Isolation of the ADP, ATP carrier as the carboxyatractylate . protein complex from mitochondria.
The procedure for the isolation from mitochondria of the undenatured ADP, ATP carrier is described. The condition of retaining the nativity are elaborated. 1. As indicator for the ADP, ATP carrier (35S)- or (3H) carboxyatractylate were used. By preloading the mitochondria with carboxyatractylate, a stable carboxyatractylate . protein complex could be retained after solubilization with Triton X-100. Among the polyoxyethylene detergents emulphogen is also solubilizing, whereas Brij and Lubrol fail to solubilize. 2. When unloaded mitochondria are solubilized the capacity for binding carboxyatractylate disappears rapidly, particularly at 20 degrees C. 3. When mitochondria are preloaded with atractylate, the binding after solubilization with Triton X-100 is considerably lower than with carboxyatractylate, indicating that the high affinity of carboxyatractylate is required for effectively protecting the protein. 4. For purification hydroxyapatite is most effective. The carboxyatractylate-protein complex appears in the pass-through whereas the bulk of other mitochondrial proteins are retained such that a 7-fold purification is obtained. The nonadsorptivity to hydroxyapatite is dependent on the undenatured state maintained in the carboxyatractylate . protein complex. 5. Subsequent gel filtration on Sepharose results in a 1.5-fold further enrichment of specific carboxyatractylate binding up to 17 mumol/g protein, corresponding to a 10-fold purification from mitochondria. This value cannot be increased with further measures. 6. At the last purification step, in sodium dodecyl sulfate polyacrylamide gel electrophoresis virtually a single band of 30 000 molecular weight is found, confirming the purity at this stage. A molecular weight of 60 000 is calculated from the carboxyatractylate binding, indicating that the carboxyatractylate protein complex consists of two 30 000 subunits. From this the protein share of the ADP, ATP carrier in beef heart mitochondria can be calculated to amount to 9.5%9 7. The intact carboxyatractylate . protein complex is protected against proteolytic degradation. The release of carboxyatractylate ensues a conformational change of protein as assayed by conformation specific antibodies, concomitant with unmasking of proteolytic site as assayed by tryptic digestion. 8. The amino acid composition indicates hydrophobicity (39% polarity) and a high content of basic amino acid such as lysine and arginine. There is 1.5 mol percent cysteine and a blocked N-terminal. 9. From the solubilized complex (35S) carboxyatractylate can be removed by carboxyatractylate, ADP and ATP but not by ITP, etc., indicating the presence of recognizing sites specific fof ADP, ATP and therefore, identity with the ADP, ATP carrier. 10. Other reported procedures for isolating the ADP, ATP carrier are shown to either fail or have lower yield than the present, original procedure.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000378:metabolism*; D002417:Cattle; D003902:Detergents; D006027:Glycosides / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000302:isolation & purification* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D011487:Protein Conformation
D000596:Amino Acids; D003902:Detergents; D006027:Glycosides; D001278:Atractyloside; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; C003853:carboxyatractyloside