The isolation and reconstitution of the ADP/ATP carrier from wild‐type Saccharomyces cerevisiae Identification of primarily one type (AAC‐2)

FEBS Letters

Knirsch, Martin¹  Gawaz, Meinrad P.¹  Klingenberg, Martin¹ 

[1] Institut für Physikalische Biochemie, Universität München, Goethestrasse 33, 8000 München 2, FRG

关键词: ADP/ATP carrier;    Cyanogen bromide cleavage;    Reconstitution;    (Saccharomyces cerevisiae);    DTE;    dithioerythritol;    BSA;    bovine serum albumin;    PMSF;    phenylmethylsulfonyl fluoride;    HTS;    hydroxyapatite;    CAT;    carboxyatractylate;    BKA;    bongrekic acid;   

DOI  :  10.1016/0014-5793(89)80577-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2. Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2. The isolated AAC is active in ADP/ATP exchange in reconsitituted liposomes with a V _max of 1100 μmol/min per g protein and K _m = 15 μM for ADP, and a V _max of 900 μmol/min per g protein and K _m = 9 μM for ATP.

【 授权许可】

Unknown