Adenine nucleotide translocase greatly increases the partition of trinitrophenyl-ATP into reduced Triton X-100 micelles.
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.
Tummino
P J
PJ
Institute of Gerontology, University of Michigan, Ann Arbor 48109.
Gafni
A
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives; D002417:Cattle; D002850:Chromatography, Gel; D003902:Detergents; D004735:Energy Transfer; D007700:Kinetics; D008433:Mathematics; D008823:Micelles; D008929:Mitochondria, Heart / Q000201:enzymology; D000226:Mitochondrial ADP, ATP Translocases / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols; D013050:Spectrometry, Fluorescence / Q000379:methods
D003902:Detergents; D008823:Micelles; D001278:Atractyloside; D011092:Polyethylene Glycols; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D017830:Octoxynol; D000226:Mitochondrial ADP, ATP Translocases; C003853:carboxyatractyloside