The use of 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate for studies of nucleotide interaction with sarcoplasmic reticulum vesicles.
The ATP analogue 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was used to study nucleotide site stoichiometry and interactions in sarcoplasmic reticulum (SR) vesicles. The TNP absorption spectrum in the visible region undergoes a specific change upon binding of the nucleotide to SR ATPase. Equilibrium binding was therefore measured by differential spectrophotometry. In parallel experiments, TNP-[gamma-32P]ATP binding was measured directly by radioisotope distribution. The maximum number of nucleotide sites was estimated to be 8 nmol/mg of protein in SR vesicles. These binding sites can be separated into two distinct groups of different affinity. Accordingly, 10-100 microM ATP displaces a maximum of only 4 nmol of TNP-ATP/mg of protein from high affinity sites which are considered to be specific for enzyme catalysis. Free TNP-ATP in aqueous solution yields a weak fluorescence signal which is slightly increased upon binding of the analogue to SR ATPase. However, a pronounced fluorescence enhancement and a spectral change are observed when ATP is added in concentrations permitting partial occupancy of the specific sites by TNP-ATP. This effect is strictly dependent on ATP utilization by the SR ATPase, inasmuch as it requires Ca2+, and it is not produced by adenyl-5'-yl-imidodiphosphate. The fluorescence enhancement is reversible upon exhaustion of added ATP. It is concluded that TNP-ATP acts as a reporter of an ATPase conformational change following enzyme phosphorylation of the catalytic site by ATP, and that the observed conformational change is operative in the mechanism of calcium site translocation for active transport. Furthermore, the sensitivity of the bound analogue to the phosphorylation reaction is likely to be related to nucleotide regulation of enzyme turnover.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases