The interaction and photolabeling of myosin subfragment 1 with 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate.
The photoprobe 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (Bz2ATP) was used to characterize the nucleotide-binding site of myosin subfragment 1 (SF1). Improved synthesis and purification of Bz2ATP are reported. 1H NMR and ultraviolet spectroscopy show that Bz2ATP is a 60:40 mixture of the 3'(2')-ribose isomers and that the epsilon M261 is 41,000 M-1 cm-1. Bz2ATP is hydrolyzed by SF1 comparably to ATP in the presence of actin or K+, NH4+, or Mg2+ ions; and the product, Bz2ADP, has a single binding site on SF1 (K'a = 3.0 X 10(5) M-1). [3H]Bz2ATP was photoincorporated into SF1 with concomitant loss of K+-EDTA-ATPase activity. Analysis of photolabeled SF1 showed that the three major tryptic peptides (23, 50, and 20 kDa) of the heavy chain fragment and the alkali light chains were labeled. The presence of ATP during irradiation protected only the 50-kDa peptide, indicating that the other peptides were nonspecifically labeled. If Bz2ATP was first trapped on SF1 by cross-linking the reactive thiols, SH1 and SH2, with p-phenylenedimaleimide, only the 50-kDa tryptic peptide was labeled. These results confirm and extend previous observations that [3H]Bz2ATP trapped on SF1 by cobalt(III) phenanthroline photolabeled the same 50-kDa peptide (Mahmood, R., and Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959). Thus, the 50-kDa peptide is labeled with or without thiol cross-linking, indicating that the relative position of SH1 and SH2 does not affect the labeling pattern.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D009682:Magnetic Resonance Spectroscopy; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010782:Photolysis; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D015879:Myosin Subfragments; D010446:Peptide Fragments; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins