MOLECULAR-SIZE OF THE FUNCTIONAL COMPLEX AND PROTEIN SUBUNITS OF THE RENAL PHOSPHATE SYMPORTER

被引:8
作者
DELISLE, MC
GIROUX, S
VACHON, V
BOYER, C
POTIER, M
BELIVEAU, R
机构
[1] UNIV MONTREAL, RECH TRANSPORT MEMBRANAIRE GRP, MONTREAL H3C 3J7, PQ, CANADA
[2] HOP ST JUSTINE, MED GENET SECT, MONTREAL H1W 2C4, PQ, CANADA
关键词
D O I
10.1021/bi00197a012
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 oligomeric structure of the rabbit renal brush-border membrane sodium/phosphate cotransporter was examined with the radiation inactivation and fragmentation technique. The size of its functional complex (its ''radiation inactivation size'') was estimated from the rate of decay of its sodium-dependent transport activity as a function of the radiation dose. A radiation inactivation size of 223 +/- 42 kDa was obtained. The polypeptide constituting the monomeric unit of the Na1+/P-i symporter was detected by immunoblotting with polyclonal anti-peptide antibodies directed against the 14 amino acid C-terminal portion of the symporter molecule. Its apparent molecular size estimated by comparison with standards following SDS-polyacrylamide gel electrophoresis was 64 000. This value is in good agreement with its known molecular mass of 51 797 Da calculated from the amino acid sequence deduced from the nucleotide sequence of its gene since this protein is probably glycosylated. The loss of labeling intensity of the polypeptide of M(r) = 64 000 was also measured as a function of radiation dose. The molecular size calculated from these data (its ''target size'') was 165 +/- 20 kDa. The target size estimated for the rat phosphate cotransporter was 184 +/- 46 kDa, and its previously reported radiation inactivation size was 234 +/- 14 kDa. These results strongly suggest that the renal Na1+/P-i cotransporter exists as an oligomeric protein; probably a homotetramer. The fact that the values obtained for the target size are about 3/4 those obtained for the radiation inactivation size of these cotransport proteins indicates that their subunits are closely associated since most of their subunits appear to be fragmented by a single ionizing radiation hit.
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页码:9105 / 9109
页数:5
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