Characterization of the 70 kDa polypeptide of the Na/Ca exchanger

被引:23
作者
Saba, RI
Bollen, A
Herchuelz, A
机构
[1] Free Univ Brussels, Sch Med, Pharmacol Lab, B-1070 Brussels, Belgium
[2] Univ Brussels, Fac Sci, Lab Appl Genet, B-1400 Nivelles, Belgium
关键词
Na/Ca exchange; heart sarcolemmal vesicles; protein purification;
D O I
10.1042/0264-6021:3380139
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1-21; NCX1, residues 393-306: and Exon F, residues 622-644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same M-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35%, from its C-terminal portion as compared with that of the wild-type exchanger.
引用
收藏
页码:139 / 145
页数:7
相关论文
共 29 条
[21]  
REEVES JP, 1985, CURR TOP MEMBR TRANS, V25, P77
[22]  
REEVES JP, 1990, INTRACELLULAR CALCIU, P305
[23]   RAPID, SENSITIVE, AND SPECIFIC METHOD FOR DETERMINATION OF PROTEIN IN DILUTE-SOLUTION [J].
SCHAFFNE.W ;
WEISSMAN.C .
ANALYTICAL BIOCHEMISTRY, 1973, 56 (02) :502-514
[24]  
SOLDATI L, 1985, J BIOL CHEM, V260, P3321
[25]   Secondary and tertiary structure changes of reconstituted P-glycoprotein - A Fourier transform attenuated total reflection infrared spectroscopy analysis [J].
Sonveaux, N ;
Shapiro, AB ;
Goormaghtigh, E ;
Ling, V ;
Ruysschaert, JM .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (40) :24617-24624
[26]   Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells [J].
VanEylen, F ;
Svoboda, M ;
Herchuelz, A .
CELL CALCIUM, 1997, 21 (03) :185-193
[27]  
VERMURI R, 1988, BIOCHIM BIOPHYS ACTA, V937, P258
[28]   FOURIER-TRANSFORM INFRARED-SPECTROSCOPY STUDY OF THE SECONDARY STRUCTURE OF THE RECONSTITUTED NEUROSPORA-CRASSA PLASMA-MEMBRANE H+-ATPASE AND OF ITS MEMBRANE-ASSOCIATED PROTEOLYTIC PEPTIDES [J].
VIGNERON, L ;
RUYSSCHAERT, JM ;
GOORMAGHTIGH, E .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (30) :17685-17696
[29]   PARTIAL-PURIFICATION OF NA+-CA2+ ANTIPORTER FROM PLASMA-MEMBRANE OF CHICK HEART [J].
WAKABAYASHI, S ;
GOSHIMA, K .
BIOCHIMICA ET BIOPHYSICA ACTA, 1982, 693 (01) :125-133