Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle.
The photoaffinity analog of ATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [alpha-32P]Bz2ATP results in covalent binding of [alpha-32P]Bz2ATP to the 450-kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146 +/- 0.087 (n = 2) was estimated. The covalent binding of [alpha-32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P = ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold. The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [alpha-32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [alpha-32P]Bz2ATP-labeled 76-kDa tryptic fragment. Digestion of either the [alpha-32P]Bz2ATP-labeled 450-kDa or the 76-kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39-kDa fragment is the precursor of the 27-kDa and 13-kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27-kDa and 13-kDa fragments of the ryanodine receptor protein.
Zarka
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Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Shoshan-Barmatz
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010777:Photochemistry; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel
D000345:Affinity Labels; D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate