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At least 19 recordsLinked to original sources
PubMed1991

Phosphorylation of cardiac and skeletal muscle calsequestrin isoforms by casein kinase II. Demonstration of a cluster of unique rapidly phosphorylated sites in cardiac calsequestrin.

Calsequestrin is an acidic Ca2(+)-binding protein of sarcoplasmic reticulum existing as different gene products in cardiac muscle and skeletal muscle. A unique feature of cardiac calsequestrin is a 31-amino acid-long COOH-terminal tail (Scott, B. T., Simmerman, H. K. B., Collins, J. H., Nadal-Ginard, B., and Jones, L. R. (1988) J. Biol. Chem. 263, 8958-8964), which is highly acidic and contains several consensus phosphorylation sites for casein kinase II. In the work described here, we tested whether this cardiac-specific sequence is a substrate for casein kinase II. Both cardiac and skeletal muscle calsequestrins were phosphorylated by casein kinase II, but cardiac calsequestrin was phosphorylated to a higher stoichiometry and at least 50 times more rapidly. The site of rapid phosphorylation of cardiac calsequestrin was localized to the distinct COOH terminus, where a cluster of three closely spaced serine residues are found (S378DEESN-DDSDDDDE-COOH). The slower phosphorylation of skeletal muscle calsequestrin occurred at its truncated COOH terminus, at threonine residue 363 (I351NTEDDDDDE-COOH). The similar sequence in cardiac calsequestrin (I351NTEDDDNEE) was not phosphorylated. Cardiac calsequestrin, as isolated, already contained 1.2 mol of Pi/mol of protein, whereas skeletal muscle calsequestrin contained only trace levels of Pi. The endogenous Pi of cardiac calsequestrin was also localized to the distinct COOH terminus. Our results indicate that the cardiac isoform of calsequestrin is the preferred substrate for casein kinase II both in vivo and in vitro.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of chicken calsequestrin deduced from cDNA: comparison of calsequestrin and aspartactin.

We 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.

Amino Acid Sequence↗
PubMed1988

Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin.

Calsequestrin is a Ca2+-binding protein located intraluminally in the junctional sarcoplasmic reticulum (SR) of striated muscle. In this study, Ca2+ binding to cardiac calsequestrin was assessed directly by equilibrium dialysis and correlated with effects on protein conformation and calsequestrin's ability to interact with other SR proteins. Cardiac calsequestrin bound 800-900 nmol of Ca2+/mg of protein (35-40 mol of Ca2+/mol of calsequestrin). Associated with Ca2+ binding to cardiac calsequestrin was a loss in protein hydrophobicity, as revealed with use of absorbance difference spectroscopy, fluorescence emission spectroscopy, and photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine. Ca2+ binding to cardiac calsequestrin also caused a large change in its hydrodynamic character, almost doubling the sedimentation coefficient. We observed that cardiac calsequestrin was very resistant to several proteases after binding Ca2+, consistent with a global effect of Ca2+ on protein conformation. Moreover, Ca2+ binding to cardiac calsequestrin completely prevented its interaction with several calsequestrin-binding proteins, which we identified in cardiac junctional SR vesicles for the first time. The principal calsequestrin-binding protein identified in junctional SR vesicles exhibited an apparent Mr of 26,000 in sodium dodecyl sulfate-polyacrylamide gels. This 26-kDa calsequestrin-binding protein was greatly reduced in free SR vesicles and absent from sarcolemmal vesicles and was different from phospholamban, an SR regulatory protein exhibiting a similar molecular weight. Our results suggest that the specific interaction of calsequestrin with this 26-kDa protein may be regulated by Ca2+ concentration in intact cardiac muscle, when the Ca2+ concentration inside the junctional SR falls to submillimolar levels during coupling of excitation to contraction.

Animals↗
PubMed1992

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.

Animals↗
PubMed1994

Sarcoplasmic reticulum calsequestrins: structural and functional properties.

Calsequestrin is the major Ca(2+)-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its alpha-helical content and the intrinsic fluorescence upon binding of Ca2+. Calsequestrin binds Ca2+ with high-capacity and with moderate affinity and it functions as a Ca2+ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca2+ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsequestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.

Amino Acid Sequence↗
PubMed1998

Cloning of the genes encoding mouse cardiac and skeletal calsequestrins: expression pattern during embryogenesis.

Calsequestrin is a low-affinity and high-capacity calcium-binding protein in the sarcoplasmic reticulum (SR). In the present study, we have cloned and sequenced mouse cardiac and skeletal calsequestrin cDNAs. The deduced amino acid sequences are highly homologous to those of other mammalian calsequestrins. As expected, the cardiac and skeletal calsequestrins are expressed specifically and exclusively in adult heart and skeletal muscles, respectively. In-situ hybridization was performed to examine the expression pattern of the calsequestrins in the developing mouse and rat embryos. During early organogenesis, the cardiac and skeletal calsequestrin transcripts were detected exclusively in the heart primordium and the myotome of somites, respectively. The cardiac calsequestrin transcripts were later detected in fetal heart and skeletal muscles, whereas the skeletal calsequestrin transcripts were only found in fetal skeletal muscles. These data suggest that the cardiac calsequestrin plays a role in the differentiation and function of heart, and in the function of fetal skeletal muscles in conjunction with the skeletal calsequestrin, but not in the early differentiation of the myotome of somites. The expression of the skeletal calsequestrin in the myotome is regulated probably by myogenin, a myogenic regulatory gene.

Amino Acid Sequence↗
PubMed1991

Frog cardiac calsequestrin. Identification, characterization, and subcellular distribution in two structurally distinct regions of peripheral sarcoplasmic reticulum in frog ventricular myocardium.

Calsequestrin is a calcium-binding protein known to sequester calcium accumulated in the sarcoplasmic reticulum (SR) of muscle cells during relaxation. In the present study, we used affinity-purified antibodies to chicken cardiac calsequestrin to identify a 60,000-Da calsequestrin in frog myocardium. Like previously identified cardiac calsequestrins, it is enriched in cardiac microsomes, it is enriched by biochemical procedures previously used to purify cardiac and skeletal calsequestrins, and it exhibits a pH-dependent shift in its apparent Mr on a two-dimensional gel system. Finally, the NH2-terminal amino acid sequence of this 60,000-Da immunoreactive protein purified by fast protein liquid chromatography was identical to that of rabbit skeletal and canine cardiac calsequestrin. Thus, we conclude that this protein corresponds to the calsequestrin isoform in frog ventricular muscle. Frog calsequestrin was localized in discrete foci present at the periphery but absent from the central regions of frog ventricular myocytes as determined by immunofluorescence labeling. Immunoelectron microscopic labeling demonstrated that calsequestrin was confined to the lumen of two structurally distinct regions of the SR, where it was localized in the subsarcolemmal region of the myofibers. One of these appeared to correspond to the terminal SR previously reported to be closely apposed to the sarcolemma of frog myofibers. The other region, although close to the sarcolemma, was not physically joined to it and appeared to correspond to corbular SR. It generally is believed that frog cardiac SR does not provide activator Ca2+ required for excitation-contraction coupling. However, the identification of a calsequestrin isoform very similar to mammalian cardiac calsequestrin that is confined to specialized regions of frog cardiac SR lends support to the idea that frog cardiac SR has the ability to store Ca2+ and thus function in some capacity in frog cardiac muscle contraction.

Amino Acid Sequence↗
PubMed1993

Ca(2+)-induced folding and aggregation of skeletal muscle sarcoplasmic reticulum calsequestrin. The involvement of the trifluoperazine-binding site.

Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca(2+)-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca(2+)-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca(2+)-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca(2+)-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca(2+)-binding by calsequestrin.

Amino Acid Sequence↗
PubMed1987

Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin.

It is proposed that smooth-muscle endoplasmic reticulum contains calsequestrin and that this protein in smooth muscle resembles cardiac calsequestrin more than the skeletal-muscle form. This proposal is based on seven similarities between the smooth-muscle protein and cardiac calsequestrin. Proteins with an Mr of 55,000 can be extracted from the membranes of smooth muscle and of cardiac muscle using 100 mM Na2CO3. The protein from smooth muscle binds to phenyl-Sepharose in the absence of Ca2+ and is released by 10 mM CaCl2, as has been observed for cardiac calsequestrin. The protein from smooth muscle comigrates with the cardiac calsequestrin on Laemmli-type SDS-polyacrylamide gel electrophoresis. The protein of Mr 55,000 from smooth muscle and cardiac calsequestrin both strain blue with the carbocyanine dye Stains-all. Both proteins present similar one-dimensional Cleveland peptide maps although minor differences might exist. From an analysis of subcellular membranes separated by sucrose gradient centrifugation it is concluded that the protein with Mr 55,000 from the smooth muscle is confined to the endoplasmic reticulum, the same subcellular structure from which, in heart muscle, calsequestrin can be isolated. Antibodies raised against canine cardiac calsequestrin bind to a protein of similar Mr in smooth-muscle endoplasmic reticulum. In addition to the calsequestrin, three other extrinsic proteins with an Mr of 130,000, 100,000 and 63,000, stain blue with Stains-all and occur in the endoplasmic reticulum of smooth muscle.

