Purification of a putative Na+ /D‐glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column

FEBS Letters

Morrison, Alison I.¹  Kitlar, Tomas¹  Deutscher, Josef¹  Kinne, Rolf¹ 

[1] Max-Planck-Institut für Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, FRG

关键词: Affinity chromatography;    Phlorizin;    Na+ /D-glucose cotransporter;    (Brush border;    Pig kidney);    BBM;    brush border membrane;    DMS;    dimethyl suberimidate;    DTT;    dithiothreitol;    Hepes;    N-(2-hydroxy-ethyl)piperazine-N′-2-ethanesulfonic acid;    HPLC;    high performance liquid chromatography;    HRP;    horseradish peroxidase;    LLC-PK1;    pig kidney cells from American Type Culture Collection strain ATCC no. CL 101;    M r;    relative molecular mass;    SDS-PAGE;    SDS-polyacrylamide gel electrophoresis;    TFA;    trifluoroacetic acid;    Tris;    tris(hydroxymethyl)aminomethane;   

DOI  :  10.1016/0014-5793(88)81315-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Phlorizin, a potent inhibitor of the Na⁺ /D-glucose cotransporter, was derivatised to 3-aminophlorizin and subsequently coupled to Affi-Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X-100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na⁺ /D-glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi-Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na⁺; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.

【 授权许可】

Unknown