The isolation and reconstitution of the ADP/ATP carrier from wild-type Saccharomyces cerevisiae. Identification of primarily one type (AAC-2).
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 reconstituted liposomes with a Vmax of 1100 mumol/min per g protein and Km = 15 microM for ADP, and a Vmax of 900 mumol/min per g protein and Km = 9 microM for ATP.
Knirsch
M
M
Institut für Physikalische Biochemie, Universität München, FRG.
Gawaz
M P
MP
Klingenberg
M
M
D001278:Atractyloside / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D003488:Cyanogen Bromide; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000302:isolation & purification* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D001278:Atractyloside; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; D003488:Cyanogen Bromide; C003853:carboxyatractyloside