Animals↗
PubMed1987

Terbium-binding properties of calsequestrin from skeletal muscle sarcoplasmic reticulum.

Calsequestrin (Mr = 40,000) is a calcium-binding protein (Kd = 1 mM, 50 sites/molecule) located within the terminal cisternae of the sarcoplasmic reticulum of skeletal muscle cells. The interaction of terbium, a calcium analog, with rabbit skeletal muscle calsequestrin was studied by fluorescence and circular dichroism spectroscopy. Direct measurement of terbium binding using a fluorescence assay for terbium revealed that calsequestrin bound approx. 30 mol of terbium per mol of protein with an affinity of approx. 7 microM. The fluorescence of terbium measured at 545 nm was enhanced dramatically upon binding to calsequestrin, reaching a maximum value at a terbium to protein ratio of 28. The excitation spectrum of protein-bound terbium and chemical modification studies revealed that energy transfer occurred between aromatic residues, including tryptophan and bound terbium. Terbium bound to calsequestrin could be removed by EGTA, or displaced by Ca2+ or La3+. In the presence of Ca2+ or La3+ terbium bound to calsequestrin with a higher apparent affinity and lower capacity. 0.1 M KCl or 5 mM MgCl2 had little effect on terbium binding. Terbium increased the intrinsic fluorescence of calsequestrin 2-fold, and increased the alpha-helical content of calsequestrin from 16 to 33%. Terbium binding induces the same conformational changes in calsequestrin as does calcium, confirming that terbium is a useful calcium analog in this system.

Animals↗
PubMed1989

Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum.

Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.

Animals↗
PubMed1991

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

Organization of calsequestrin-positive sarcoplasmic reticulum in rat cardiomyocytes in culture.

The sarcoplasmic reticulum (SR) regulates the levels of cytoplasmic free Ca2+ ions in muscle cells. Calsequestrin is a major Ca(2+)-storing protein and is localized at special sites in the SR. To investigate the development of calsequestrin-positive SR and its interaction with the cytoskeleton, we examined the distribution of calsequestrin in cultured cardiomyocytes from newborn rats by immunofluorescence with anticalsequestrin and antitubulin antibodies and rhodamine-phalloidin. In frozen sections of neonatal rat heart, anticalsequestrin immunostaining was apparent as cross-striations at Z-lines. When newborn cardiomyocytes were isolated, calsequestrin-positive SR was disorganized and was apparent as small vesicles beneath the sarcolemma, whereas myofibrils accumulated in the center of the cells. As the cells spread in culture, calsequestrin-positive vesicles spread to the periphery of the cytoplasm, becoming associated with the developing myofibrils. In mature cells, calsequestrin was closely associated with myofibrils, showing cross-striations at the Z-lines. Double-labeling using anticalsequestrin and antitubulin antibodies demonstrated that the distribution of calsequestrin-positive structures was similar to that of the microtubular arrays. When the microtubules were depolymerized by nocodazole at an early stage, the extension of the SR to the cell periphery was inhibited. In mature cardiomyocytes, nocodazole appeared not to affect the distribution of the SR. These results indicate that the calsequestrin-positive SR in cardiomyocytes is organized at the proper sites of myofibrils during myofibrillogenesis and that the microtubules might serve as tracts for the transport of components of the SR.

Animals↗
PubMed1991

Purification and characterization of a calsequestrin-like calcium-binding protein from carp (Cyprinus carpio) sarcoplasmic reticulum.

1. A calsequestrin-like calcium-binding protein was purified from carp sarcoplasmic reticulum by column chromatographies using DEAE-cellulose and Butyl-Toyopearl 650S. 2. The mol. wt was estimated to be 50 kDa, which was larger than that of rabbit calsequestrin (42 kDa). 3. Carp calsequestrin-like protein bound Ca2+ with a higher affinity (apparent Kd = 400 microM) and lower capacity (25 mol/mol) compared with rabbit calsequestrin (1 mM and 40-50 mol/mol, respectively). 4. Anti-carp calsequestrin-like protein rabbit antiserum reacted with rabbit calsequestrin in immunoblotting analysis. 5. Carp calsequestrin-like protein was rich in acidic amino acids, as was rabbit calsequestrin.

Amino Acids↗
PubMed1976

Assembly of the sarcoplasmic reticulum. Biosynthesis of calsequestrin in rat skeletal muscle cell cultures.

