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The isolation and reconstitution of the ADP/ATP carrier from wild‐type Saccharomyces cerevisiae Identification of primarily one type (AAC‐2)
Author(s) -
Knirsch Martin,
Gawaz Meinrad P.,
Klingenberg Martin
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80577-0
Subject(s) - saccharomyces cerevisiae , isolation (microbiology) , identification (biology) , biochemistry , chemistry , biophysics , biology , yeast , microbiology and biotechnology , botany
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.