence reported for aspartactin, with the exception of a single amino acid difference (ileu vs. val) which may be animal strain-related. Based on these data, calsequestrin and aspartactin are the same protein
Characterization of calsequestrin of avian skeletal muscle.
No full textA 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, an intracellular calcium-binding protein of skeletal muscle sarcoplasmic reticulum, is homologous to aspartactin, a putative laminin-binding protein of the extracellular matrix
No full textCalsequestrin was isolated from chicken fast-twitch skeletal muscle, and partial amino terminal sequence was determined. The sequence (NH2) EEGLNFPTYDGKDRVIDLNE shows high identity with known mammalian calsequestrins contained in the Protein Identification Resource data bank (1). Most importantly, this 20 amino acid sequence shares complete identity with the amino terminus of aspartactin, a putative laminin-binding protein of the extracellular matrix (2, 3). The possible relationship of aspartactin to calsequestrin is discussed
Dual role of calsequestrin as substrate and inhibitor of casein kinase-1 and casein kinase-2
No full textCalsequestrin from different muscle tissues and species has been phosphorylated by casein kinase-1 and casein kinase-2, in the conditions previously reported by Cala and Jones (J. Biol. Chem. 266, 391-398, 1991). Results indicates that rabbit cardiac and skeletal calsequestrin and frog skeletal calsequestrin are phosphorylated by both casein kinase-1 and casein kinase-2, at variance with chicken skeletal calsequestrin which is a poor substrate for both enzymes. We also observed that chicken calsequestrin is able to inhibit phosphorylation of cardiac calsequestrin, as well as other specific substrates, when added together to the assay medium
Fibre type specific expression of skeletal and cardiac muscle myotonin kinase
No full textMyotonin kinase (MtPk) is the protein coded by the Myotonic Distrophy (DM) gene. The primary structure of MtPK has been deduced from the DNA sequence and indicates that the gene product is a protein kinase, most likely a Ser/Thr kinase. The presence of a polymorphic CTG-repeat expansion in pathological genes has been indicated as the molecular lesion leading to the DM phenotype. The bulk of information on this protein is inferred from genetics and molecular biology studies, whereas few data are available on the native protein. We previously reported (Salvatori et al., Biochem. Biophys. Res. Commun., 203, 1365-70, 1994) that MtPK is associate to membranes derived from terminal cisternae of the sarcoplasmic reticulum, from where it can be removed by alkali-treatment, similarly to other proteins like for instance, dystrophin, which is easily removed from sarcolemma at alkaline pH. We also reported (Salvatori et nl., 1. Muscle Res. Cell Mot& 16, 190, 1994) that MtPK is localized to the I- band and that quantitative differences exist among fast and slow skeletal and cardiac muscles. We decided to reinvestigate the aspect of fibre typing to search for any difference in reactivity. With the help of antibodies directed against specific markers like myosin heavy chains, we found that, in skeletal muscle, MtPK was maximally expressed in type I fibres, as expected, but also that type IIB fibres were unreactive and type IIA fibres had an intermediate level of reactivity. This seems to suggest a possible link between fibre metabolism and MtPK expression, Moreover, to better localize the Mtl’K within muscle fibres we isolated single skinned fibres for the observation with a laser confocal microscope. Results show that both slow and fast fibres exhibited the same banding pattern transverse to the long axis of the fibre, that the reactivity was due to regularly spaced spots and that the signal is reinforced in the correspondence of the external membrane. In the cardiac muscle fibres the staining seems to be rather different in that, the immunoreactivity appears to be confined to the intercalated disks
Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties.
No full textIt had been previously demonstrated that endoplasmic reticulum membranes from rat hepatocytes contain a major calsequestrin-like protein, on account of electrophoretic and Stains All-staining properties (Damiani et al., J. Biol. Chem. 263, 340-343). Here we show that a Ca2+-binding protein sharing characteristics in size and biochemical properties with this protein is likewise present in the isolated endoplasmic reticulum from human liver. Human calsequestrin-like protein was characterized as 62 kDa, highly acidic protein (pl 4.5), using an extraction procedure from whole tissue, followed by DEAE-Cellulose chromatography, that was originally developed for purification of skeletal muscle and cardiac calsequestrin. Liver calsequestrin-like protein bound Ca2+ at low affinity (Kd = 4 mM) and in high amounts (Bmax = 1600 nmol Ca2+/mg of protein), as determined by equilibrium dialysis, but differed strikingly from skeletal muscle calsequestrin for the lack of binding to phenyl-Sepharose resin in the absence of Ca2+, and of changes in intrinsic fluorescence upon binding of Ca2+. Thus, these results suggest that liver 62 kDa protein, in spite of its calsequestrin-like Ca2+-binding properties, does not contain a Ca2+-regulated hydrophobic site, which is a specific structural feature of the calsequestrin-class of Ca2+-binding proteins
Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions
No full textMyotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes
Localization of protein kinase C in skeletal muscle T-tubule membranes
No full textMembrane fractions enriched in transverse tubules, either predominantly free or junctional, sarcoplasmic reticulum subfractions and purified sarcolemmal preparations have been isolated from rabbit skeletal muscle and examined for their contents of protein kinase C. Using activity measurements and immunoblotting methods, we have been able to detect substantial amounts of endogenous protein kinase C in T-tubules membranes and to a lesser extent, in muscle sarcolemma. Protein kinase C was found to be highest in junctional T-tubules and to be virtually absent from sarcoplasmic reticulum-derived membrane fractions. Immunofluorescence staining of muscle fibers is consistent with a T-tubule localization of the kinase. The T-tubule-associated protein kinase C enzyme phosphorylates several potentially important membrane proteins
- …
