Vol. August
170,
No. 76,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 1089-1095
1990
Amino Acid Sequence of Chicken Calsequestrin Deduced from c DNA: Comparison of Calsequestrin and Aspartactin Paul J. Yazaki*, Sergio Salvatori”, and A. Stephen Dahms* *Departments
of Chemistry, Molecular Biology Institute, and Rees-Stealy Research Foundation, San Diego State University, San Diego, CA 92182
“National Research Council Unit for Muscle Biology and Physiopathology, Pathology, University of Padua, Padua, Italy Received
June
26,
Institute of General
1990
We have previously reportedthe aminoterminal sequenceof adult chicken calsequestrin,an intraluminal Ca2+-binding protein isolatedfrom fast-twitch skeletalmuscle.The partial sequence showedhomology with mammaliancalsequestrinscontained in the PIR data bank and complete identity with the amino terminusof a putative laminin-binding protein of the extracellular matrix, aspartactin.Basedon thesedata, oligonucleotide primerswere synthesizedfor PCR amplification and direct DNA sequencing.We report herein the primary sequenceof chicken calsequestrin, deducedfrom cDNA. The sequencehasbeenverified by amino acid sequencingof internal tryptic peptides. Importantly, the data show the primary structure of calsequestrinto be identical to the amino acid sequencereported for aspartactin,with the exception of a singleamino acid difference (ileu vs. val) which may be animal strain-related.Basedon thesedata, calsequesttinandaspartactin are the sameprotein. 0 1990Rcademic Press.Inc.
Calsequestrinis the major Ca2+-binding protein involved in the sequestrationof Ca2+ within the terminal cistemaeof the skeletalmusclesarcoplasmicreticulum and possesses the ability to bind 50 Ca2+per molecule, with moderateaffinity (Kd = 1 mM) (l-4). Upon nerve stimulation, a rapid depolarization of the transversetubule occursand, by an asyet unknown mechanism,Ca2+ is released from its association with calsequestrin and exits the lumen of the sarcoplasmic reticulum, thereby initiating musclecontraction (5). Calsequestrinwasfirst isolatedfrom rabbit fast twitch skeletal muscle(1) and hasbeen well characterizedat the biochemical level (6, 7), yet the exact mechanismof Ca2+-bindinghasnot beenelucidated.Severalinvestigatorshave reportedthat chicken skeletalmusclecalsequestrinbindsonly one half the amountof Ca2+ascomparedto rabbit skeletalmusclecalsequestrin(g-10). The primary amino acid sequenceof the alternatively spliced rabbit neonataland adult fast-twitch skeletalmusclecalsequestrins have beendeterminedby cDNA cloning (1 I, 12); the cDNA sequencefor canine cardiac calsequestrinhas also beendetermined, and it is encoded asa separategene(13). ABBREVIATIONS: PIR, Protein Identification Resource;PCR, polymerasechain reaction; DNA, deoxyribonucleic acid; cDNA, complementarydeoxyribonucleic acid; mRNA, messenger ribonucleic acid.
1089
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
We have recently reported the partial amino terminal sequence of the mature form of chicken skeletal muscle calsequestrin and have shown common identity with known mammalian calsequestrin isoforms (14). Strikingly, we have found complete sequence identity for the twenty amino acids that form the mature amino terminus of adult skeletal calsequestrin with the amino terminus of a putative laminin-binding protein of the extracellular matrix, aspartactin (1516). In this report, we present the primary sequence of adult chicken fast-twitch skeletal muscle calsequestrin
as deduced from the level of cDNA
sequence data. The results confii
and verified by partial internal amino acid
our previous suggestion that calsequestrin and aspartactin are
indeed the same protein. MATERIALS
AND METHODS
SR vesicles from adult chicken pectoralis muscle were isolated by a Ca2+ oxalate loading procedure, followed by sucrose gradient ultracentrifugation (17), while an enriched terminal cistemae fraction was isolated by the method of Saito (18). The purification of calsequestrin is described in detail by Damiani et al. (10); briefly, partially purified membrane vesicles or column purified calsequestrin was electrophoresed on a SDS-PAGE (lo-20%) gradient and electroblotted onto Immobilon-P (Millipore) (19). A single Mr 51,000 band was identified by a rabbit antichicken calsequestrin IgG fraction (9), metachromatic staining with the dye Stain-All (20), and Ca2+-binding determined by a 45Ca-gel overlay technique (10). Purified calsequestrin was subjected to limited proteolytic digestion by trypsin (21). Blotted undigested calsequestrin and tryptic peptide fragments were subjected to automated microsequencing analysis on an Applied Biosystems (Foster City) model 470A protein sequencer (22). Oligonucleotides were synthesized on an Applied Biosystems model 380 DNA synthesizer (Foster City), based on backtranslation of the amino terminus of the mature form of calsequestrin (EEGLNFPTYDGKDRVIDLNE) and a known mammalian calsequestrin conserved carboxy terminal consensus sequence (INTEDDD). Polyadenylated mRNA was isolated from the breast muscle of adult chicken (23). Poly(A) mRNA (0.5 ug) and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Sciences) in the presence of 50 pmoles of the 3’ primer (corresponding to the calsequestrin consensus sequence, 5’ GTCATCGTCITCAGTATT 3’) was used to synthesize single-stranded cDNA. The mixture was heated to 95 oC for 10 minutes to denature the reverse transcriptase and with the addition of 50 pmoles of the 5’ primer (which corresponds to the amino terminal sequence, 5’ GAAGAAGGACTGAATITCCC 3’) and AmphiTaq ( Perkin ElmerKetus) to this mixture, second strand synthesis was carried out at 50 oC for 45 min. The reaction was placed in DNA Thermocycler (Perkin Elmer Cetus), and amplified (30 cycles of 94 oC X 1 min., 37 oC X 2 min., 72 OC X 5 min.) (24). The reaction mixture, electrophoresed on a 1% agarose gel, yielded a band of approximately 1.1 kb upon staining with ethidium bromide. This band was excised and eluted with Geneclean (BiolOl Inc.), and portions were used as template for asymmetric PCR in both orientations (0.5 pmoles / 50 pmoles of respective primers, 30 cycles of 94 eC X 1 min., 50 eC X 2 min., 72 oC X 3 min.) (25). The asymmetric PCR product was combined with 1 mL of water, spun and washed twice through a Centricon 30 microconcentrator (Amicon). This procedure was repeated using an internal primer (corresponding to nucleotides 498-518, determined by DNA sequencing; 5’ GCTGAACACTTCCAGCCATAT 3’) and a 3’ primer (complementary to the untranslated region of aspartactin, 5’ AGGCTCTCTGATGAAGGGACC 3’), in order to amplify the entire open reading frame. Determination of the DNA sequence, employing initial and internal primers, was conducted by dideoxy chain termination (26) using Sequenase (U.S. Biochemical). Sequence data was analyzed by searching PIR (27) data banks utilizing the FASTA (28) and Staden (29) programs, and multiple sequence alignments were performed using the NEWAT program (30) on a VAX 6230. RESULTS AND DISCUSSION We have previously reported the partial amino terminal sequence of the mature form of chicken calsequesnin, isolated from breast fast-twitch skeletal muscle (14). Backtranslation of this 1090
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
partial amino acid sequence (EEGLNFPTYDGKDRVIDLNE) and a mammalian calsequestrin carboxy terminal consensus sequence (INTEDDD) were employed to synthesize oligonucleotides to be used as PCR primers. Reverse transcription of adult chicken breast muscle polyadenylated mRNA employing the 3’ primer, denaturation, and second strand synthesis with the addition of the 5’ primer and Taq polymerase were carried out and then amplified by PCR (24). The product of this amplification was a 1.1 kb band based upon ethidium bromide staining of an agarose gel. The double-stranded product was gel isolated, and aliquots were used as template for asymmetric PCR. These asymmetric products were directly sequenced using the respective amplification primer and additional internal sequencing primers (25). The result of this DNA sequencing was a partial (92 %) open-reading frame coding for a protein with high identity to calsequestrin isoforms whose partial sequences are contained in the PIR (27) data bank. Importantly, the predominant segment of the open-reading frame of intraluminal chicken calsequestrin shows virtually complete identity with a corresponding region of the putative extracellular
l,aminin-binding
protein, chick aspartactin, with the exception of 4 base changes.
Aspartactin has been identified as an extracellular matrix protein which when electrophoresed and transferred to nitrocellulose has the ability to bind 1251-laminin; immunoelectron microscopic results using a conjugated secondary antibody system were interpreted to indicate that aspartactin was located at or near the plasma membrane or was associated with the extracellular matrix (15), although this is subject to other interpretations (14). In order to determine the complete open reading frame, an internal sequencing primer and a primer utilizing the 3’ untranslated region of aspartactin was used for reverse transcription,
PCR
and direct sequencing (25). This 0.75 kb band showed an identical 117 nucleotide overlap with the previously sequenced segment. Combining these two DNA fragments resulted in an open reading frame of 1164 nucleotides coding for 387 amino acids, shown in Figure 1. Computer-predicted physical characteristics are: 1) an estimated isoelectric point of 3.84; 2) molecular weight of 45,060 dahons; 3) no transmembranous segments; and 4) one probable N-linked glycosylation site for this glycoprotein. In comparing the complete open reading frame of chick aspartactin cDNA (14, 16), there were a total of 5 nucleotide differences (at positions 114, 385, 429, 720 and 117 1). Four base differences resulted in no amino acid change; base 385 codes for Ileu12g in calsequestrin vs. Va1129 in aspartactin. This minor conservative amino acid difference may be due to animal strain variation, since aspartactin was cloned from a day-l 1 chick embryo library, while calsequestrin was isolated from adult chickens of a different strain (17). Based upon these results, the two sequences can be considered identical and aspartactin is calsequestrin. As additional proof, we explored sequencing of internal tryptic fragments of chicken calsequestrin. We purified chicken calsequestrin (10) and subjected it to limited tryptic proteolysis. Two internal tryptic fragments, shown in Figure 1, were blotted onto Immobilon-P and subjected to direct amino acid sequencing (22); internal sequencing results, corresponding to nucleotide spans 136-201 and 694-747, show an exact identity of the corresponding amino acid sequences of calsequestrin to aspartactin. The results leave little doubt that the cDNA sequenced codes for the intracellular and intraluminaI protein, calsequestrin. Analysis of chicken calsequestrin shows that it, like mammalian calsequesnin, is highly acidic, with no EF hand or known calcium binding structures. The Ca2+-binding domain has been 1091
Vol.
170,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
GluGluGlyLeuAsnPheProThrTyrAspGlyLysAspArqValIleAspLeuAsnGluLysAsnTyrLysHisAlaLeuLysLysTyr GAAGAAGGACTGAATTTCCCCACGTATGATGGGAAAGACCGAGTGATCGACCTGAACGAGAAGAACTACAAGCATGCCCTGAAGAAGTAT ),,,>,,>,)),,,,)),),) 10
20
30
40
50
60
70
80
90
AspMetLeuCysLeuLeuPheHisGluProValSerSerAspArqValSerGlnLysGlnPheGlnMetThrGluMetValLeuGluLeu GACATGCTTTGTCTGCTCTTCCATGAGCCTGTGAGCTCCGACAGGGTGTCCCAGAAGCAGTTCCAGATGACGGAGATGGTCCTGGAGCTG >,>>>>>>,>> 460
470
480
490
500
510
520
AspLysGlyValAlaLysLysLeuGlyLeuLysMetAsnGluValAspPheTyrGluProPheMetAspGluProValHisIleProAsp GACAAAGGGGTTGCCAAGAAACTGGGCCTGAAGATG~CGAGGTGGACTTCTATGAACCATTCATGGATGAGCCTGTTCACATCCCCGAT 550
560
570
580
590
600
LysProTyrThrGluGluGluLeuValGluPheValLysGluHisLysA~gAlaThrLeuArqLysLeuArqProGluAspMetPheGlu AAACCTTACACAGAAGAGGAGCTGGTTGAATTTGTGAAGGAGCCACCTTGCGGAAGCTGCGCCCAGAAGACATGTTTGAG 640
650
660
670
680
690
ThrTrpGluAspAspMetGluGlyIleHisIleValAlaPheAlaGluGluAspAspProAspGlyPheGluPheLeuGluIleLeuLys ACATGGGAGGATGACATGGAAGGAATCCATATCCATATCGTGGCCTTTGCTGAAGAAGATGACCCAGATGGCTTTGAGTTCCTGG~ATCCTGAAG 730
740
750
760
770
780
790
800
810
890
900
980
990
GlnValAlaArqAspAsnThrAspAsnProASpLeuSerIleValTrpIleAspProAspAspPheProLeuLeuIleThrTyrTrpGlu CAGGTTGCCAGGGACAACACTGATAATCCTGACCTGAGCATTGTGTGGATTGACCCTGACGACTTTCCTCTGCTCATCACTTACTGGGAG 820
830
840
850
860
870
880
f LysThrPheLysIleAspLeuPheArqProGlnIleGlyIleValAsnValThrAspAlaAspSerVdlTrpMetGluIleArqAspAsp AAGACCTTCAAGATTGACCTGTTCAGGCCGCAGATTGGGATTGTG~TGTCACAGACGCTGACAGTGTCTGGATGGAGATCAGAGATGAT 910
920
930
940
950
960
970
AspAspLeuProThrAlaGluGluLeuGluAspTrpIleGluAspValLeuSerGlyLysIleAsnThrGluAspAspAspAspAspAsp GATGACCTGCCCACAGCCGAGGAGCTGGAGGACTGGATAGAGGATGTGCTTTCTGGGAAGATAAATACTGAAGACGATGACGATGATGAC (‘(((((
170,
No. 76,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 1089-1095
1990
Amino Acid Sequence of Chicken Calsequestrin Deduced from c DNA: Comparison of Calsequestrin and Aspartactin Paul J. Yazaki*, Sergio Salvatori”, and A. Stephen Dahms* *Departments
of Chemistry, Molecular Biology Institute, and Rees-Stealy Research Foundation, San Diego State University, San Diego, CA 92182
“National Research Council Unit for Muscle Biology and Physiopathology, Pathology, University of Padua, Padua, Italy Received
June
26,
Institute of General
1990
We have previously reportedthe aminoterminal sequenceof adult chicken calsequestrin,an intraluminal Ca2+-binding protein isolatedfrom fast-twitch skeletalmuscle.The partial sequence showedhomology with mammaliancalsequestrinscontained in the PIR data bank and complete identity with the amino terminusof a putative laminin-binding protein of the extracellular matrix, aspartactin.Basedon thesedata, oligonucleotide primerswere synthesizedfor PCR amplification and direct DNA sequencing.We report herein the primary sequenceof chicken calsequestrin, deducedfrom cDNA. The sequencehasbeenverified by amino acid sequencingof internal tryptic peptides. Importantly, the data show the primary structure of calsequestrinto be identical to the amino acid sequencereported for aspartactin,with the exception of a singleamino acid difference (ileu vs. val) which may be animal strain-related.Basedon thesedata, calsequesttinandaspartactin are the sameprotein. 0 1990Rcademic Press.Inc.
Calsequestrinis the major Ca2+-binding protein involved in the sequestrationof Ca2+ within the terminal cistemaeof the skeletalmusclesarcoplasmicreticulum and possesses the ability to bind 50 Ca2+per molecule, with moderateaffinity (Kd = 1 mM) (l-4). Upon nerve stimulation, a rapid depolarization of the transversetubule occursand, by an asyet unknown mechanism,Ca2+ is released from its association with calsequestrin and exits the lumen of the sarcoplasmic reticulum, thereby initiating musclecontraction (5). Calsequestrinwasfirst isolatedfrom rabbit fast twitch skeletal muscle(1) and hasbeen well characterizedat the biochemical level (6, 7), yet the exact mechanismof Ca2+-bindinghasnot beenelucidated.Severalinvestigatorshave reportedthat chicken skeletalmusclecalsequestrinbindsonly one half the amountof Ca2+ascomparedto rabbit skeletalmusclecalsequestrin(g-10). The primary amino acid sequenceof the alternatively spliced rabbit neonataland adult fast-twitch skeletalmusclecalsequestrins have beendeterminedby cDNA cloning (1 I, 12); the cDNA sequencefor canine cardiac calsequestrinhas also beendetermined, and it is encoded asa separategene(13). ABBREVIATIONS: PIR, Protein Identification Resource;PCR, polymerasechain reaction; DNA, deoxyribonucleic acid; cDNA, complementarydeoxyribonucleic acid; mRNA, messenger ribonucleic acid.
1089
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
We have recently reported the partial amino terminal sequence of the mature form of chicken skeletal muscle calsequestrin and have shown common identity with known mammalian calsequestrin isoforms (14). Strikingly, we have found complete sequence identity for the twenty amino acids that form the mature amino terminus of adult skeletal calsequestrin with the amino terminus of a putative laminin-binding protein of the extracellular matrix, aspartactin (1516). In this report, we present the primary sequence of adult chicken fast-twitch skeletal muscle calsequestrin
as deduced from the level of cDNA
sequence data. The results confii
and verified by partial internal amino acid
our previous suggestion that calsequestrin and aspartactin are
indeed the same protein. MATERIALS
AND METHODS
SR vesicles from adult chicken pectoralis muscle were isolated by a Ca2+ oxalate loading procedure, followed by sucrose gradient ultracentrifugation (17), while an enriched terminal cistemae fraction was isolated by the method of Saito (18). The purification of calsequestrin is described in detail by Damiani et al. (10); briefly, partially purified membrane vesicles or column purified calsequestrin was electrophoresed on a SDS-PAGE (lo-20%) gradient and electroblotted onto Immobilon-P (Millipore) (19). A single Mr 51,000 band was identified by a rabbit antichicken calsequestrin IgG fraction (9), metachromatic staining with the dye Stain-All (20), and Ca2+-binding determined by a 45Ca-gel overlay technique (10). Purified calsequestrin was subjected to limited proteolytic digestion by trypsin (21). Blotted undigested calsequestrin and tryptic peptide fragments were subjected to automated microsequencing analysis on an Applied Biosystems (Foster City) model 470A protein sequencer (22). Oligonucleotides were synthesized on an Applied Biosystems model 380 DNA synthesizer (Foster City), based on backtranslation of the amino terminus of the mature form of calsequestrin (EEGLNFPTYDGKDRVIDLNE) and a known mammalian calsequestrin conserved carboxy terminal consensus sequence (INTEDDD). Polyadenylated mRNA was isolated from the breast muscle of adult chicken (23). Poly(A) mRNA (0.5 ug) and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Sciences) in the presence of 50 pmoles of the 3’ primer (corresponding to the calsequestrin consensus sequence, 5’ GTCATCGTCITCAGTATT 3’) was used to synthesize single-stranded cDNA. The mixture was heated to 95 oC for 10 minutes to denature the reverse transcriptase and with the addition of 50 pmoles of the 5’ primer (which corresponds to the amino terminal sequence, 5’ GAAGAAGGACTGAATITCCC 3’) and AmphiTaq ( Perkin ElmerKetus) to this mixture, second strand synthesis was carried out at 50 oC for 45 min. The reaction was placed in DNA Thermocycler (Perkin Elmer Cetus), and amplified (30 cycles of 94 oC X 1 min., 37 oC X 2 min., 72 OC X 5 min.) (24). The reaction mixture, electrophoresed on a 1% agarose gel, yielded a band of approximately 1.1 kb upon staining with ethidium bromide. This band was excised and eluted with Geneclean (BiolOl Inc.), and portions were used as template for asymmetric PCR in both orientations (0.5 pmoles / 50 pmoles of respective primers, 30 cycles of 94 eC X 1 min., 50 eC X 2 min., 72 oC X 3 min.) (25). The asymmetric PCR product was combined with 1 mL of water, spun and washed twice through a Centricon 30 microconcentrator (Amicon). This procedure was repeated using an internal primer (corresponding to nucleotides 498-518, determined by DNA sequencing; 5’ GCTGAACACTTCCAGCCATAT 3’) and a 3’ primer (complementary to the untranslated region of aspartactin, 5’ AGGCTCTCTGATGAAGGGACC 3’), in order to amplify the entire open reading frame. Determination of the DNA sequence, employing initial and internal primers, was conducted by dideoxy chain termination (26) using Sequenase (U.S. Biochemical). Sequence data was analyzed by searching PIR (27) data banks utilizing the FASTA (28) and Staden (29) programs, and multiple sequence alignments were performed using the NEWAT program (30) on a VAX 6230. RESULTS AND DISCUSSION We have previously reported the partial amino terminal sequence of the mature form of chicken calsequesnin, isolated from breast fast-twitch skeletal muscle (14). Backtranslation of this 1090
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
partial amino acid sequence (EEGLNFPTYDGKDRVIDLNE) and a mammalian calsequestrin carboxy terminal consensus sequence (INTEDDD) were employed to synthesize oligonucleotides to be used as PCR primers. Reverse transcription of adult chicken breast muscle polyadenylated mRNA employing the 3’ primer, denaturation, and second strand synthesis with the addition of the 5’ primer and Taq polymerase were carried out and then amplified by PCR (24). The product of this amplification was a 1.1 kb band based upon ethidium bromide staining of an agarose gel. The double-stranded product was gel isolated, and aliquots were used as template for asymmetric PCR. These asymmetric products were directly sequenced using the respective amplification primer and additional internal sequencing primers (25). The result of this DNA sequencing was a partial (92 %) open-reading frame coding for a protein with high identity to calsequestrin isoforms whose partial sequences are contained in the PIR (27) data bank. Importantly, the predominant segment of the open-reading frame of intraluminal chicken calsequestrin shows virtually complete identity with a corresponding region of the putative extracellular
l,aminin-binding
protein, chick aspartactin, with the exception of 4 base changes.
Aspartactin has been identified as an extracellular matrix protein which when electrophoresed and transferred to nitrocellulose has the ability to bind 1251-laminin; immunoelectron microscopic results using a conjugated secondary antibody system were interpreted to indicate that aspartactin was located at or near the plasma membrane or was associated with the extracellular matrix (15), although this is subject to other interpretations (14). In order to determine the complete open reading frame, an internal sequencing primer and a primer utilizing the 3’ untranslated region of aspartactin was used for reverse transcription,
PCR
and direct sequencing (25). This 0.75 kb band showed an identical 117 nucleotide overlap with the previously sequenced segment. Combining these two DNA fragments resulted in an open reading frame of 1164 nucleotides coding for 387 amino acids, shown in Figure 1. Computer-predicted physical characteristics are: 1) an estimated isoelectric point of 3.84; 2) molecular weight of 45,060 dahons; 3) no transmembranous segments; and 4) one probable N-linked glycosylation site for this glycoprotein. In comparing the complete open reading frame of chick aspartactin cDNA (14, 16), there were a total of 5 nucleotide differences (at positions 114, 385, 429, 720 and 117 1). Four base differences resulted in no amino acid change; base 385 codes for Ileu12g in calsequestrin vs. Va1129 in aspartactin. This minor conservative amino acid difference may be due to animal strain variation, since aspartactin was cloned from a day-l 1 chick embryo library, while calsequestrin was isolated from adult chickens of a different strain (17). Based upon these results, the two sequences can be considered identical and aspartactin is calsequestrin. As additional proof, we explored sequencing of internal tryptic fragments of chicken calsequestrin. We purified chicken calsequestrin (10) and subjected it to limited tryptic proteolysis. Two internal tryptic fragments, shown in Figure 1, were blotted onto Immobilon-P and subjected to direct amino acid sequencing (22); internal sequencing results, corresponding to nucleotide spans 136-201 and 694-747, show an exact identity of the corresponding amino acid sequences of calsequestrin to aspartactin. The results leave little doubt that the cDNA sequenced codes for the intracellular and intraluminaI protein, calsequestrin. Analysis of chicken calsequestrin shows that it, like mammalian calsequesnin, is highly acidic, with no EF hand or known calcium binding structures. The Ca2+-binding domain has been 1091
Vol.
170,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
GluGluGlyLeuAsnPheProThrTyrAspGlyLysAspArqValIleAspLeuAsnGluLysAsnTyrLysHisAlaLeuLysLysTyr GAAGAAGGACTGAATTTCCCCACGTATGATGGGAAAGACCGAGTGATCGACCTGAACGAGAAGAACTACAAGCATGCCCTGAAGAAGTAT ),,,>,,>,)),,,,)),),) 10
20
30
40
50
60
70
80
90
AspMetLeuCysLeuLeuPheHisGluProValSerSerAspArqValSerGlnLysGlnPheGlnMetThrGluMetValLeuGluLeu GACATGCTTTGTCTGCTCTTCCATGAGCCTGTGAGCTCCGACAGGGTGTCCCAGAAGCAGTTCCAGATGACGGAGATGGTCCTGGAGCTG >,>>>>>>,>> 460
470
480
490
500
510
520
AspLysGlyValAlaLysLysLeuGlyLeuLysMetAsnGluValAspPheTyrGluProPheMetAspGluProValHisIleProAsp GACAAAGGGGTTGCCAAGAAACTGGGCCTGAAGATG~CGAGGTGGACTTCTATGAACCATTCATGGATGAGCCTGTTCACATCCCCGAT 550
560
570
580
590
600
LysProTyrThrGluGluGluLeuValGluPheValLysGluHisLysA~gAlaThrLeuArqLysLeuArqProGluAspMetPheGlu AAACCTTACACAGAAGAGGAGCTGGTTGAATTTGTGAAGGAGCCACCTTGCGGAAGCTGCGCCCAGAAGACATGTTTGAG 640
650
660
670
680
690
ThrTrpGluAspAspMetGluGlyIleHisIleValAlaPheAlaGluGluAspAspProAspGlyPheGluPheLeuGluIleLeuLys ACATGGGAGGATGACATGGAAGGAATCCATATCCATATCGTGGCCTTTGCTGAAGAAGATGACCCAGATGGCTTTGAGTTCCTGG~ATCCTGAAG 730
740
750
760
770
780
790
800
810
890
900
980
990
GlnValAlaArqAspAsnThrAspAsnProASpLeuSerIleValTrpIleAspProAspAspPheProLeuLeuIleThrTyrTrpGlu CAGGTTGCCAGGGACAACACTGATAATCCTGACCTGAGCATTGTGTGGATTGACCCTGACGACTTTCCTCTGCTCATCACTTACTGGGAG 820
830
840
850
860
870
880
f LysThrPheLysIleAspLeuPheArqProGlnIleGlyIleValAsnValThrAspAlaAspSerVdlTrpMetGluIleArqAspAsp AAGACCTTCAAGATTGACCTGTTCAGGCCGCAGATTGGGATTGTG~TGTCACAGACGCTGACAGTGTCTGGATGGAGATCAGAGATGAT 910
920
930
940
950
960
970
AspAspLeuProThrAlaGluGluLeuGluAspTrpIleGluAspValLeuSerGlyLysIleAsnThrGluAspAspAspAspAspAsp GATGACCTGCCCACAGCCGAGGAGCTGGAGGACTGGATAGAGGATGTGCTTTCTGGGAAGATAAATACTGAAGACGATGACGATGATGAC (‘(((((