Temporal patterns of biosynthesis of the sarcoplasmic reticulum protein, calsequestrin, were analyzed and compared with rates of ATPase synthesis in primary cultures of rat skeletal muscle cells. Rates of synthesis were measured by the incorporation of radioactive leucine into the isolated proteins. Cells at various stages of differentiation were incubated for 2 h with tritium-labeled leucine and extracted with detergent. The extracts were incubated with antibodies specific against calsequestrin or the ATPase and immunoprecipitates were separated by disc gel electrophoresis. Incorporation of radioactivity into bands identified as calsequestrin or the ATPase was analyzed by counting of gel slices. In Dulbecco's modified Eagles medium (DME medium) containing 0.1 volume of horse serum and 0.005 volume of chick embryo extract, the cells began to fuse after about 50 h in culture, forming multinucleated myotubes. Calsequestrin synthesis was barely detectable after 24 h in culture. After 44 h, before fusion of myoblasts began, the rate of calsequestrin synthesis increased severalfold. The rate of synthesis continued to increase until about 72 h and then diminished. If cells were transferred at 44 h to DME medium containing 0.2 volume of fetal calf serum and 0.08 volume of chick embryo extract, fusion was delayed by about 20 h. In this medium the rate of calsequestrin synthesis diminished after a peak at 44 h but, by contrast, the rate of synthesis of the ATPase increased dramatically following fusion at about 80 h. If cells were transferred at about 40 h to DME medium containing 0.1 volume of horse serum and only 60 muM Ca2+ the cells did not fuse and, again, the rate of calsequestrin synthesis was diminished after a peak at about 40 h. By contrast the rate of ATPase synthesis increased sharply in spite of the lack of fusion. Both proteins were degraded with a half-life of about 20 h. These studies show that the synthesis of calsequestrin, an extrinsic membrane protein, and the ATPase, an intrinsic protein of the same membrane, are synthesized under separate control.

Adenosine Triphosphatases↗
PubMed1989

Plant cells contain calsequestrin.

Calsequestrin is a high capacity low affinity Ca2+-binding protein thought to be essential for the function of the intracellular rapid releasable Ca2+ pool of a variety of animal cells. Here we show that two types of plant tissues, cultured Streptanthus tortuosus cells and spinach leaves, contain a form of calsequestrin. In subcellular fractions of S. tortuosus cells, Stains-all staining reveals a metachromatically blue-staining 56,000-Da protein enriched in the microsomal fraction. This protein shares several biochemical characteristics with animal calsequestrin: 1) it changes its apparent molecular weight with the pH; 2) it is able to bind 45Ca2+ on nitrocellulose transfers; and 3) it is recognized by antibodies against canine cardiac calsequestrin. Calsequestrin was also identified in spinach leaves using a direct extraction procedure that was developed for muscle calsequestrin. Thus, our results demonstrate that plant cells contain calsequestrin within a subcellular membrane fraction. These results also suggest that calsequestrin is an ubiquitous protein rather than being limited only to animal cells.

Animals↗
PubMed1988

Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning.

cDNA cloning was used to deduce the complete amino acid sequence of canine cardiac calsequestrin, the principal Ca2+-binding protein of cardiac junctional sarcoplasmic reticulum. Cardiac calsequestrin contains 391 amino acid residues plus a 19-residue amino-terminal signal sequence. The molecular weight of the mature protein, excluding carbohydrate, is 45,269. Cardiac calsequestrin is highly acidic, and a striking feature is the enrichment of acidic residues (60%) within the 63 carboxyl-terminal residues. No part of the sequence contains EF hand Ca2+-binding structures. The photo-affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to localize the Ca2+-regulated hydrophobic site to amino acid residues 192-223. The cardiac and skeletal muscle isoforms of calsequestrin (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171), although the products of different genes, are 65% identical, are acidic, and share one glycosylation site. However, cardiac calsequestrin has several unique features. First, it has a 31-amino acid extension at its carboxyl terminus (residues 361-391), which contains 71% acidic residues and a second glycosylation site. Second, its mRNA contains a second open reading frame with the capacity to code for a 111-amino acid protein. Third, contrary to the restricted expression of the fast skeletal isoform, cardiac calsequestrin mRNA is present in both cardiac and slow skeletal muscle, but not in fast skeletal muscle. We conclude that the deduced amino acid sequence of cardiac calsequestrin is consistent with its ability to bind large amounts of Ca2+ (40 mol of Ca2+/mol of calsequestrin). The protein probably binds Ca2+ by acting as a charged surface rather than by presenting multiple discrete Ca2+-binding sites.

Amino Acid Sequence↗