31P NMR saturation-transfer study of the in situ kinetics of the mitochondrial adenine nucleotide translocase.
The exchange of intramitochondrial ATP (ATP(in)) for extramitochondrial ATP (ATP(out)) was measured by using 31P NMR spectroscopy over a range of temperatures in isolated rat liver mitochondria oxidizing glutamate and succinate in the presence of external ATP but no added ADP (state 4). The rate of this exchange is more than an order of magnitude faster than rates reported previously that were determined by using isotopic techniques in the presence of oligomycin, the potent ATPase inhibitor. Differences are ascribed in part to the low levels of matrix ATP present in oligomycin-treated mitochondria. The addition of oligomycin to mitochondrial suspensions decreases intramitochondrial ATP levels from 17 +/- 3 (SEM) nmol/mg of protein in state 4 to 1.51 +/- 0.1 nmol/mg of protein in the presence of inhibitor at 8 degrees C. Simultaneously, transporter flux falls from 960 +/- 55 nmol/min.mg to undetectable levels (less than 300 nmol/min.mg). Although transport rates are much faster when measured by saturation-transfer than by conventional isotopic methods, the enthalpy values obtained by determining the effect of temperature on flux are very similar to those reported in the past that were determined by using isotopic techniques. Intramitochondrial ATP content regulates the rate of the ATP(in)/ATP(out) exchange. At 18 degrees C, the concentration of internal ATP that produces half-maximal transport rate is 6.6 +/- 0.12 nmol/mg of mitochondrial protein. The relationship between substrate concentration and flux is sigmoidal and is 90% saturated at 11.3 +/- 0.18 nmol/mg of mitochondrial protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Masiakos
P T
PT
Department of Cellular, Milton S. Hershey Medical Center, College of Medicine, Pennsylvania State University, Hershey 17033.
Williams
G D
GD
Berkich
D A
DA
Smith
M B
MB
LaNoue
K F
KF
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D010758:Phosphorus; D051381:Rats; D011919:Rats, Inbred Strains
D010758:Phosphorus; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases