Studies on the Adenine Nucleotide Translocase from Rat Liver Mitochondria.

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Abstract
Solubility of mitochondrial membranes in various solvent systems was determined quantitatively. The most effective agent was the anionic detergent, sodium dodecylsulfate, which solubilizes 90% of the protein at the concentration of 0.1% followed by Triton X-100 (70%), sodium deoxycholate (60%), Brij 56 (50%) and guanidine hydrochloride (40%) at a concentration of 2 M. Affinity chromatography of a clear 0.1% sodium dodecylsulfate solution of digitonized mitochondria on Sepharose 4B containing carboxyatractylate always resulted in the separation of 2 fractions, 1 of which was not retained by the column and the other which could be obtained after elution with 2% sodium dodecylsulfate. The retained protein showed a high binding specificity for ATP and [3H]atractylate when compared with the unretained fraction. The amount of bound [3H]atractylate or carboxyatractylate-sensitive binding of ATP was 10.5 .+-. 4 nmol/mg protein, and 22 .+-. 8 nmol/mg protein, respectively. The major component within the retained fraction, comprising 85% of the total weight, was protein, followed by phospholipids (14%) and approximately 1% triglycerides. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed a major (95%) and a minor (5%) component with an apparent MW of 26,000 .+-. 1000 and 8300 .+-. 400, respectively. The gels did not stain for carbohydrates. Ultracentrifugal analysis showed 1 symmetrical boundary. Double immunodiffusion analysis gave a single precipitin line with the corresponding antiserum. [14C]ADP exchange of digitonin particles was completely inhibited by an antiserum to the carboxyatractylate binding protein fraction, whereas the adenine nucleotide transport of intact mitochondria remained unaffected. In the presence of specific immunoglobulins state-3 respiration rate of digitonin particles was prolonged and reduced by approximately 25%. State-4 respiration rate was unaffected.