Biophysical Journal 63: 1071-1080 (1992)
© 1992 the Biophysical Society

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Adenine nucleotide translocase greatly increases the partition of trinitrophenyl-ATP into reduced Triton X-100 micelles.

Institute of Gerontology, University of Michigan, Ann Arbor 48109.

ABSTRACT

The presence of adenine nucleotide translocase (ANT) was found to greatly enhance the partitioning of the ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) into reduced Triton X-100 micelles. The protein's effect was studied through the quenching of fluorescence of purified ANT, irreversibly inhibited by carboxyatractyloside (CAT), solubilized in reduced Triton X-100 micelles. The dependence of quenching of the protein's time-resolved tryptophan fluorescence on TNP-ATP concentration was measured and found to follow a Stern-Volmer mechanism. However, the calculated quenching constant was too large to be accounted for by the aqueous TNP-ATP concentration. Experiments were therefore conducted to determine the partitioning of the quencher between the three phases present: aqueous, protein-free micelle, and protein micelle; a system also described by the equation of Omann, G. M., and M. Glaser (1985. Biophys. J. 47:623-627.). By measuring the dependence of the apparent quenching rate constant on the protein concentration and protein/micelle ratios, this equation was used to calculate both the quencher partition coefficient into protein-free micelles (Pm) and into protein-micelles (Ppm), as well as the bimolecular quenching rate constant (kpm) in protein micelles. From the quenching experiments, kpm = 5.0 x 10(8)M-1s-1,Pm = 290 and pyrene quenching experiment to be 325, and by a rapid filtration experiment to be 450. Clearly, the presence of the integral membrane protein ANT-CAT in reduced Triton X-100 micelles greatly increases the partition of TNP-ATP into the micelle. ANT alters the properties and thus, the structure of the detergent micelle, which has direct implications for the use of detergent micelles as a model system for membrane proteins and may indicate that analogous effects occur in the mitochondrial membrane.