The ADP/ATP carrier from yeast (AAC‐2) is uniquely suited for the assignment of the binding center by photoaffinity labeling

FEBS Letters

Mayinger, Peter¹  Winkler, Edith¹  Klingenberg, Martin¹ 

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

关键词: ADP/ATP carrier;    Photoaffinity labeling;    Azido-ATP;    2-;    Azido-ATP;    8-;    (Saccharomyces cerevisiae);    C10E5;    n-decylpentaoxyethylene;    C12E8;    n-dodecyloctaoxyethylene;    octyl-POE;    n-octylpolyoxyethylene;    PMSF;    phenylmethylsulfonyl fluoride;    SDS-PAGE;    SDS-polyacrylamide gel electrophoresis;    CAT;    carboxyatractyloside;    YWM;    yeast wildtype mitochondria;   

DOI  :  10.1016/0014-5793(89)80576-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The ADP/ATP carrier from yeast was photoaffinity-labeled in mitochondria with 2-azido-[α-³²P]ATP in a binding-center-specific, i.e. carboxyatractylate-sensitive, manner. After isolation, fragmentation possibilities unique for the yeast AAC-2 could be exploited to assign the insertion to a narrow range of the sequence. The CNBr fragment 115–210 contained all the incorporated label which corresponds to the second domain within the triple-domain primary structure of the AAC. With hydroxylamine cleavage directed to the Asn 171-Gly 172 site, all the label was found in the C-terminal 16 kDa fragment. Thus the 2-azido-ATP incorporation is clearly delimited to the 172–210 segment. 8-Azido-[α-³²P]ATP could be site-specifically incorporated only in isolated AAC since it has a much lower affinity for AAC than 2-azido-ATP. The label was also exclusively found in the 172–210 region. With both forms no incorporation into the C-terminal region was found, as claimed for bovine AAC. The labeled segment contains Lys 179 and 182 which are homologous to bovine Lys 162 and 165 and which have been proposed to be in the translocation path.

【 授权许可】

Unknown